bioremediation of biodiesel and diesel contaminated
TRANSCRIPT
BIOREMEDIATION OF BIODIESEL AND DIESEL CONTAMINATED SOIL
BY PSEUDOMONAS PUTIDA
QUIN ANAK EMPARAN
A thesis submitted in
fulfillment of the requirement for the award of the
Degree of Master of Civil Engineering
Faculty of Civil and Environmental Engineering
Universiti Tun Hussein Onn Malaysia
MAY 2015
vi
ABSTRACT
As occurs to the diesel fuel, the commercialization of biodiesel and their diesel blends
can cause environmental damages due to accidental spillage. Presence of these
contaminants containing polyaromatic hydrocarbons (PAHs) in soil is toxic to humans,
plants and soil microorganisms due to their recalcitrant and mutagenic or carcinogenic
properties. Therefore, this study was conducted to suggest a new technique of treatment
to clean up the biodiesel and diesel contaminated soil by Pseudomonas putida. Spill
simulations with biodiesel, diesel and their blends in sandy gravel soil were performed
according to previous study with some modification. Briefly, 200 mL of Pseudomonas
putida was inoculated into soil samples: B5 (5% biodiesel + 95% diesel), B20 (20%
biodiesel + 80% diesel), B50 (50% biodiesel + 50% diesel), B100 (100% biodiesel) and
D100 (100% diesel). As a control sample, there is no addition of biodiesel and diesel
into the sample. All samples were stored in the incubator at 35 ºC throughout the 24
days of treatment. Samples were analyzed for: soil particle size, moisture content, pH,
total nitrogen (TN), orthophosphate, sulfate, total organic carbon (TOC), soxhlet
extraction of PAHs and enumeration of Pseudomonas putida. The measurement of all
testing parameters was carried out at interval of three days starting from Day 0 to Day
24 of bioremediation period. Results showed that the highest removal of total nitrogen
(TN), orthophosphate, sulfate, total organic carbon (TOC) and PAHs were observed in
the sample B100 with up to 70.43%, 69.47%, 68.08%, 97.66% and 96.28% removal,
respectively. The degradation rates of PAHs and survival of Pseudomonas putida were
also observed highest in the sample B100 with up to 0.149 mg/kg/day and 60 × 106
cfu/g, respectively. Based on these overall findings, it can be verified that the sample
B100 has the higher biodegradability than other samples. According to results, it can
conclude that, the capability and effectiveness of Pseudomonas putida as oil-
biodegradable agent in soil bioremediation were proved and bioremediation of
contaminated samples may be considered as a successful and feasible practice.
vii
ABSTRAK
Seperti yang berlaku pada bahan api diesel, pengkomersialan biodiesel dan campuran
biodiesel/diesel boleh menyebabkan pencemaran alam sekitar yang berpunca daripada
tumpahan secara tidak sengaja. Kehadiran bahan pencemar ini mengandungi
poliaromatik hidrokarbon (PAHs) dalam tanah adalah toksik kepada manusia, tumbuhan
dan mikroorganisma kerana sifat degil dan mutagenik atau karsinogenik. Maka, kajian
ini dijalankan untuk mencadangkan satu teknik baru dalam merawat tanah yang tercemar
dengan biodiesel dan diesel menggunakan Pseudomonas putida. Simulasi tumpahan
biodiesel dan diesel ke atas tanah kerikil berpasir dijalankan. Secara ringkas, 200 mL
Pseudomonas putida dimasukkan ke dalam sampel tanah: B5 (5% biodiesel + 95%
diesel), B20 (20% biodiesel + 80% diesel), B50 (50% biodiesel + 50% diesel), B100
(100% biodiesel) and D100 (100% diesel). Sebagai sampel kawalan, tiada penambahan
biodiesel dan diesel ke dalam sampel tanah. Semua sampel dieram dalam inkubator
selama 24 hari. Semua sampel dianalisis untuk: saiz zarah tanah, ujikaji kandungan
kelembapan, pH, nitrogen, ortofosfat, sulfat, karbon organik, pengekstakkan
poliaromatik hidrokarbon (PAHs) dan pengiraan pembiakkan Pseudomonas putida.
Ujikaji dilakukan setiap selang tiga hari bermula Hari 0 sehingga Hari 24. Data analisis
dan keputusan menunjukkan peratus penyingkiran tertinggi nitrogen, ortofosfat, sulfat,
karbon organik dah poliaromatik hidrokarbon (PAHs) didapati di dalam sampel B100
masing-masing mencapai 70.43%, 69.47%, 68.08%, 97.66% dan 96.28% penyingkiran.
Kadar penguraian PAHs dan kebolehterusan hidup Pseudomonas putida didapati juga
paling tinggi di dalam sampel B100 sehingga masing-masing mencapai 0.149
mg/kg/hari dan 60 × 106
cfu/g. Berdasarkan keseluruhan data analisis dan keputusan,
dapat disahkan bahawa sampel B100 mempunyai penguraian tertinggi daripada sampel-
sampel lain. Boleh disimpulkan bahawa kebolehan dan kecekapan Pseudomonas putida
sebagai agen pengurai minyak di dalam rawatan sampel tanah secara biologi telah
terbukti berkesan dan berjaya dilaksanakan.
viii
CONTENTS
TITLE i
DECLARATION ii
DEDICATION iii
WORK FORMED FROM JOINTLY AUTHORED
PUBLICATIONS
iv
ACKNOWLEDGEMENTS v
ABSTRACT vi
ABSTRAK vii
CONTENTS viii
LIST OF TABLES xvi
LIST OF FIGURES xvii
LIST OF SYMBOLS AND ABBREVIATIONS xx
LIST OF APPENDICES xxiii
CHAPTER 1 INTRODUCTION 1
1.1 Background 1
1.2 Problems Statement 4
1.3 Objectives of Study 6
ix
1.4 Scopes of Study 6
1.5 Limitation of Study 7
1.6 Thesis Outline
8
1.7 Conclusion 8
CHAPTER 2 LITERATURE REVIEW 9
2.1 Introduction 9
2.2 Soil and Soil Chemistry 9
2.3 Soil Sandy Characterization 10
2.3.1 Soil Texture 10
2.3.2 Soil Pores 11
2.3.3 Soil Moisture Content 11
2.3.4 Soil pH 12
2.3.5 Soil Nutrient 13
2.3.5.1 Total Nitrogen 14
2.3.5.2 Orthophosphate 15
2.3.5.3 Sulfate 16
2.3.5.4 Total Organic Carbon 17
2.4 Biodiesel 17
2.4.1 Advantages of Biodiesel 19
2.5 Petroleum Diesel 19
x
2.6 Polycyclic Aromatic Hydrocarbons 20
2.7 Mechanisms in Bioremediation 22
2.7.1 Bioaugmentation 22
2.7.2 Biostimulation 25
2.8 Bioremediation Techniques 26
2.9 In Situ Bioremediation 26
2.9.1 Intrinsic Bioremediation 26
2.9.2 Engineered In Situ Bioremediation 27
2.9.3 Bioventing 27
2.9.4 Biosparging 27
2.10 Ex Situ Bioremediation 28
2.10.1 Solid Phase Treatment 28
2.10.1.1 Landfarming 28
2.10.1.2 Composting 29
2.10.1.3 Soil Biopiles 29
2.10.2 Slurry Phase Treatment 29
2.10.2.1 Bioreactors for Bioremediation of Soil 30
2.11 Advantages of Bioremediation 30
2.12 Bioremediation of Polyaromatic Hydrocarbons in Oil
Contaminated Soil
31
2.13 Factors Affecting Bioremediation 31
xi
2.13.1 Soil Moisture 32
2.13.2 Soil pH 32
2.13.3 Oxygen Content 33
2.13.4 Nutrient Content 34
2.13.5 Temperature 35
2.14 Microorganisms in Soil 36
2.14.1 Aerobic Bacteria 37
2.14.2 Anaerobic Bacteria 38
2.14.3 Ligninolytic Fungi 39
2.14.4 Methylotrophs Bacteria 40
2.15 Bacteria as Oil-biodegradable Agent 40
2.16 Microbial Degradation Pathways of Polyaromatic
Hydrocarbons
41
2.17 Pseudomonas putida 42
2.17.1 Characterization and Significance of
Pseudomonas putida
42
2.17.2 Genome Structure of Pseudomonas putida 44
2.17.3 Bio-conversion and Degradation of Non-natural
Chemicals by Pseudomonas putida
45
2.18 Industrial Application of Pseudomonas putida Strain 45
2.18.1 Bio-based Materials 45
xii
2.18.2 Bioconversion and De Novo Synsthesis of
Chemicals
46
2.18.3 Phamaceuticals and Agrochemicals 48
2.19 Disadvantages of Pseudomonas putida 48
