ABSTRACT
Contamination of soil by organic compounds, especially hydrocarbon, is prevalent in
industrialized and oil producing countries of the world. About 1.7 to 8.8 million metric
tons of oil is released into the water and soils every year. This may pose a great threat to
the environment and human being at large. This study evaluated the efficiency of organic
wastes (biowastes) as supplementations for remediation of diesel fuel contaminated soil.
Three organic wastes [tea leaf (TL), Soybean cake (SC) and Potato skin (PS)] and two
economically viable plants (Dracaena reflexa and Podocarpus polystachyus) were
utilized to evaluate the biodegradation of diesel fuel in soil contaminated with different
concentrations of oil. For biodegradation studies, soils were treated with 20%, 15%, 10%
and 5% (w/w) diesel fuel and amended with 10% and 5% TL, SC and PS. Completely
randomized design was used for a period of 126 days under laboratory condition. At the
end of 126 days, soil polluted with 20% diesel oil and amended with 5% TL recorded the
lowest percentage of oil degradation (14.5%) and diesel utilizing bacteria at 30×105
colony-forming units (CFU) per gram of soil. The highest rate of biodegradation (95%)
was recorded in soil polluted with 5% diesel oil and amended with 10% SC with the
count of diesel utilizing bacteria at 210×105 CFU/g. First order kinetic showed that soil
amended with SC had the highest rate of oil degradation and illustrates the least half-life
for all the diesel fuel concentrations. Bioremediation of diesel fuel contaminated soil
with biomass amendments was monitored for a period of one year under natural
condition. Result indicates complete biodegradation of C8 to C16 and remarkable
biodegradation of C16 to C22 hydrocarbon fractions in contaminated soil amended with
SC. In phytoremediation study, contamination of soil with 2.5% and 1% diesel fuel and
amended with 5% of the three different organic residues was monitored for a period of
270 days under laboratory and natural conditions. About 98.8%, 90.3% and 19% oil loss
i
was recorded in soil amended with SC, polluted with 1%, 2.5% and 5% diesel oil planted
with D. reflexa, respectively. However, diesel contaminated soil with Dracaena but
without organic wastes recorded 62%, 52.4% and 8.5% for 1%, 2.5% and 5%
contamination, respectively under laboratory condition. Also 91%, 84% and 13.8% oil
loss was recorded in soil amended with SC, polluted with 1%, 2.5% and 5% diesel oil
with P. polystachyus, respectively. The remediation process was influenced by oil
concentration and organic biomass added. However, D. reflexa and P. polystachyus root
did not accumulate hydrocarbons from the soil, thus indicating that the mechanism of the
oil degradation was via phytovolatilization or rhizodegradation. Phytoremediation of cocontamination of soil with heavy metals (80 ppm Zn and 60 ppm Pb) and 2.5% diesel
fuel was amended with 5% organic waste was studied for a period of 180 days.
Significant bioaccumulation of Pb and Zn in the root and stem of Dracaena plant was
observed. At the end of 180 days, 16.53 mg/kg and 12.2 mg/kg of Zn accumulation in
root and stem while 16.7 mg/kg and 9.8 mg/kg of Pb in root and stem of D. reflexa was
recorded, respectively. However, 11.8 mg/kg and 9.8 mg/kg bioaccumulation of Zn and
Pb was observed in root of P. polystachyus. Potential of five diesel utilizing bacterial
(DUB) isolates (Stenotrophomonas acidaminiphila, Bacillus lichenifomis, Brevibacillus
parabrevis, Ochrobactrum tritici, Pesedomonas citronellolis) from oil-contaminated soil
to degrade diesel fuel was studied in broth culture for 35 days at 320C. At the end of the
incubation period higher percentage degradation was recorded for Bacillus lichenifomis
(45.8%).
