production of polyhydroxybutyrate during the...
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PRODUCTION OF POLYHYDROXYBUTYRATE DURING THE
TREATMENT OF PALM OIL MILL EFFLUENT
ZULKAFLI BIN MAT RANI
UNIVERSITI TEKNOLOGI MALAYSIA
i
PRODUCTION OF POLYHYDROXYBUTYRATE DURING THE
TREATMENT OF PALM OIL MILL EFFLUENT
ZULKAFLI BIN MAT RANI
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Engineering (Environmental)
Faculty of Chemical Engineering
Universiti Teknologi Malaysia
JUNE 2014
iii
Dedicated with love and gratitude to Bonda, Allahyarham Ayahanda, Isteri (Rohana),
Anakanda (Zafirah Amni, Zahidah Amni, Zahirah Amni, Muhd Zakwan, Muhd Zul
Aiman Adib, Muhamad Zul Iqbal Hafiz), Kekanda and Adinda
iv
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to the following individuals who
have helped me in the completion of my thesis:
Dato’ Prof. Dr. Ir. ZainiUjang, former Vice Chancellor, UniversitiTeknologi
Malaysia for his encouragement and support.
Assoc. Prof. Dr Zainura Zainon Noor, my supervisor, for her timely
guidance, endless patience and constant encouragement.
Ir. Dr. Mohd Fadhil Md Din, for his valuable guidance, discussion and
comments.
The management and staff of PPNJ Kahang Palm Oil Mill especially the
laboratory staff for their full cooperation, help and technical assistance.
Laboratory staff at IPASA Universiti Teknologi Malaysia for the analysis and
technical assistance.
My wife, daughters and sons for their encouragement, patience and
endurance.
v
ABSTRACT
Palm oil mill effluent (POME) is one of the most problematic waste,
generated through out the year from the palm oil mill industry. The excessive
production of this effluent without proper control results in serious environmental
problems. However POME can be a cheap carbon source to produce bioplastic, like
polyhydroxyalkanoate (PHA) or polyhydroxybutyrate (PHB) in the activated POME
treatment system. In this study the sludge in anaerobic pond A1, anaerobic pond A2
and facultative pond F1 in the treatment plant at PPNJ, Kahang palm oil mill, were
used as substrates to produce maximum amount of PHB. By using a 10 liters of
laboratory reactor, the existing bacteria propagated in growth phase and the
optimum formation of PHB in the bacterial cell was studied during accumulation
phase under feast and famine process. The range of volatile fatty acid (VFA) was
11.43 mg/l to 577.22 mg/L for the scope of this study. Analytical measurement and
PHB quantification were conducted by using gas chromatography. The highest
production of PHB was found at anaerobic pond A1 (0.281 mt per day process and
0.0003 mt/mt fresh fruit bunch), at concentration of 0.11 g/L.
vi
ABSTRAK
Efluen kilang minyak sawit (POME) yang dikeluarkan semasa pemprosesan
buah sawit kini masih memberi masalah kepada industri sawit. Pengeluaran yang
berlebihan jika tidak dikawal boleh mengakibatkan masalah besar terhadap alam
sekitar. Namun begitu POME telah dikenalpasti boleh digunakan sebagai punca
karbon yang murah untuk pengeluaran bioplastik, seperti polihidroksialkanoit
(PHA) dan polihidroksibutirat (PHB). Dalam kajian ini enapcemar dari kolam
anaerobik A1, kolam anaerobik A2 dan kolam fakultatif F1, semasa proses rawatan
efluen di kilang Sawit PPNJ Kahang, digunakan sebagai substrat untuk menghasilkan
jumlah PHB yang maksima. Dengan menggunakan bioreaktor makmal berkapasiti
10 liter, bakteria sedia ada di kolam dibiakkan melalui fasa pertumbuhan, dan
pembentukan PHB secara optima di dalam sel bakteria telah dikaji semasa fasa
penumpukan di dalam proses ‘feast and famine’. Julat asid lemak tak menentu
(VFA) di dapati dari 11.43 mg/L hingga 577.22 mg/L. Jumlah PHB terhasil di
analisa dengan menggunakan gas kromatografi. Pengeluaran PHB boleh dihasilkan
dengan kuantiti maksima dari kolam anaerobik A1 (0.281 metrik tan setiap hari
buah sawit diproses dan 0.0003 metrik ton bagi setiap metrik ton buah sawit
diproses), pada kepekatan PHB 0.11 g/L.