2.20 Previous Study on PAHs Contaminated Soil by
Pseudomonas putida
49
2.21 Hydrocarbon Extraction 53
2.22 Gas Chromatography – Mass Spectrometry 54
2.23 Quality Standard Guidelines for Soil Contamination 55
2.24 Conclusion 57
CHAPTER 3 METHODOLOGY 58
3.1 Introduction 58
3.2 Sampling Location and Technique 58
3.2.1 Soil 58
3.2.2 Biodiesel 59
3.2.3 Diesel 60
3.2.4 Pseudomonas putida 60
3.2.5 Chemicals 61
3.3 Research Methodology Overview 62
3.4 Standard Method of Experimentations 63
3.5 Preparation of Sample at Different Proportions 63
xiii
3.6 Physico-chemical Test 65
3.6.1 Soil Particle Size Test 65
3.6.2 Moisture Content Test 66
3.6.3 pH Test 67
3.6.4 Total Organic Carbon and Total Nitrogen Test 68
3.6.4.1 Standard Preparation for Total Carbon 70
3.6.4.2 Standard Preparation for Inorganic Carbon
70
3.6.4.3 Standard Preparation for Total Nitrogen 70
3.6.5 Sulfate and Orthophosphate Test 70
3.7 Optimization for Extraction 71
3.8 Soxhlet Extraction 72
3.9 Gas Chromatography – Mass Spectrometry 72
3.9.1 Preparation of PAHs Mixture Standard Solution 73
3.10 Microbiology Laboratory Works 74
3.10.1 Preparation of Nutrient Broth 74
3.10.2 Preparation of Nutrient Media (CHROM Agar
Pseudomonas)
77
3.10.3 Optimization and Pseudomonas putida Culture
Broth
78
3.11 Bacteria Count 78
3.12 Data Collection 81
xiv
3.13 Data Analysis 81
3.14 Conclusion 81
CHAPTER 4 RESULTS AND ANALYSIS 82
4.1 Introduction 82
4.2 Characterization of soil at UTHM 82
4.3 Characterization of Pseudomonas putida by Field
Emission Scanning Microscopy (FESEM)
83
4.4 Physico-chemical Test 83
4.4.1 Soil Particle Size Distribution 83
4.4.2 Effect of Bioremediation on Moisture Content 85
4.4.3 Effect of Bioremediation on pH 87
4.4.4 Effect of Bioremediation on Total Nitrogen 88
4.4.5 Effect of Bioremediation on Orthophosphate 91
4.4.6 Effect of Bioremediation on Sulfate 93
4.4.7 Effect of Bioremediation on Total Organic Carbon 95
4.5 Result of Optimization for PAHs Extraction 97
4.6 Effect of Bioremediation on Polyaromatic Hydrocarbons 97
4.7 Result of Optimization on Growth of Pseudomonas
putida
119
4.8 Survival of Pseudomonas putida 119
4.9 Conclusion 123
xv
CHAPTER 5 CONCLUSION AND SUGGESTIONS 124
5.1 Research Summary 124
5.2 Conclusion and Contribution from This Study 125
5.3 Suggestions for Further Study 127
REFERENCES 129
APPENDICES 150
VITA 184
xvi
LIST OF TABLES
2.1 Technical properties of biodiesel and diesel 19
2.2 Priority PAHs as listed by US EPA 21
2.3 Environmental conditions affecting degradation 32
2.4 Types of hydrocarbon-degrading microbes 36
2.5 Bacteria having biodegradation potential for xenobiotics 41
2.6 Summarized degradation rate of PAHs by Pseudomonas putida 52
2.7 Maximum permissible limit concentration for PAHs in soil at
different countries
56
3.1 Summarized of Standard Method and Equipment 63
xvii
LIST OF FIGURES
2.1 Effect of pH on the availability of nutrients important in plant growth
and microorganisms
13
2.2 Structures of US EPA‟s 16 priority pollutants PAHs 21
2.3 General pathways for the bacterial degradation of PAHs 42
3.1 Shovel was pushed into the soil to a depth of 10-15 cm 59
3.2 Biodiesel was obtained from Biodiesel Pilot Plant, UTHM 59
3.3 Diesel was purchased from Petron gas station, Parit Raja 60
3.4 Pseudomonas putida in dry culti loops 60
3.5 The 16 EPA-PAHs mixture standard solution 61
3.6 The d10-anthracene and 2-fluorobiphenyl 61
3.7 Summarized of research of methodology 62
3.8 Spill simulation with biodiesel and diesel as well as inoculation of P.
putida into soil.
63
3.9 The samples were stored in the incubator shaker for 24 days of
incubation time
65
3.10 Soil samples were passed through the stack of sieves 66
3.11 The dried soil samples were weighted by analytical balance 67
3.12 Measurement of pH by pH meter 68
3.13 Measurement of TOC and TN by TOC and TN analyzer 69
3.14 Standard solution of TC, IC and TN 69
3.15 Measurement of Orthophosphate and Sulfate by Ion Chromatography
System
71
3.16 Soil samples were extracted by Soxhlet Extractor 72
3.17 Measurement of PAHs by GC-MS 73
3.18 PAHs mixture standard solution at 0.5, 1, 3 and 5 ppm 74
xviii
3.19 Medium powder and the nutrient broth were ready to sterile by
Hiramaya Autoclave Sterilizer
75
3.20 P. putida in dry culti loop was put into warm nutrient broth 75
3.21 P. putida in culture broth after being incubated in the incubator shaker
at 35 °C for 9 days
76
3.22 Crystal violet was added into the sample on slide 76
3.23 Gram stain of Pseudomonas putida cells in blue-green colour 77
3.24 Agar powder and the media agar was solidified and ready to store in
freezer
78
3.25 Soil samples were shaken by digital shaking machine 79
3.26 Enumeration bacterial dilution procedure 80
3.27 The growth of P .putida was counted by Total Colony Chamber 80
4.1 Grain size distribution of soil 84
4.2 Result percentage of moisture content 86
4.3 Result of pH 88
4.4 The removal of total nitrogen by P. putida as oil-biodegradable agent 90
4.5 The removal of orthophosphate by P. putida as oil-biodegradable agent 92
4.6 The removal of sulfate by P. putida as oil-biodegradable agent 94
4.7 The degradation rates of total organic carbon by P. putida as oil-
biodegradable agent
97
4.8 Result of total PAHs in each sample 98
4.9 The degradation rates of PAHs compounds by P. putida as oil-
biodegradable agent in sample D100
103
4.10 The degradation rates of PAHs compounds by P. putida as oil-
biodegradable agent in sample B5
104
4.11 The degradation rates of PAHs compounds by P. putida as oil-
biodegradable agent in sample B20
105
4.12 The degradation rates of PAHs compounds by P. putida as oil-
biodegradable agent in sample B50
106
4.13 The degradation rates of PAHs compounds by P. putida as oil-
biodegradable agent in sample B100
107
xix
4.14 The degradation rates of naphthalene by P. putida as oil-biodegradable
agent in sample D100, B5, B20, B50 and B100
111
4.15 The degradation rates of acenaphthylene by P. putida as oil-
biodegradable agent in sample D100, B5, B20, B50 and B100
112
4.16 The degradation rates of acenaphthene by P. putida as oil-
biodegradable agent in sample D100, B5, B20, B50 and B100
113
4.17 The degradation rates of fluorene by P. putida as oil-biodegradable
agent in sample D100, B5, B20, B50 and B100
114
4.18 The degradation rate of phenanthrene by P. putida as oil-biodegradable
agent in sample D100, B5, B20, B50 and B100
115
4.19 The degradation rates of anthracene by P. putida as oil-biodegradable
agent in sample D100, B5, B20, B50 and B100
116
4.20 The degradation rates of fluoranthene by P. putida as oil-biodegradable
agent in sample D100, B5, B20, B50 and B100
117
4.21 The degradation rates of pyrene by P. putida as oil-biodegradable agent
in sample D100, B5, B20, B50 and B100
118
4.22 The summarized for enumeration of Pseudomonas putida (106 cfu/g)
over days
122
xx
LIST OF SYMBOLS AND ABBREVIATIONS
°C Celcius degree
µm Micrometer
B10 10% biodiesel + 90% diesel
B100 100% biodiesel
B2 2% + 98% diesel
B20 20% + 80% diesel
B5 5% biodiesel + 95% diesel
B50 50% biodiesel + 50% diesel