In conclusion, the results of these studies illustrated the potential of SC and the two
plants (D. reflexa and P. polystachyus) as a good option for enhanced remediation of
hydrocarbon-contaminated soil.
ii
ABSTRAK
Pencemaran tarah oleh sebatian/bahanorganik terutamanya hidrokarbon adalah sangat
lazim dinegara perindustrian yang menghasilhan minyak. Kira-kira 1.7 hingga 8.8 juta
tan metrik minyak dilepaskan ke dalam air dan tanah setiap tahun. Pada umumnya, ini
membawa ancaman kepada alam sekitar dan manusia. Kajian ini menilai kecekapan sisa
organik sebagai pelengkap pada pemulihan tanah tercemar oleh bahan api diesel. Tiga
sisa organik [teh daun (TL), kek soya (SC) dan kulit kentang (PS)] dan dua tumbuhan
berdaya maju (Dracaena reflexa dan Podocarpus polystachyus) telah digunakan untuk
menilai tahap biodegradasi bahan api diesel dalam tanah yang tercemar menggunakan
kepekatan minyak berbeza. Tahap biodegradasi tanah telah dirawat dengan 20%, 15%,
10% dan 5% (w / w) bahan api diesel dan dipinda dengan 10% dan 5% TL, SC dan PS.
Ini telah dikaji secara rawak di bawah reka bentuk lengkap bagi tempoh 126 hari di
bawah keadaan makmal. Di penghujung hari yang ke-126, kadar peratusan terendah
degradasi minyak adalah (14.5%) dalam tanah yang tercemar dengan minyak diesel
sebanyak 20%, Seterusnya, dipinda menggunakan TL dan diesel pada 5% dengan kiraan
bakteria pada 30 × 105 pembentukan unit koloni (CFU ) bagi setiap gram tanah. Kadar
tertinggi biodegradasi (95%) dalam tanah tercemar dengan 5% minyak diesel direkod
dan dipinda dengan 10% SC dalam kiraan bakteria sebanyak 210 × 105 CFU / g.
Keputusan hasil kinetik tertib pertama menunjukkan bahawa tanah yang dipinda
menggunakan
SC
mempunyai
kadar
degradasi
minyak
paling
tinggi
serta
menggambarkan separuh hayat yang berkurang bagi semua kepekatan bahan api diesel.
Bioremediasi tanah yang tercemar dengan minyak diesel dan pindaan sisa organik
dipantau untuk tempoh satu tahun di bawah keadaan semula jadi. Keputusan
menunjukkan bahawa biodegradasi penuh C8 hingga C16 dan biodegradasi luar biasa C16
ke C22 merupakan pecahan hidrokarbon di dalam tanah tercemar yang dipinda dengan
SC.
Dalam kajian fitopemulihan, pencemaran tanah pada 2.5% dan 1%, dipinda dengan 5%
daripada tiga bahan buangan organik yang berbeza dan dipantau bagi tempoh 270 hari di
iii
bawah keadaan makmal dan semulajadi. Kadar kehilangan minyak direkod pada 98.8%,
90.3% dan 19% dalam tanah yang dipinda dengan SC. Tanah tersebut tercemar dengan
1%, 2.5% dan 5% minyak diesel dengan D. reflexa. Walaubagaimanapun, dalam tanah
tercemar yang mengandungi Dracaena tanpa sisa organik, tahap pencemaran masingmasing dicatat pada 62%, 52.4% dan 8.5% 1%, 2.5% dan 5% di bawah keadaan makmal.
Kadar kehilangan minyak direkod pada 91%, 84%, dan 13.8% dalam tanah yang
dipinda dengan SC, tercemar pada 1%, 2.5% dan 5% minyak diesel dan P. polystachyus.
Proses pemulihan dipengaruhi oleh faktor kepekatan minyak dan sisa organik biomas.