vii
TABLE OF CONTENTS
CHAPTER TITLE PAGE
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xv
LIST OF SYMBOLS xvi
LIST OF APPENDICES xvii
1 INTRODUCTION 1
1.1 Background of the Study 1
1.2 Problem Statement 4
1.3 Objectives of the Study 4
1.4 Scope of the Study 5
1.5 Significance of the Study 6
1.6 Thesis Layout 6
viii
2 LITERATURE REVIEW 8
2.1 Introduction 8
2.2 Palm Oil industry 9
2.2.1 Palm Oil Milling 9
2.2.2 Palm Oil Industry in Malaysia 14
2.3 Environmental Consideration 17
2.3.1 Characteristics of POME and its treatment system 19
2.4 Polyhydroxybutyrate and Polyhydroxyalkanoate 21
2.4.1 PHB and PHA as Bioplastic from Microorganism 21
2.4.2 Polyhydraxyalkanoates (PHA) from Waste Water 25
2.5 Recovery of PHAs from Palm oil derivative and POME 26
3 METHODOLOGY 29
3.1 The Framework of Study 29
3.2 Site 30
3.3 Materials and Method. 33
3.3.1 Laboratory Reactor 33
3.3.2 Pre-extraction Method 36
3.3.2.1 Innoculation 36
3.3.2.2 Growth Phase 37
3.3.2.3 Accumulation Phase. 38
3.3.2.4 Feast and famine period for biosludge
harvesting
39
3.4 PHB Extraction and Analytical Method 41
3.4.1 Extraction 41
3.4.2 PHB Quantification by Gas Chromatography (GC)
Method
45
3.5 Specific calculations 48
3.5.1 Concentration of PHB by using Gas
Chromatography
48
3.5.2 Amount of POME generated and PHB produced 48
4 RESULTS AND DISCUSSION 51
4.1 Introduction 51
4.2 Point of Sampling 52
ix
4.2.1 Anaerobic Pond No 1 (A1) 52
4.2.2 Anaerobic Pond No 2 (A2) 55
4.2.3 Facultative Pond No 1 (F1) 56
4.3 Growth and Accumulation Phase 57
4.3.1 Growth Phase 57
4.3.2 Accumulation Phase 58
4.3.2.1 Accumulation of PHB in Sample Pond A1
and the Best Time to Harvest Sample
59
4.3.2.2 Accumulation of PHB in Sample Pond A2
and the Best Time to Harvest Sample
65
4.3.2.3 Accumulation of PHB in Sample Pond F1
and the Best Time to Harvest Sample
70
4.4 Statistical Analysis of Data Obtained 75
4.5 Biomass Harvesting Time and Period 75
4.6 PHB Quantification 76
5 CONCLUSION AND RECOMMENDATIONS 81
5.1 Conclusion 81
5.2 Recommendations 83
REFERENCES 84
Appendices A-J 89-174
x
LIST OF TABLES
TABLE NO. TITLE PAGE
2.1 Number of Mills and processing Capacity for the
year 2010 (Tonnes FFB/year)
11
2.2 Oil Palm Planted Area: 2010 (HECTARES).-
Source MPOB
15
2.3 Yield of FFB, 2010 (tonnes/hectare) 16
2.4 Discharge standard for palm oil mill effluents
(Environmental Quality Act 1974)
18
2.5 The characteristics of Palm Oil Mill Effluent 19
2.6 Polyhyroxyalkanoates (PHA ) family 22
2.7 PHA production from various waste water 26
3.1 Detailed description of volume and hydraulic
retention time of ponds (calculation based on
normal daily production)
32
3.2 Composition in growth phase inoculation for
microbial population
37
3.3 Fill, react and decant process during accumulation
phase
38
3.4 The process and average PHA production from the
selected studies
39
3.5 Analytical measurement performed during 8 hours
cycle
40
4.1 Characteristics of POME in the Anaerobic Pond
No 1 (A1)
54
4.2 Characteristic of POME in the Anaerobic Pond
No 2 (A2)
55
4.3 Characteristic of POME in the Facultative Pond F1 56
4.4 Percentage of sludge in growth phase 57
xi
4.5 Data recorded in experiment batch I (growth phase
process)
58
4.6 Total number of experiment done for growth phase
in batch 2 in laboratory reactor
58
4.7 Total number of experiment done for feast and
famine process in the laboratory reactor.
59
4.8 Batch 3, mixing of substrate from pond A1 and
observation.
60
4.9 Results of the selected experiment for sample A1. 60
4.10 Batch experiment showing mixing rate of substrate
from pond A2 sample and its observation
65
4.11 Selected result of experiments from the samples of
A2
66
4.12 Batch of experiment mixing rate of substrate from
pond F1 and its observation.