BS British Standard
C Carbon
Cc Coefficient of Curvature
Cfu/mL Colony-forming unit per Milliliter
Cfu/g Colony-forming unit per Gram
CO Carbon Monoxide
CO2 Carbon Dioxide
CPO Crude Palm Oil
Cu Uniformity Coefficient
D100 100% diesel
DNA Deoxyribonucleic Acid
DBKL Dewan Bandaraya Kuala Lumpur
xxi
EPA Environmental Protection Agency
EU European Union
FTK Faculty of Engineering Technology
g Gram
GC-MS Gas Chromatography-Mass Spectrometry
H Hydrogen
H2O Water
H2SO4 Sulfuric Acid
HCl Hydrochloric Acid
IC Inorganic Carbon
kg Kilogram
KOH Potassium Hydroxide
Kow Octanol-water Partition
LC Liquid Chromatography
mg/L Milligram per Liter
mL Milliliters
MPOB Malaysian Palm Oil Board
N Nitrogen
NOx Nitrogen Oxides
NaOH Sodium Hydroxide
O2 Oxygen
P Phosphorus
pa Vapour Pressure
PAHs Polyaromatic Hydrocarbons
xxii
PCBs Polychorinated Biphenyls
PM Particulate Matter
ppm Part per Millions
rpm Revolutions per Minute
S Sulfur
SFC Supercritical Fluid Chromatography
TC Total Carbon
TN Total Nitrogen
TOC Total Organic Carbon
US EPA United States Environmental Protection Agency
USA United States of America
USCS Unified Soil Classification System
UTHM Universiti Tun Hussein Onn Malaysia
w Weight
xxiii
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Summarized Removal of Individual PAH in Diesel Oil Only 150
B Table for Characteristics Study of Physico-Chemical 155
C Table for Characteristics Study of PAHs 156
D Table for Percentage Recovery of PAHs at 60 °C 157
E Table for Percentage Recovery of PAHs at 80 °C 158
F Table for Percentage Recovery of PAHs at 100 °C 159
G Table for Percentage Recovery of PAHs at 120 °C 160
H Growth of P. putida for 24 Days of Incubation Time 161
I Effect of sample on moisture content in soil bioremediation by
P. putida
162
J Effect of sample on pH in soil bioremediation by P. putida 163
K Effect of sample on TN in soil bioremediation by P. putida 164
L Effect of sample on orthophosphate in soil bioremediation by P.
putida
165
M Effect of sample on sulfate in soil bioremediation by P. putida 166
N Effect of sample on TOC in soil bioremediation by P. putida 167
O Effect of sample on naphthalene in soil bioremediation by P.
putida
168
P Effect of sample on acenaphthylene in soil bioremediation by P.
putida
169
Q Effect of sample on acenaphthene in soil bioremediation by P.
putida
170
R Effect of sample on fluorene in soil bioremediation by P. putida 171
xxiv
S Effect of sample on phenanthrene in soil bioremediation by P.
putida
172
T Effect of sample on anthracene in soil bioremediation by P.
putida
173
U Effect of sample on fluoranthene in soil bioremediation by P.
putida
174
V Effect of sample on pyrene in soil bioremediation by P. putida 175
W Preparations for Biodiesel, Diesel, Their Blends and P. putida
Soil Mixture
176
X Field Emission Scanning Electon Microscopy (FESEM) of P.
putida in the Soil Samples.
179
Y Milestones/ Gant Chart of Research Activities 182
1
CHAPTER 1
INTRODUCTION
1.1 Background
Alternative fuels for diesel engines have attracted more and more attentions in the fuel
market due to the depletion of fossil fuel resources in the world feedstock and the
worsening of air pollution problems caused by the emissions from motor vehicles. An
alternative fuel to petroleum diesel must be technically feasible, competitive,
environmentally acceptable and easily available. Currently, the best candidate for a
petroleum diesel fuel substitution is biodiesel. Based on „Eurostat‟ data there are 484.8
million tons of oil products used in the European Union (EU) in 2003, which is by 2%
higher than in 2002 (Lapinskiene and Martinkus, 2007). In order to meet the
requirements of the Kyoto protocol (1997) and Madrid declaration (1994) several
alternatives source of energy especially biodiesel are extensively used as an alternative
to diesel fuel used in vehicles transport sectors of many European and American
countries. Therefore, the production and consumption of biodiesel fuel constantly
increases.
Similar properties of biodiesel and diesel fuel as well as the requirements of the
fuel strategy of the EU (Biofuels Directive 2003/30/EU) have resulted in the emergence
of mixtures of both types of fuels (biodiesel and their diesel blends) in the markets of
many countries. Since then, biodiesel fuels have attracted an increasing attention
worldwide as blending components or direct replacements for diesel fuel in diesel
engines. It can be legally blended and used in many different proportions such as B2
2
(2% biodiesel + 98% diesel), B5 (5% biodiesel + 95% diesel), B20 (20% biodiesel +
80% diesel) and B100 (100% biodiesel) (Demirbas, 2009).
Since the early 21st
century onwards, USA and other states such as Australia,
Brazil, Canada, Germany, China, South Korea and also Malaysia begun to mandate that
all diesel fuel sold at the petrol stations should contain at least 2% of biodiesel. In
Malaysia, an actual engine trial was managed by Malaysian Palm Oil Board (MPOB) on
B2, B5 and B10 of processed liquid palm oil/petroleum diesel blends (Cheng et al.,
2005). Government agencies such as Dewan Bandaraya Kuala Lumpur (DBKL) and
Armed Forces started using B5 in February 2009 which involved 3,900 diesel vehicles
with no adverse effects reported to date.
The B5 Programme has been implemented in stages in Malaysia beginning 1st
June 2011 for retail stations in the Central Region starting with Putrajaya. By 1
November 2011, B5 are available in all other retail stations in the Central Region
Malacca, Negeri Sembilan, Wilayah Persekutuan Kuala Lumpur and Selangor.
Companies such as Petronas, Shell, Chevron, Petron and BHPetrol have provided the B5
biodiesel in 1,150 petrol stations with a usage of 110,000 tonnes per year.
The use of commercial pure biodiesel fuel and their diesel blends in diesel
engines significantly reduces the particulate matter (PM), carbon dioxide (CO2), carbon
monoxide (CO), sulfur (S) and polyaromatic hydrocarbons (PAHs) compared to the
petroleum diesel fuel (Demirbas, 2009). These benefits are good evident that pure
biodiesel and their diesel blends will be used in the vehicles transport sector driven by
diesel engines in the near future; however this means new threats to the environment in
case of their spillage.
Soil contamination with products made from petroleum and their blends with
biodiesel are the major global concern today due to their serious hazard to human health,
causes organic pollution of soil, surface water as well as ground water which limits its
use, causes economic loss, environmental problems, and decreases the agricultural
productivity of the soil (Wang et al., 2008). The concern stems primarily from health
risks, from direct contact with the contaminated soil, vapors from the contaminants, and
from secondary contamination of water supplies within and underlying the soil. The
most noticeable sources of contamination are released from manufacturing and refining
3
installations, leakage of underground storage tanks and accidents during transportation
of the oil.