Malah, akar D. reflexa dan P. polystachyus, tidak mengumpul sisa hidrokarbon dari
tanah, sekali gus menunjukkan bahawa mekanisme degradasi minyak adalah melalui
fitoremediasi. Proses fitoremediasi tanah yang tercemar dengan logam berat (80 ppm Zn
dan 60 ppm Pb) dan tanah yang tercemar dengan 2.5% bahan api diesel, dipinda dengan
5% sisa organik bagi kajian selama 180 hari. Biaokumulasi logam Pb dan Zn dalam akar
dan batang tumbuhan Dracaena diperhatikan. Pada akhir tempoh 180 hari, masingmasing sebanyak 16.53 mg / kg dan 12.2 mg / kg pengumpulan Zn diperhatikan dalam
akar dan batang manakala 16.7 mg / kg dan 9.8 mg / kg Pb diperhatikan dalam akar dan
batang D. reflexa. Walau bagaimanapun, hanya 11.8 mg / kg dan 9.8 mg / kg logam Zn
dan Pb diperhatikan dalam akar P. polystachyus. Potensi lima bakteria menggunakan
diesel pencilan (DUB) (Stenotrophomonas acidaminiphila, Bacillus lichenifomis,
Brevibacillus parabrevis, Ochrobactrum tritici, Pesedomonas citronellolis) dari tanah
yang tercemar dengan minyak diperhatikan selama 35 hari pada 320 °C bagi mengkaji
pengurangan minyak diesel. Pada hari terakhir tempoh pengeraman, kadar peratusan
degradasi yang tinggi direkod bagi Bacillus lichenifomis pada 45.8%.
Kesimpulannya, keputusan kajian ini menggambarkan potensi SC serta dua tumbuhan
iaitu (Dracaena reflexa dan Podocarpus polystachyus) sebagai pilihan terbaik bagi
meningkatkan daya pemulihan tanah tercemar dengan hidrokarbon.
iv
ACKNOWLEDEGMENT
First and foremost, I praise God, the almighty for the gift of life, health and providing me
this opportunity and granting me the capability to proceed successfully. This research has
been extremely fulfilling, not only because of how much I learned, but especially
because it may eventually contribute to the preservation of the environment and assist in
the development of better strategies for approaching environmental contamination.
I would like to express my great gratitude to Professor Dr. Agamuthu Periathamby, for
his guidance, stimulating suggestions and encouragement helped me to overcome
difficulties during the research and writing of this thesis. Thank you for your guide and
without your guide this research will not be completed and well organized.
My appreciation goes to the University of Malaya, for providing the research grant
(PS300/2010B, PV055/2011B and FP014/2010A) to fund this research.
My sincere thanks go to Assistant Professor Dr. Babak Motesharezadeh and Dr. Abioye
O. Peter, for their support and time when I had questions or needed advice. Furthermore,
I would also like to thank Dr. Roslanzairi Mostapa, for his assistance and allowing me
use the facilities and instrumentation at the Malaysian Nuclear Agency.
This thesis is dedicated to my family. My deepest appreciation and love goes to my
mother and brother, Fakhrosadat and Ehsan, for their support and patient throughout my
study outside the country and always encourage me to be best. Thank you very much.
Last but not least, I would like to thank all my lab mates in solid waste laboratory for
their co-operation and advices. Thank you and I love you all.
v
TABLE OF CONTENTS
ABSTRACT
i
ABSTRAK
iii
ACKNOWLEDGEMENT
v
TABLE OF CONTENTS
vi
LIST OF FIGURES
xv
LIST OF TABLES
xxxviii
LIST OF PLATES
xxxiv
LIST OF ABBREVIATIONS
xxxiii
CHAPTER 1
INTRODUCTION
1.1
Significance of petroleum hydrocarbons
1
1.2
Risk of spills for the environment and human health
3
1.3
Environmental biotechnology to diesel fuel clean up
5
1.4
Problem statement of diesel
8
1.5
Aim and Objectives
11
1.6
Research plan
12
CHAPTER 2
2.1
2.2
LITERATURE REVIEW
Overview of Petroleum Hydrocarbons
13
2.1.1 Structure and chemistry of PAHs
13
2.1.2 Diesel fuel toxicity and its composition
16
Weathering Processes of petroleum sources
vi
18
2.3
Biodegradation of pollution contaminated sites