70
4.13 The sample result of experiment from pond F1 71
4.14 The best time for harvesting the sample 75
4.15 The results of dry sludge from samples A1, A2 and
F1
76
4.16 Percentage concentration of PHB (summary of GC
results)
78
4.17 Percentage of PHB (summarize of GC results),
calculated raw PHB per day and raw PHB per FFB
processed.
79
xii
LIST OF FIGURES
FIGURE NO. TITLE PAGE
1.1 Effluent treatment system at PPNJ Kahang Palm
Oil Mill
3
2.1 Complete Process of Palm Oil Milling 12
2.2 The components of FFB after Sterilization 13
2.3 The world major exporters of oils & fats, 2010 17
2.4 General Structure of Polyhydroxyalkanoates
(PHA)
22
2.5: Scanning and transmission electron
microphotographs of the source for PHA
(Source: DUKE university iGem team -2009).
23
2.6 Fatty acid pathway of E.coli enhancing PHA
production (Adapted from Park and Lee, 2003)
23
2.7 PHA granule 24
3.1 Framework set up for overall process 30
3.2 Flow diagram of PPNJ Kahang Palm oil mill
effluent treatment plant and point of sampling
31
3.3 Anaerobic and facultative ponds at PPNJ
Kahang Palm Oil Mill
33
3.4 Schematic diagram of laboratory reactor 34
3.5 The components of laboratory reactor 35
3.6 The picture of laboratory reactor 35
3.7 Flow diagram for biosludge extraction 42
3.8 Samples harvested from laboratory reactor after
feast-famine period
43
3.9 Biomass sludge before centrifuge 43
3.10 Biomass sludge after centrifuge 44
xiii
3.11 Dried sludge 44
3.12 Samples digested at 100ºC for two hours using
reflux digester
45
3.13 The sample of PHA detection in Gas
Chromatography
46
3.14 The calibrated sample of poly (3-
hydroxybutyrate)
46
3.15 GC method for the determination of PHB 47
4.1 PPNJ Kahang Palm Oil Mill 53
4.2 Close up view for anaerobic pond No 1 (A1) 53
4.3a DO and pH (data from Table 4.9) taken at 27
April 2009
61
4.3b VFA, COD, and SS (data from Table 4.9) taken
at 27 April 2009.
61
4.4 The superimpose of VFA pattern in 8 hours
under feast and famine condition for sample
pond A1
62
4.5 The superimpose of COD pattern in 8 hours
under feast and famine condition for sample
taken from pond A1.
62
4.6 The average of VFA and COD pattern in 8 hours
under feast and famine condition for sample
taken from pond A1.
63
4.7(a-e) Average of VFA, COD, pH, DO and SS pattern
in 8 hours under feast and famine condition in
sample pond A1.
64
4.8a Patern of pH and DO during feast and famine
(from Table 4.11)
66
4.8b Patern of VFA, COD and SS during feast and
famine (from Table 4.11)
67
4.9 The superimpose of VFA in 8 hours under feast
and famine condition in sample pond A2.
67
4.10 The superimpose of COD pattern in 8 hours
under feast and famine condition in sample A2.
68
4.11 Average of VFA and COD pattern in 8 hours
under feast and famine condition in sample pond
A2.
68
4.12(a-e) Average of VFA, COD,pH, DO and SS pattern
in 8 hours under feast andfamine condition in
sample pond A2.
69
4.13a Pattern of pH and DO during feast and famine
(from Table 4.13)
71
xiv
4.13b Pattern of VFA, COD, and SS during feast and
famine (from Table 4.13).
72
4.14 The superimpose of VFA pattern in 8 hours
under feast and famine condition in sample pond
F1.
72
4.15 Superimpose of COD pattent in 8 hours under
feast and famine condition in sample taken
from F1
73
4.16 Average of VFA and COD pattern in 8 hours
under feast and famine condition in sample taken
from pond F1
73
4.17(a-e) Average of VFA, COD, pH, DO. SS, and COD
pattern in 8 hours under feast and famine
condition in sample pond F1.
74
4.18 Dried biosludge extracted for each of the pond
sample.