It has been known that many microorganisms are capable of using petroleum
hydrocarbons as a sole source of carbon and energy for growth in aqueous environment.
They are ubiquitous in the environment and are able to degrade the different types of
hydrocarbon, as contained in oil products (Ron and Rosenberg, 2002). In fact, the
degradation of hydrocarbon by microorganisms depends to their molecular weight,
metabolism of microorganisms and also environmental conditions. Several factors such
as temperature, pH, amount of nutrient supply and oxygen content have influenced the
efficiency and degradation rate of hydrocarbons (Vidali, 2001).
Pseudomonas putida (P. putida) is a safe strain which mean it could be used to
clone genes from other soil-inhibiting bacteria. Certain strains of P. putida are not
pathogenic to environment and human health due to lack of certain genes including
enzymes that digest cell membranes and well of human and plants. The optimum
temperature of its growth is 35 ºC (Kucerova, 2006). P. putida is tolerant to xenobiotics
especially PAHs and play a vital role in the treatment of petroleum contaminated soil
(Haritash and Kaushik, 2009). It occurs in various environmental niches because of its
metabolic versatility and low nutritional requirement (Timmis, 2002). Recently, many
studies on degradation of hydrocarbon by bacterium consortia, including P. putida have
been carried out because of its high capability to degrade recalcitrant substances and
inhibiting xenobiotics. The reason is due to the P. putida can adapt to diverse substrates
and posses some catabolic pathways capable of acting on recalcitrant substances
(Poblete-Castro et al., 2012).
A goal of bioremediation is to degrade the hazardous contaminants into carbon
dioxide (CO2) and water (H2O) (Seo et al., 2009) by microorganisms. Many researchers
found that P. putida has the ability to degrade a great variety of organic contaminants.
Therefore, this ability has been put to use in bioremediation by using P. putida to
degrade PAHs. The use of this pure P. putida strain is preferable to some
other Pseudomonas species capable of such degradation as it is a safe species of
bacteria, unlike P. aeruginosa for example, which is an opportunistic human pathogen
(Badrunnisa et al., 2012).
4
1.2 Problems Statement
As occurs to the diesel fuel, the commercialization of biodiesel and their diesel blends
containing PAHs on the market of many countries would resulted towards
environmental damages due to accidental spills such as leakage from underground
storage tank, pipelines and container tank truck or rail accidents.
There are several hundred combination of PAHs exist, but up to 16 PAHs are
considered priority pollutants in terms of health effects: naphthalene (NAPH),
acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), phenanthrene (PHE),
anthracene (ANT), fluoranthene (FLT), pyrene (PYR), chrysene (CHR),
benzo(a)anthracene (BaA), benzo(a)pyrene (BaP), benzo(b)fluoranthene (BbF),
benzo(k)fluoranthene (BkF), indeno(1,2,3-cd)pyrene (IP), dibenz(a,h)anthracene (DahA)
and benzo(g,h,i)Perylene (BghiP) by US EPA and EU (Lundstedt et al., 2003; Nasseri et
al., 2010). According to Contaminated Land Management and Control Guidelines No. 1:
Malaysia Recommended Site Screening Levels for Contaminated Soil Land (SIRIM,
2008) PAHs are also listed as hazardous contaminants that have potential in causing
unacceptable human health risk.
Continuous low-level inputs are not often noticed, and possibly pose a serious
threat to the environment as contamination accumulates and subsequently penetrates and
disperses from contaminated soil to underground and surface water. This contaminant
affects and alters parameters of soil and water that could influence biological activities
of native microbes in the contaminated areas. In fact, presence of these contaminants
containing PAHs in soil is toxic to humans, plants and soil microorganisms.
Furthermore, there is serious concern due to their recalcitrant and mutagenic or
carcinogenic properties (Mrozik et al., 2003). Moreover, exposure of many living
organisms including humans to PAHs may lead to elevated levels of deoxyribonucleic
acid (DNA) mutation, reproductive defects, and increased risk of cancer and other
adverse health effects through diverse pathways, such as ingestion of plants that uptake
soil contaminants and also leaching of compound from contaminated soil to ground and
surface water used as drinking water (Wilcke, 2007). In addition, once in soil, the PAHs
5
in particular accumulate in soil where they may be retained for many years due to their
hydrophobicity and persistence (Nadal et al., 2004).
In 20th
century, brownfield sites over the world are on the rise. The Brownfields
Law defines a brownfield site as real property, the expansion, redevelopment, or reuse of
which may be complicated by the presence or potential presence of a hazardous
substances and contaminants. The Brownfields Law also expressly includes property
contaminated with petroleum, mine scarred land, and former sites of methamphetamine
laboratories. Many of brownfield or polluted sites have been reclaimed for construction
or other beneficial purposes and the pollutant from the sites might come from PAHs
family. Therefore, these hazardous substances and contaminants especially PAHs create
a world-wide problem of contaminated soil that importantly requires decontamination.
Due to a great disruption of soil quality and limited effectiveness to remove
hydrocarbons from contaminated sites by physico-chemical treatments (Boopathy,
2000), bioremediation is a promising and cost effective technological development
environmental-friendly for cleaning up soil contaminated with PAHs. In addition,
bioremediation has a greater public acceptance, with regulatory encouragement.
Bioremediation is a technique based on the action of microorganisms, in which
hazardous contaminants will be degraded into “non toxic” substances such as carbon
dioxide (CO2), water (H2O) and biomass (Mariano et al., 2008). Biodegradation
technologies first proved practical on a wider scale during the Exxon Valdez oil spill
took place in Alaska. Since then, they have been used more frequently in many countries
(Kucerova, 2006).
Increasing attention has been paid to bacteria, which act as efficient tools in the
bioaugmentation process. Among the Pseudomonas species, P. putida, P. aeruginosa
and P. alcaligenes are more thoroughly investigated and their effectiveness as
bioremediation agents for hydrocarbons bioremediation is well established (Adeline et
al., 2009). Therefore, to reduce the level of contaminants in soil, this study conducted
the soil bioremediation with P. putida as oil-biodegradable agent. This study helps us to
know whether the bioremediation technique can be used in soil contamination by
biodiesel and diesel whereas P. putida was potentially utilized as a great oil-
biodegradable agent. Therefore, this sustainable method of bioremediation shall be
6
considered and provide more option in order to remediate oil-contaminated soil in the
future.
1.3 Objectives of Study
The main objective of this study is to suggest a new technique of biotreatment, namely
bioremediation by Pseudomonas putida in treating soil contaminated with biodiesel and
diesel.
To achieve the main objective of this study, there are some specific objectives need
to be fulfilled, which are:
i. To determine the removal of total nitrogen, orthophosphates, sulfates and total
organic carbon in bioremediation of soil contamination by Pseudomonas putida.
ii. To analyze the biodegradability of biodiesel, diesel and their blends at different
ratio based on removal and degradation rate of selected PAHs in soil
bioremediation.
iii. To evaluate the survival of Pseudomonas putida as oil-biodegradable agent in
bioremediation for biodiesel and diesel soil blend.
1.4 Scopes of Study
This study consists of field activities and laboratory works. Field activities involved the
sampling of soil, biodiesel and diesel samples.
The soil samples were taken from inside campus area. The soil samples were
collected from the surface of 10-15 cm deep layer of soil.
The biodiesel is palm-based biodiesel, which is produced by transesterification
with methanol and obtained from Biodiesel Pilot Plant, Faculty of Engineering
Technology (FTK), UTHM.
The refined fossil fuel used throughout this study was petroleum diesel fuel (EN
590: 2004). It was purchased from Petron gas station at Parit Raja, Johor.
7
Laboratory works consists of three stages of experiments as shown in Figure 3.7.
The stages of laboratory activities are categorized into characteristics study,
biodegradation study and then analysis of experimental works.