2.3.1 Biodegradation of organic compounds by bacteria
2.4
2.5
2.6
Mechanism of petroleum hydrocarbon degradation
20
21
24
2.4.1 Degradation of hydrocarbon fractions
26
2.4.2 Biodegradation of Aromatic compounds
27
2.4.3 Biodegradation of Benzene
28
2.4.4 Biodegradation of n-alkane
32
Factors affecting biodegradation
35
2.5.1 Oxygen
36
2.5.2 Soil properties
37
2.5.3 Nutrient availability
37
2.5.4 Moisture
39
2.5.5 Temperature
39
2.5.6 Acidity or Alkalinity
41
2.5.7 Bioavailability of hydrocarbon
41
2.5.8 Chemical properties of hydrocarbon
42
2.5.9 Concentration of Petroleum hydrocarbon
43
Remediation techniques
44
2.6.1 Current clean up techniques
47
i. Soil washing with solvents and water
48
ii. Soil vapor extraction
49
iii. Solidification/stabilization
50
vii
2.6.2 Biological technology/ Bioremediation
50
i. In situ bioremediation
53
ii. Bioventing
54
iii. Biosparging/air sparging
55
iv. Ex situ bioremediation
56
v. Bioreactor
56
vi. Land farming
57
vii. Composting and addition of composting materials
57
viii. Biopiling
59
2.6.3 Bioremediation strategies
61
i. Monitored natural recovery
61
ii. Biostimulation approach
61
iii. Bioaugmentation approach
62
2.6.4 Cost of remediation
67
2.6.5 Phytoremediation (Phytotechnology)
68
2.6.6 Costs of phytoremediation
71
2.6.7 Methods of phytoremediation application
71
i. In-situ phytoremediation
71
ii. In-vivo phytoremediation
72
iii. In-vitro phytoremediation
72
2.6.8 Mechanisms of phytoremediation
i. Phytoaccumulation/ Phytoextraction
viii
72
75
ii. Phytodegradation/ Phytotransformation
76
iii. Phytostabilization
78
iv. Rhizodegradation
79
v. Phytovolatilization
81
2.6.9 Influence of Environmental Factors on Phytoremediation
83
2.6.10 Interaction between plants and microorganisms
83
2.6.11 Plant Selection Criteria
84
2.6.12 Plant spices used in this study
87
i. Dracaena reflexa
87
ii. Podocarpus polystachyus
88
2.7
Biodegradation Kinetics
89
2.8
Stable isotopes: A tool to monitor biodegradation process
93
2.9
Heavy metals and PAHs as mixed contaminations
97
CHAPTER 3
METHODS AND MATERIALS
3.1
Collection of soil, diesel fuel, organic wastes and plant materials
100
3.1.1
Organic wastes used in this study
101
i. Spent Tea leaf
101
ii. Soybean cake (SC)
102
iii. Potato skin (peel)
104
3.2
Physicochemical analysis of soil and organic wastes
105
3.2.1
Diesel fuel characteristics
106
3.3
Biostimulation methodology under laboratory condition
106
ix
3.3.1 Experimental set- up
106
3.3.2 Sampling and analysis
107
i. Measurement of residual total petroleum hydrocarbon
107
in soil
ii. PAHs extraction by hydroxypropyl[b]cyclodextrin 108
(Bioavailability)
iii. Measurement of dehydrogenase activity
108
iv. Soil respiration
109
v. Isolation and identification of bacterial diesel degraders 109
vi. Measurement of pH value
111
vii Seed germination toxicity test
111
viii. Gas Chromatography analysis of residual degraded
112
diesel fuel
3.4
3.5
ix. Biodegradation efficiency calculation
112
x. kinetics of diesel removal and Half- Life
112
xi. Measurement of stable isotope carbon (δ 13C)
113
Biostimulation methodology under natural condition
114
3.4.1 Sampling
115
Phytoremediation methodology used in this study
115
3.5.1 Physicochemical analysis of soil, diesel fuel and organic 115
wastes
3.5.2 Experiment set-up under laboratory and natural conditions
115
3.5.3 Sampling and analysis
116
x
i. Plant biomass
3.6
116
Phytoremediation of heavy metals in diesel fuel contaminated soil
117
3.6.1 Selection of heavy metals concentration
117
3.6.2 Preparation of co- contaminated soil
117
3.6.3 Sampling and analysis of samples
118
3.6.4 Analysis of heavy metals in soil and plants
118
3.6.5 Calculations of Translocation factor and Bioconcentartion 119
factor
3.6.6 Rate of metal uptake by plants
120
3.7
Biodegradation studies with microorganisms isolated