76
4.19 GC’s results for STANDARD PHB 77
4.20 GC’s results for Anaerobic 1 (sample 1) 78
xv
LIST OF ABBREVIATIONS
AN - Ammonical Nitrogen (mmol/l or mg/l)
CDW - cell dry weight (g/l)
COD - chemical oxygen demand c-mmol/l or mg/l)
CPO - crude palm oil
DO - dissolved oxygen (Oxygen Saturated) (mg/l)
DOE - Department of Environment
EFB - Empty fruit bunch
GC - Gas Chromatography
HRT - Hydraulic retention time
MPOB - Malaysian Palm Oil Board
Mt - Metric tone
PHA - Polyhydroxyalkanoates
PHB - Poly-β- hydroxybutyrate
POME - Palm oil mill effluent
SBR - Sequencing batch reactor
SS - Suspended Solid
Temp - Temperature (º C)
VFA - Volatile Fatty Acid
xvi
LIST OF SYMBOLS
CO² - carbon dioxide (mmol/l or mg/l)
HAc - acetic acid (mg/l)
HBt - butyric acid (mg/l)
HPr - propionic acid (mg/l)
NOз - nitrate (mg/l or mmol/l)
O² - oxygen (mg/l or mmol/l)
PO4 - phosphate (mg/l or mmol/l)
xvii
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Sample calculation for optimum recovery of
PHB from 60mt FFB/hr capacity Palm Oil Mill
89
B Characteristics of POME in the effluent
treatment pond A1, A2, and F1, at PPNJ Kahang
Palm Oil Mill
92
C Accumulation of PHB (feast and famine
processed) for sample obtained from pond A1
106
D Accumulation of PHB (feast and famine
processed) for sample obtained from pond A2
122
E Accumulation of PHB (feast and famine
processed) for sample obtained from pond F1
132
F Gas chromatography results for sample obtained
from pond A1
153
G Gas chromatography results for sample obtained
from pond A2 and F1
159
H Analysis of correlation for overall obtained datas 168
I Drawing representing overall study and results 169
J Mass balance (for processing 1200mt FFB/day) 170
1
CHAPTER 1
INTRODUCTION
1.1 Background of the Study
Biomaterials are natural products that are widely used in biotechnological
applications. They are biodegradable, environmental friendly and do not cause toxic
effects.
Bioplastics are polymers and are considered to be a special type of bio-
material. Micro-organisms generate a source of bioplastics and biopolymers from
renewable sources. Although it is currently more expensive than plastic derived
from petrochemicals, bacteria have proved to help yield bioplastics with comparable
properties. The most widely used microbial bioplastics are polyhydroxyalkanoates
(PHA), polyhydroxybutyrate (PHB) and other different monomers and derivatives.
Palm oil mill effluent (POME) is a liquid waste, generated during the
processing of fresh fruit bunches to produce crude palm oil and palm kernel. POME
is non-toxic in nature but must be treated to acceptable quality before it can be
discharged into the water course. The biological treatment of POME comprises both
2
anaerobic and aerobic treatment. POME can serve as a cheap substrate for
commercial scale production of PHB/PHA as it is available in huge quantities from
406 mills present in Malaysia as listed in Table 2.1.
Fresh POME or mixed raw effluent consists of 95 to 96% water, 0.6 to 0.7%
oil, 4 to 5% total solids and 2 to 4% of suspended solids and without proper
treatment it contributes to a major source of water pollution in Malaysia.
POME has been reported to be suitable for PHA production (Hassan et al.,
1997, 2002; Md Din et al., 2004, 2006, Mumtaz et al., 2010). Fresh POME is
usually present in the complex form containing mainly triacyglycerides,
diacylglycerides, monoacylglycerides and some other derivatives like fatty acids
(Alias and Tan, 2005).These form cannot be directly utilized by PHA producing
bacterial species for PHA synthesis. Hydrolysis and acetogenesis processes in
anaerobic treatment have been proposed to reduce the POME characteristics and also
converting undissolved compound of POME to simpler short–chain VFAs (Volatile
Fatty Acids) compounds like acetic, butyric, and propionic acids. The VFAs will be
utilized by PHA- producer for PHA production (Lee and Yu, 1997). Hassan et al.,
(1997, 2002) found that 7 g/l of organic acids could be obtained from POME and can
be utilized for PHA production with pure culture. The high organic acids contained
in POME are fit to be used as carbon source to produce PHA/PHB.
PPNJ Kahang Palm Oil Mill is owned by Pertubuhan Peladang Negeri Johor.
Its processing capacity of 60 mt fresh fruit bunch (FFB) per hour generating POME
as a waste is average at about 800mt per day. The POME has to be treated according
to the Department of Environment’s Regulations. In the mill, POME is treated in
two anaerobic ponds, three aerobic ponds, followed by extended aeration plant for
polishing before final discharge to river as shown in Figure 1.1.
3
Figure 1.1 Effluent treatment system at PPNJ Kahang Palm Oil Mill
Considering that about 300,000 mt of fresh fruit bunch is processed a year, as
budgeted for every year, PPNJ Kahang Palm oil Mill has approximately 210,000 mt
of POME per year that could be used as a cheap carbon source in producing
PHB/PHA.