First of all, spill simulation with pure biodiesel, diesel and their blends: B100
(100% biodiesel), D100 (100% diesel), B5 (5% biodiesel + 95% diesel), B20 (20%
biodiesel + 80% diesel) and B50 (50% biodiesel + 50% diesel) into the soil samples
were carried out at laboratory. As control sample, there is no addition of biodiesel and
diesel into the sample.
Once the mixture of soil and biodiesel as well as diesel was done, P. putida
(ATCC 49128) in culture broth was subsequently inoculated into the polluted soil
samples and used as oil-biodegradable agent in bioremediation of PAHs-contaminated
soil.
The testing parameters involved in this study are soil particle size, moisture
content, pH, total nitrogen (TN), orthophosphate, sulfate, total organic carbon (TOC),
and extraction of PAHs as well as survival of P. putida.
1.5 Limitation of Study
This study was carried out to determine the removal of TN, orthophosphate, sulfate and
PAHs by P. putida pure strains (ATCC 49128) from soil samples contaminated with
biodiesel, diesel and their blends only; B100, B50, B20, B5 and D100.
Nowadays, most of soil was contaminated with the accidental spillage of several
refined petroleum products, but this study is limited to the spillage of diesel and also
from palm-based biodiesel only.
The biodegradation process was also limited to eight PAHs namely, naphthalene,
acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and
pyrene. Thus, results from this study are based on laboratory experiments.
Next, no experiments were conducted to study the pathway degradation for each
contaminant. Results from this study will be compared against studies reported in
literature appropriate.
8
1.6 Thesis Outline
This thesis is represented in 5 chapters as described below;
Chapter 1 introduces the study, the objectives and scopes that needed to be
achieved to address the problem. Chapter 2 explores the literature of various researches
carried out prior to this research. Chapter 3 explains the research methodology used in
carrying out the research from sampling methods, extraction, determination of physico-
chemical parameters, PAHs and survival of P. putida in samples and result analysis.
Chapter 4 and chapter 5 show the data analyses and interpretation of the research.
Chapter 6 summarizes the results of the research and provides recommendations for
future research.
1.7 Conclusion
Soil pollution problem is a big issue in our country and the major source for soil
pollution is from accidental spillage of biodiesel, diesel and their blends containing
PAHs due to leakage of underground storage tank and container tank truck accidents.
This study focused on the PAHs pollution in the contaminated soil and the ability of P.
putida to remove the pollutants thereby becoming as the oil-biodegradable agent for
removing the pollutants from the environment.
9
CHAPTER II
LITERATURE REVIEW
2.1 Introduction
Over the past decades, the desires to establish the own national energy of every country
and develop the fuel fossil resources have resulted in the development of fuel
technologies that based on agriculture materials as feedstock of biodiesel such as
soybean oil, kernel and crude palm oil. Biodiesel can be used alone or blended with
petroleum diesel in diesel engine as they have similar characteristics, but biodiesel has
lower exhaust emissions.
There is a great demand for alternative sources of petroleum-based fuel,
especially diesel fuel due to the decreasing of the world‟s petroleum reserves and the
increasing environmental and health concerns in case of their spillage. Biodiesel, a
renewable and biodegradable fuel, has recently been considered as the best option for a
diesel fuel substitution because of it environmentally friendliness and subsequently can
be used in conventional diesel engine.
2.2 Soil and Soil Chemistry
Soil is an integrated mixture of inorganic (mineral), organic matter (decomposing
biomass), water and air, called as the four soil constituents. The chemistry of these soil
components, their composition, structures, mutual interactions, and interactions with the
biospheres, hydrospheres and atmospheres, are called as soil chemistry (Kim, 2011).
10
2.3 Soil Sandy Characterization
Soil is classified in three main categories: heavy, medium and light. Sandy soil belongs
to the light category due to its light weight, grainy texture. Soil may also be described as
coarse or fine, a coarse-textured soil has more sand, whereas a fine-textured soil has
more clay (Sylvia et al., 2005).
Coarse-grained soils have sand or gravel components. The sand or gravel might
be combined with silt and clay. A sample is considered coarse-grained if contained less
than 50% fine-grained materials. It is considered gravel if the percentage of gravel is
greater than the percentage of sand. It is sand if the percentage of gravel is equal to or
less than the percentage of sand (Hmtri, 1997). The sample is well-graded if it has a
wide range of particles sizes and substantial amounts of the intermediate particle sizes. It
is poorly graded if it consists mostly of one size (uniformly graded) or if it has a wide
range of sizes with some of the intermediate grain size missing.
2.3.1 Soil Texture
Soil texture is an indicator for the ease of detachment of soil particles. The basic soil
textural class names used by USDA are defined in terms of the relative amounts of sand,
silt, and clay determined by laboratory grain size analysis. Soil texture depends on the
composition of sand, silt and clay. For the USDA system classification, sand particles
are the largest, ranging in diameter size from 0.05 mm (very fine sand) to 2 mm (very
coarse sand) and have a gritty texture to it (loose and single-grained) (Banon, 2008).
Sandy soil is the lightest of all the soils and therefore is prone to both water and wind
erosion if no plant life exists in it. They tend to be rounded or irregular, which creates
large pore spaces between particles. Sand particles have a low capacity to hold water and
nutrients.
Clay particles are the smallest mineral particles and are less than 0.002 mm in
size (Banon, 2008). Clay particles are generally flat, plate-like, and fit closely together.
They have the largest surface area, which facilitates the adsorption of water and
nutrients to the clay particles. In between, ranging in size from 0.002 mm to 0.05 mm,
11
are silt particles (Banon, 2008). Like sand, silt particles are irregularly shaped and are
generally nutrient poor. They affect soil behavior, including the retention capacity for
nutrients and water. Sand and silt are the products of physical weathering, while clay is
the product of chemical weathering. Clay content has retention capacity for nutrients and
water. Clay soils resist wind and water erosion better than silty and sandy soils, because
the particles are more tightly joined to each other (Banon, 2008).
2.3.2 Soil Pores
Soil pores play a major role in water and air movement. Also, soil microorganisms
reside in pores. Coarse-textured (sandy) soils have higher bulk densities and less total
pore space (35% to 50%) than fine-textured (clay) soils that have lower bulk densities
and more pore spaces (40% to 60%). The size of the pores, however, is just as important
as the total quantity of pore space.
As classified by the Soil Science Society of America (1997), there are five
classes of pore sizes such as macropores (>75µm), mesopores (30-75µm), micropores
(5-30 µm), ultramicropores (0.1-5 µm), and cryptopores (<0.1 µm).
Large pores characteristically allow the rapid movement of soil gases and soil
water. Sandy soils have less total pore space, but those spaces are mostly macropores;
thus, sandy soils usually drain rapidly. In contrast, clayey soils have more total pore
space, but these spaces are mostly small pores, micropores and smaller. Soils high in
clay usually drain slowly because the small pores restrict the water flow. Therefore,
sandy soil has a relatively low water-holding capacity and a clayey soil has a relatively
high water-holding capacity.
2.3.3 Soil Moisture Content
Water is primary medium that transports nutrients and contaminants and major
environmental factor biological availability (William, 2012). Water in soil occupies pore
spaces that arises from the physical arrangement of the particulate solid phase,
12
competitively and often concurrently with the soil gas phase. While hidden from casual
view, highly substantial volumes of water are commonly stored in soils.
Soil microorganisms which are beneficial also rely on the water holding
characteristics of soils for their growth. On the other hand, soil water is a highly
dynamic entity, exhibiting substantial variation in both time and space. Changes in soil
water content and its energy status affect many soil properties including strength,
compactibility and penetrability, and may cause changes in the bulk density of swelling
soils (William, 2012). The liquid phase characteristics affect the soil gaseous phase and
the rates of exchange between these phases, as well as other important soil properties
such as the hydraulic conductivity which governs the rate of water and soluble chemical
flow.
2.3.4 Soil pH
Soil pH has often been called the master variable of soils and greatly affects numerous
soil chemical reactions and process. It is important measurement in deciding how acid
soil is, and can be expressed as pH = - log [H+] (Donald, 2003). Soils that have a pH < 7
are acid, those with a pH > 7 are alkaline, and those with a pH of 7 are neutral. In acid
soils, more H+ than OH
− ions is present. On the other hand, an alkaline soil has in its soil
solution more OH− than H
+ ions. A soil with a neutral reaction contains equal amounts
of H+
and OH−
ions.
Most plants grow best in soil with a slightly acid reaction. In this pH range,
nearly all plant nutrients are available in optimal amounts (Donald, 2003). Therefore,
soil pH significantly affects the availability of plant nutrients and microorganisms
(Figure 2.1). Most microbial species can survive only within a certain pH range as well
as affects availability of nutrients. In strongly acid soils, aluminum (Al), iron (Fe) and
manganese (Mn) may exist in toxic quantities because of their increased solubility. It is
known that high toxicity of Al in the soil solution causes stunted roots and tops in
susceptible plants. However, as pH increases, and alkaline reaction then occurs, the soils
would contain low amounts of soluble Al, Fe and Mn due to the formation and
subsequent precipitation of insoluble Al, Fe and Mn hydroxides.
13
Figure 2.1: Effect of pH on the availability of nutrients important
in plant growth and microorganisms (Donald, 2003)
2.3.5 Soil Nutrients
Sandy soil is an acidic soil type, which many plants prefer, but its general lack of
nutrients makes it a less than desirable growing medium on its own. Adding organic
matter, such as compost from yard or manure, will provide the nutrients plants need to
thrive in this soil type.
Macro-nutrients are essential for plant and microorganisms‟ growth and
development. Carbon, oxygen, and hydrogen are the basic constituents of organic
matter. Carbon and oxygen are obtained from air during photosynthesis. Hydrogen is
derived from water in the soil. Other macro-nutrients such as nitrogen, phosphorus and
sulfur are obtained from dissolved minerals in the soil solution and serve various
physiological roles.
14
2.3.5.1 Total Nitrogen
Nitrogen (N) in soil has been studied for centuries and is still the most studied element
in soil chemistry, microbiology, and fertility. Nitrogen is unique because it is found in
soil as both reduced and oxidized species, as part of organic molecules, and as oxidized
gaseous species, as well as in the elemental form (i.e., nitrogen gas). Many nitrogen
species are soluble in water and tend to be found in soil solution as attached to soil
particles. Nitrogen occurs in both organic and inorganic forms in soil.
Nitrogen chemistry in soils is the changes of nitrogen during organic reactions,
oxidation of organic nitrogen to N2 and N2O (denitrification) or to NO3−(mineralization),
and the reduction of N2 to amino acids (nitrogen fixation) (Bohn et al., 2002). Nitrate is
stable only in under strongly oxidizing conditions, and amino N, under strongly reducing
conditions. Nitrogen moves between the various soil compartments easily, depending on
the soil environmental condition. These movements are often outlined as the nitrogen
cycles (Conklin, 2005). The slowest and most energy-intensive part of the circle is the
“fixation” of nitrogen gas (N2) forming various nitrogen compounds with either oxygen,
hydrogen, forming ammonia, or with carbon to form amino acids. Once these initial
compounds are formed, subsequent changes in nitrogen species are rapidly and easily
accomplished.
Nitrogen is leached from soils as nitrate; ammonium (NH4+) is retained by cation
exchange but is oxidized to nitrate if not absorbed by plants and microbes (Bohn et al.,
2002). Nitrate leaching is a potential pollution hazard to environmental media.
Denitrification and avid plant and microbial uptake of nitrogen tend to minimize the
NO3− concentrations of soil solutions. Nitrite and nitrate occur in all soils in the soil
solution. Although nitrite in the environment is of concern, its occurrence is usually
limited because the oxidation of nitrite to nitrate is more rapid than the oxidation of
ammonia to nitrite. Both nitrite and nitrate move readily in soil, and nitrate is available
to plants as source of nitrogen and can move to plant roots in water.
15
2.3.5.2 Orthophosphate
Phosphate chemistry is of greatest interest in acid soils. The phosphate species found is
dependent in large measure on pH but it also dependent on organic matter. Lower soil
pH (e.g, 4-7) favors monobasic phosphate, while higher pH (i.e., 7-10) favors dibasic
phosphate. A monobasic salt refers to a salt, which has only one atom of an univalent
metal. And dibasic salt means having two univalent metal ions. In this case, the
univalent metal ion is the sodium cation. Since these are salts, they readily dissolve in
water and produce alkaline solutions. These compounds are commercially available in
hydrous and anhydrous forms. Monobasic and dibasic sodium phosphate together is very
important in biological systems as a buffer. Phosphate is increasingly unavailable as soil
acidity increases, due to the retention by Al and Fe. However, as the soil acidity
increases, the Al and Fe become more soluble, and thus as soil pH decreases, its
“phosphate fixing power” increases. This means that Al and Fe react with phosphate to
form insoluble and plant unavailable Fe and Al phosphate species (Bohn et al., 2002).
Under basic conditions, high concentration of Calcium (Ca) exists and insoluble calcium
phosphates form. The rate of phosphate loss from soils by weathering is about the same
as the overall weathering rate, so the total amount of phosphate in soils tends to remain
constant throughout soil development. The decay of phosphate in organic matter by
passes the inorganic soil fraction to some extent and maintains some phosphate
availability over the range of soil pH.
Phosphate (PO43−
) is only oxidation state in soils and plants. Phosphite (PO33−
)
and phosphine (PH3) compounds of lower phosphorus have not been found in soils. The
dihydrogen phosphate (H2PO4−) and hydrogen phosphate (HPO4
2−) are predominant at
the pH of soil solutions. The phosphate mineral apatite (Ca5 (OH, F) (PO4)3) is common
in rock minerals. In acids soils, most solid-phase phosphate associated with Fe and Al
and their hydroxides. In basic soils, phosphate is associated with Ca in apatite-like form.
Phosphate fixation is appreciable in all soils especially in coarse-textured soils and is
particularly strong in soils rich in amorphous Fe and Al hydroxides or allophone (in
volcanic soils).
16
2.3.5.3 Sulfate
Although sulfur is less volatile than carbon and nitrogen, organic decay in soils may
release a little sulfur as methyl mercaptan (CH3SH), dimethyl sulfide (CH3SCH3),
dimethyl disulfide (CH3SSCH3) and hydrogen sulfide (H2S) but the release is rare in
aerobic soils. The organic sulfur gases are the distinctive odor from paper mills. Within
the soil, these gases oxidize, or the sulfide reacts strongly with transition metal ions in
soils and precipitates as iron sulfide (FeS2), manganese (ii) sulfide (MnS) and other
transition metal sulfides rather than escaping to the atmosphere (Bohn et al., 2002). If
soil emissions of H2S and the organic sulfide gases were substantial, their strong odors
would be noticeable.
Sulfate is the stable sulfur oxidation state in aerobic soils, and sulfide is stable in
anaerobic soils. Elemental sulfur is rare naturally in soils but is sometimes added to soils
as an amendment and sulfides are common in many mining wastes. When elemental
sulfur and sulfides are exposed to oxygen, they oxidize to H2SO4. Soil acidities as high
as pH 2 may persist until the sulfide or sulfur has all been oxidized and leached away.
Major sulfur inputs to soils include atmospheric SO2 and its various oxidation
products from coal combustion, petroleum processing, ore smelting, and oil sulfate from
sea spray. Most of atmosphere‟s sulfur falls near the areas where it is produced. The
fallout occurs both as acid rain and as direct plant absorption; direct soil absorption of
atmospheric sulfur is minor in humid and temperate regions. In arid regions SO2 and its
oxidation products, H2SO3 and H2SO4, are absorbed directly and rapidly by the basic
soils and their dust. In regions of sulfur-deficient soils, atmospheric sulfur at low
concentration can benefit plants. Benefits from the low concentration, however, must be
weighed against the associated acidification of freshwater, phytotoxicity, health hazards,
smog and building deterioration at the higher concentrations near the sources.