The aim of this study is to determine the pond and the maximum amount of
PHB that can be produced from the entire system of effluent treatment plant at PPNJ
Kahang Palm oil mill. The amount of PHB produced will be calculated base on one
tonne of FFB processed, as well as per day of FFB processed at PPNJ Kahang Palm
oil mill.
4
1.2 Problem Statement
The problem statements in this study are:
i. PPNJ Kahang Palm Oil Mill with processing capacity of 60 mt
FFB/hour, discharges 48 mt POME/hour or 960 mt of POME/day to
anaerobic and facultative ponds for treatment. The POME, which is
rich in organic carbon, could be used as a cheap carbon source to
produce biodegradable plastic (PHA/PHB). This could be useful in
converting palm oil mill waste to wealth.
ii. Where and how maximum PHB can be produced from the actual
treatment system, need to be studied. In PPNJ Kahang palm oil mill,
there are two anaerobic ponds (no 1 and 2) followed by three aerobic
ponds at different range of BOD, pH and VFA concentrations. The
maximum quantities of PHB to be produced per day and per metric
tonne of FFB processed could be calculated and identified.
iii. Anaerobic bacteria from the existing anaerobic pond are possible to
be used as PHB producer, without the used of any additional inocula.
As stated in previous studies, bacteria have to be properly conditioned
under feast and famine to produce higher amounts of PHB.
1.3 Objectives of the Study
The objectives of this study are:-
a. To determine the pond which produces maximum amount of PHB (in
metric tonne PHB per metric tonne of fresh fruit bunch processed) in the
actual POME treatment system at PPNJ Kahang Palm Oil Mill.
5
b. To quantify the optimum concentration of PHB produced in the actual
POME treatment system in percentage on sample and in g/L.
c. To determine the quantity of PHB that can be produced per day, from the
processing of 60 mt FFB/hour (PPNJ Kahang Palm Oil Mill), and the
maximum PHB that can be produced from one tonne of fresh fruit bunch
processed , where POME is the sole carbon source.
1.4 Scope of the Study
The scopes of this study are as follows:
i. Samples for this study were taken at the potential ponds along the
POME treatment system at PPNJ Kahang Palm Oil Mill, both
anaerobic and aerobic treatment, for maximum recovery of PHB.
ii. 10 liters laboratory scale reactor was used and operated based on
sequential batch reactor (SBR) system. Aeration time needed was
studied and set for feast and famine condition to get optimum
recovery of PHB at every point sample.
iii. Analytical measurement and PHB quantifications were conducted at
UTM laboratory according to the standard methods used for the
examination of water and waste water (APHA, 2000).
6
1.5 Significance of the Study
Three important aspects which will be beneficial in achieving the
objectives of this study are:
i) Investigation of abundance POME, which could be useful in converting it to
harmless and beneficial substrates (biodegradable plastic). This study will
convert oil palm industry waste to wealth.
ii) PHB extraction from POME will contribute to palm oil industry in
diversifying their non-food uses at a low cost of production (by using
available free carbon source from the estimated 70 million tonne available
POME per year).
iii) POME is a major cource of water pollution in Malaysia. From the total
amount of fresh fruit bunch (FFB) processed at palm oil mills, 80 percent of
POME is generated. Thus, this study provides an alternative mean to reduce
the pollution load due to the separation of solid biomas and carbon uptake to
produce PHB.
1.6 Thesis Layout
The remaining part of the thesis is organized into four chapters as follow:
i. Chapter 2 contains literature review related to the topics discussed
in this study. This chapter explains the existence of POME during
the processing of fresh fruit bunch to produce crude palm oil and
palm kernel, and the characteristic of POME to serve as substrate
and to extract PHA/ PHB. Description of PHA/PHB as
biodegradable plastic generated by the microorganism from waste
7
water (as carbon source), and the potential of PHA/PHB as a
revenue for palm oil industry are also discussed in this chapter.
ii. Chapter 3 presents the implementation of the study in terms of
detailed theory and methodology, including processes and
equipment.
iii. Chapter 4 examines the results of the study at every point of
sample collection along the POME treatment system. Specifically,
this chapter discusses the inoculation technique, statistical data
analysis of the obtained data and PHB quantification.
iv. Chapter 5 presents the overall finding, achievements and
conclusions of the study. The conclusions are drawn from the
experience gained throughout this study. The significance of the
findings and achievements that could be used for considerations of
future work is also discussed.
84
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