Sulfate anions are retained only weakly by soils, but the retention increases with
soil acidity. Sulfate anions are absorbed readily by plants and incorporated into biomass.
Hence, biomass and soil organic matter (SOM) constitute large sulfur reservoirs at the
earth‟s surface. The sulfate content of soils increases with aridity and with salt
accumulation.
17
2.3.5.4 Total Organic Carbon
Total organic carbon (TOC) is the carbon (C) stored in the soil organic matter (SOM).
SOM is the organic fraction of soil exclusive of non-decomposed plant and animal
residues. Organic carbon enters the soil through decomposition of plant and animal
residues, living and dead microorganisms as well as soil biota. Other sources of organic
carbon can be manure or mulch application. Turnover rate for different types of soil
organic carbon (plant residues, particulate organic matter, soil microbial biomass and
humus) can vary from one month to thousands of years depending on composition.
In fact, organic carbon is the main source of energy for microorganisms (USDA,
2009). This occurs faster when the soil is moist and warm.A direct effect of poor organic
carbon is reduced microbial biomass, activity, and nutrient mineralization due to a
shortage of energy sources. Scarce of organic carbon results in less diversity in soil biota
with a risk of the food chain equilibrium being disrupted, which can cause disturbance in
the soil environment (e.g., plant pest and disease increase, accumulation of toxic
substances). Practices that increase organic carbon include continuous application of
manure and compost and also use of cover crops.
2.4 Biodiesel
Biodiesel is composed of methyl or ethyl esters of long chain fatty acids with low
structural complexity as oleate, palmitate, stearate, linoleate, myristate, laureate and
linolenate derived from a variety of vegetable oil sources such as palm oil, soybean,
peanut, coconut, sun-flower, cotton, babassu and castor oil and also from animal fats
(Pinto et al., 2005). It is most commonly produced through a transesterification process.
Alkaline catalysts (e.g., sodium hydroxide, NaOH or potassium hydroxide, KOH) or
acidic catalysts (e.g., hydrochloric acid, HCL or sulfuric acid, H2SO4) must be added to
the alcohol in order to obtain high ester yield in short reaction (Canakci and Gerpen,
2003).
18
Biodiesel is pure, or 100% biodiesel fuel. It is referred to as B100 or “neat” fuel.
A biodiesel blend is pure biodiesel blended with petroleum diesel. Biodiesel and diesel
blends are referred to as BXX. The XX stand for the amount of biodiesel in their blends
(Demirbas 2009).
Table 2.1 shows the technical properties of biodiesel and diesel. Biodiesel is a
clear amber-yellow liquid with a viscosity similar with petroleum diesel. Biodiesel is
non-flammable and non-explosive with a higher flash point of 423 K as compared to
petroleum diesel (337 K). Unlike petroleum diesel, biodiesel has higher combustion
efficiency, lower sulfur and polyaromatic hydrocarbons, higher cetane number, and
higher biodegradability (Mudge and Pereira, 1999; Ma and Hanna, 1999; Knothe et al.,
2006; Speidel et al., 2006; Leme et al., 2012).
Biodiesel production has received an increasing attention worldwide in the recent
past as a nonpolluting fuel. However, this assertion has been based on its
biodegradability and reduction in exhaust emissions of toxic gases only. Assessments of
water and soil biodiesel pollution are still limited.
Leme et al., (2012), studied the biodiesel soil pollution, aiming at analyzing its
cytotoxic, mutagenic and genotoxic potentials via in vitro tests with cultured mammalian
cells. There are 8 single PAHs such as naphthalene, acenaphthylene, fluorene,
phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene,
benzo(e)pyrene, benzo(e)acephenanthrylene, benzo(k)fluoranthene, benzo(a)pyrene,
dibenzo(a,h)anthracene, benzo(g,h,i)perylene and indene(1,2,3-c,d)pyrene were detected
in biodiesel contaminated samples. They concluded that, even though biodiesel fuel is
considered as an environmental friendly alternative to petroleum diesel in conventional
diesel engines, biodiesel still can promote genotoxic and mutagenic affects by inducing
chromosomal aberration (CA) and base-pair substitution mutations. Thus, taking into
account that Leme et al., (2012) highlighted harmful effects on organisms exposed to
biodiesel contaminated soils, the designation of this biofuel as an environmental friendly
fuel should be carefully reviewed to assure environmental quality.
19
Table 2.1: Technical properties of biodiesel and diesel
(adapted from Cheng et al., 2005; Demirbas, 2009)
Property Details
Common name Biodiesel Diesel
Common chemical name Fatty acid (m)ethyl ester Petrodiesel
Chemical formula range C14-C24 methyl esters C10H20-C15H28
Viscosity range (mm2/s, at 313 K) 3.3-5.2 3.918 - 3.974
Density range (kg/m3, at 288 K) 860-894 780-832
Boiling point range (K) > 475 > 453
Flash point range (K) 420-450 333.15-350.15
Distillation range (K) 470-600 365.4 - 365.9
Vapor pressure (mm Hg, at 295 K) < 5 < 75
Solubility in water More soluble than diesel Less soluble than biodiesel
Physical appearance Light to dark yellow/brown Amber and bright yellow
Biodegradability More biodegradable than diesel Less biodegradable
2.4.1 Advantages of Biodiesel
The biggest advantage of biodiesel as diesel fuel is environmentally friendliness that it
has over petroleum diesel. The other advantages of biodiesel are its portability, ready
availability, renewability, lower sulfur and phosphate as well as aromatic hydrocarbon,
higher biodegradability and lubricity, and lower toxicity than petroleum diesel (Knothe
et al., 2006; Leung et al., 2006). The sulfur content of petrodiesel is 20 to 50 times that
of biodiesel. Biodiesel degrades about four times faster than petroleum diesel. Compared
to diesel, it contains more oxygen which improves the biodegradation process and
further leading to an increased level of quick biodegradation (Demirbas, 2009).
2.5 Petroleum Diesel
Among hydrocarbon pollutants, diesel is a liquid produced from refined petroleum
product that frequently reported as soil contaminants leaking from storage and pipelines
or released in accidental spills. Diesel is a complex mixture of saturated and aromatic
hydrocarbons. For the most part, diesel comprises aliphatic hydrocarbons, but it also
contains polycyclic aromatic hydrocarbons such as naphthalene, fluorene and
phenanthrene (Eriksson et al., 2001).
20
Petroleum diesel containing polycyclic aromatic hydrocarbon (PAHs) raises
substantial concern because of their widely known toxic potential such as mutagenic,
carcinogenic, teratogenic, photo-induced toxicity and endocrine-disrupting activities
(Wernersson, 2003). High concentration of PAHs can be harmful and toxic to microbes‟
activity in diesel-contaminated soil and then inhibit bacterial degradation.
2.6 Polycyclic Aromatic Hydrocarbons
Polycyclic aromatic hydrocarbons (PAHs), also known as polynuclear aromatic
hydrocarbons are group of chemical compounds containing carbon (C) and hydrogen
(H) atoms, composed of two or more fused aromatic rings in linear, angular and cluster
arrangements (Mrozik et al., 2003) as shown in Figure 2.2. They are formed during a
range of human activities, including incomplete combustion of carbon-based fuels and
pyrolysis of organic matter (Gan et al., 2009). PAHs are also formed in the environment
media by natural process such as volcanic eruptions and forest fires. In addition, they are
contained in substances such as crude oil, coal and tar deposits. All these activities lead
to their ubiquitous environmental distribution, where their stability and persistence is
governed by their chemical and physical properties on the contaminated soil.
There are several hundred different combinations of PAH exist, but up to 16
compounds as listed in Table 2.2 have been identified as hazardous contaminants by the
United States Environmental Protection Agency, USEPA (Lundstedt et al., 2006).
Pollutants are chosen for this list because of potential for toxicity and frequency of
occurrence in hazardous sites. Figure 2.2 shows the diagram of molecular structures of
these PAHs. The distinguishing features of these compounds are that they are highly
hydrophobic. PAHs which consist of fused benzene rings are hydrophobic in nature with
very low water solubility and high octanol-water partition coefficient (Kow). Due to this,
they tend to adsorb easily into the organic matter of soil particles rendering them less
susceptible to biological and chemical degradation result in forming persistence
micropollutants in the environment. Prolonged aging time in contaminated soil promotes
the sequestration of PAHs molecules into micropores and increases the recalcitrance of
21
PAHs towards treatment. Due to its large holding capacity for pollutants, soil acts as the
long-term sink and also major repository of PAHs in the environment.
Table 2.2: Priority PAHs as listed by USEPA
(adapted from Lundset et al., 2006)
PAH name Number of
ring
Molecular weight
(g/mole)
Solubility in water
(mg/L)
Log Kow
Naphthalene 2 128.17 31 3.37
Acenaphthylene 3 152.2 16.1 4.00
Acenaphthene 3 154.21 3.8 3.92
Fluorene 3 166.22 1.9 4.18
Phenanthrene 3 178.23 1.1 4.57
Anthracene 3 178.23 0.045 4.54
Fluoranthene 4 202.26 0.26 5.22
Pyrene 4 202.26 0.132 5.18
Chrysene 4 228.29 0.0015 5.91
Benzo(a)anthracene 4 228.29 0.011 5.91
Benzo(a)pyrene 5 252.32 0.0038 5.91
Benzo(b)fluoranthene 5 252.32 0.0015 5.80
Benzo(k)fluoranthene 5 252.32 0.0008 6.00
Indeno(1,2,3-cd)pyrene 6 276.34 0.062 6.50
Benzo(g,h,i)perylene 6 276.34 0.00026 6.50
Dibenz(a,h)anthracene 6 278.35 0.0005 6.75
Figure 2.2: Structures of US EPA‟s 16 priority pollutants PAHs
(Lundset et al., 2006)
22
2.7 Mechanisms in Bioremediation
The most basic approaches for bioremediation of PAHs currently being practiced are
bioaugmentation and biostimulation. Bioaugmentation method involves addition of
highly concentrated and specialized populations of specific microorganisms indigenous
or exogenous into a contaminated soil in order to increase the rate of contaminants
biodegradation in the affected soil due to the density of contaminant-specific degraders
have been increased (Abdulsalam and Omale, 2009). Besides that, microorganisms
especially bacteria tend to degrade hydrocarbons into carbon dioxide (CO2) and water
(H2O) rather than assimilate carbon cell growth even no nutrients are added to the soil.
Due to this benefit, bioaugmentation seems to be a good approach for clean up oil-
contaminated soil in worldwide. On the other hand, biostimulation is the addition of
nutrients such as nitrogen (N), phosphorus (P) as well as sulfur (S) to stimulate naturally
occurring microbial populations at hasted rate (Tsai et al., 2009). Carbon (C) is the most
basic form of nutrient required for living microorganisms; however bacteria also need
nitrogen, phosphorus and sulfur to ensure effective degradation of the oil. In fact, these
nutrients are the basic building blocks of life and allow microbes to create the enzymes
to break down the contaminant compounds (Vidali, 2001). They are also an essential
energy source for biodegradation.
2.7.1 Bioaugmentation
Bioaugmentation is the applications of indigenous or genetically modified
microorganisms to polluted hazardous sites in order to accelerate the removal of
undesired compounds (Mrozik and Piotrowska-Seget, 2010). This method should be
applied in soils with low or non-detectable number of contaminant-degrading microbes,
containing compounds requiring multi-processes remediation, including processes
detrimental or toxic to microbes and for small-scales sites on which cost of non-
biological methods exceed cost for bioaugmentation. Besides that, the introduction of
microorganisms into soil is recommended for sites polluted with hazardous compounds
requiring long acclimation and adaptation period of time.
23
Successful soil augmentation requires knowledge on type and level of
contaminants as well as the suitable strains of microorganisms or their consortia. The
selection of proper culture should take into consideration some features of
microorganisms. They must be fast growth, easy cultured, able to withstand high
concentrations of contaminants and survive well in a wide range of environmental
conditions. Particularly attractive are „heirloom‟ microorganisms that are maintained and
handed down for many years and are specifically modified for bioaugmentation purpose
(Thompson et al., 2005). For remediation of areas contaminated with PAHs it is
necessary to use strains able to produce surfactants to make PAHs more accessible
(Gentry et al., 2004). There are several approaches that allow selection of
microorganisms useful for bioaugmentation. Bacteria may be isolated from
contaminated soils and after culturing under laboratory conditions pre-adapted pure
bacterial strains return to the same contaminated soil. This approach is called
reinoculation of soil with indigenous microorganisms. The second possibility is selection
of appropriate microorganisms from contaminated sites with similar contaminants that
are present in soils submitted to clean-up by these selected microorganisms. In most
experiments, strains suitable for bioremediation were isolated from aromatic
hydrocarbon-contaminated areas or industrial wastewater treatment plants (Xiaojun et
al., 2008). Most experiments dealing with bioaugmentation were performed using gram-
negative bacteria belonging to genus Pseudomonas, Flavobacterium, Sphingobium,
Alcaligenes Haluska and Achromobacter (Mrozik and Piotrowska-Seget, 2010).
However, one of the most difficult issues associated with bioaugmentation is
survival of strains introduced to soil. It has been observed that number of exogenous
microorganisms has decreased shortly after soil inoculation. Many studies have shown
that both abiotic (temperature, moisture content and pH) and biotic factors influence the
effectiveness of bioaugmentation (Bento et al., 2005). Moreover, aeration, nutrient
content and soil type also determine the efficiency of bioaugmentation.
According to Qing et al., (2007), investigating the effect of temperature and pH
on nitrophenolic pesticide degradation by inoculated Burkholderia sp. FDS-1, found that
the optimal parameters for bacteria activity were 30 °C and slightly alkaline pH, whereas
10 and 50 °C and highly acidic condition were unsuitable for pesticide detoxification.
24
Water potential has been reported to have significant influence on survival and
degradative activity of Pseudomonas stutzeri P16 lux AB4 in sterile and non-sterile soil
amended with phenanthrene (Mashreghi and Prosser, 2006). They found that at 750 and
500 kPa, differences in bacterial counts and activity were greater as compared with that
at 30 kPa. They explained the obtained results by the fact that low soil water contents
decreased activity of bacteria due to diffusional limitation of substrate supply and
adverse physiological effects associated with cell dehydration. Haluska et al., (1995),
studying the degradation of PCB in different soils by inoculated hydrocarbon-degrading
strain Alcaligenes xylosoxidans, stated that both total organic matter and content of
aromatic carbon in humic acids played an important role in survival and activity of
inoculants. They observed that A. xylosoxidans exhibited the best survival in soil
containing an intermediate amount of organic carbon whereas PCB degradation was the
most efficient in soil with the high content of organic carbon.
Biotic factors, including competition between indigenous and exogenous
microorganisms for limited carbon sources as well as antagonistic interactions and
predation by protozoa and bacteriophages, also play vital roles in the final stages of
bioaugmentation. All these interactions potentially decrease the number of introduced
cells (Sorensen et al., 1999). A comparison of usefulness of bioaugmentation,
biostimulation and bioattenuation in degradation of total petroleum hydrocarbons in
diesel-contaminated soil after 12 weeks of experiment was made by Bento et al., (2005).
The researchers reported that bioaugmentation was the most effective method as
compared with biostimulation and bioattenuation in the removal of petroleum
hydrocarbons. In this soil the percentages of petroleum hydrocarbons removal reached
the values of 70%, 59% and 38% for bioaugmentation, biostimulation and
bioattenuation, respectively.
Biougmentation approaches appear to have a great potential for aromatic
compounds remediation. The most important step in successful bioaugmentation is
selection of proper microbial strains. The most effective elimination of contaminants
may be achieved by using microbial inoculants isolated from environments where
contamination had occurred over several decades. Last but not least, the success of
129
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