120
3.8
Statistical analysis
121
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Characterization of soil and amendments
122
4.2
Evaluation of bioremediation under lab condition
124
4.2.1 Biodegradation of diesel fuel (5 % pollution)
124
4.2.2 Biodegradation of diesel fuel (10% pollution)
129
4.2.3 Biodegradation of diesel fuel (15% pollution)
134
4.2.4 Biodegradation of diesel fuel (20% pollution)
139
4.2.5 Kinetics model and Half- Life of biodegradation
144
4.2.6 Microbial population of soil polluted with 5% diesel fuel
148
4.2.7 Microbial population of soil polluted with 10% diesel fuel
154
4.2.8 Microbial population of soil polluted with 15% diesel fuel
155
4.2.9 Microbial population of soil polluted with 20% diesel fuel
159
xi
4.2.10 Bioavailable fraction in total content of analyzed PAHs
163
in diesel fuel contaminated soil
4.2.11 pH of soil contaminated with 5, 10, 15 and 20% diesel fuel
167
4.2.12 Seed germination toxicity test
172
4.2.13 Monitoring bioremediation using CO2 produced
175
4.2.14 Dehydrogenase activity (DHA)
186
4.2.15 Correlation between CO2 evolution, Dehydrogenase activity 190
and TPH degradation
4.2.16
4.3
4.4
13
C stable isotope analysis
192
4.2.17 Biodegradation Efficiency (BE)
194
Results of bioremediation studies under field condition
197
4.3.1 Biostimulation of diesel fuel contaminated soil
197
4.3.2 Extraction and analysis of residual diesel by GC/MS
201
Result of phytodegradation of soil contaminated with diesel oil using 212
D. reflexa under laboratory condition
4.4.1 Loss of diesel fuel in soil planted with D. reflexa
212
4.4.2 Bacterial count
216
4.4.3 pH of soil in D. reflexa remediation under laboratory 219
condition
4.4.4 Plant growth and
condition
Biomass production under laboratory 221
4.4.5 Plant uptake of hydrocarbons
4.5
225
Results of phytodegradation of soil contaminated with diesel using 226
P.polystachyus under laboratory conditions
xii
4.6
4.5.1 Loss of diesel fuel in soil planted with P.polystachyus
226
4.5.2 Bacterial count
229
4.5.3 pH of soil in P.polystachyus remediation
233
4.5.4 Plant growth and biomass production
234
4.5.5 Plant uptake of hydrocarbons
237
Result of phytodegradation of soil contaminated with diesel using 237
D.reflexa under natural condition
4.6.1 Loss of diesel fuel in soil planted with D.reflexa
238
4.6.2 Bacterial count
241
4.6.3 pH of soil in D.reflexa remediation under natural condition
245
4.6.4 Response of D. reflexa to oil population under natural 246
condition
4.7
Phytodegradation
of
soil
contaminated
with
diesel
using 247
P.polystachyus under natural condition
4.7.1 Loss of diesel fuel in soil planted with P.polystachyus
247
4.7.2 Bacterial count
250
4.7.3 pH of soil in the P.polystachyus remediation
253
4.7.4 Bioaccumulation of heavy metals by Dracaena reflexa
255
4.7.5 Translocation and Bioconcentration factors of Zn and Pb in
259
D. reflexa treatment
4.7.6 Rate of metal uptake by Dracaena reflexa
262
4.7.7 Bioaccumulation of heavy metal by Podocarpus
263
polystachyus
4.7.8 Translocation and Bioconcentartion factor of metals in P.
polystachyus
xiii
267
4.7.9 Rate of metal uptake by Podocarpus polystachyus
4.8
Biodegradation test using bacteria isolated from diesel-contaminated
269
270
soil
4.9
Comparison results of phytoremediation studies with Dracaena and
272
Podocarpus
4.10
General discussion
CHAPTER 5
272
CONCLUSION AND RECOMMENDATIONS
5.1
Conclusion
275
5.2
Recommendations
277
REFERENCES
280
APENDIX A Chemical composition of Diesel fuel
311
APENDIX B Methodology for biostimulation
312
APENDIX C Germination index calculation
313
APENDIX D Methodology for phytodegradation
314
APENDIX E Experimental Design for phytoremediation study
315
under laboratory and natural conditions
APENDIX F Methodology for phytodegradation of co-contaminated soil
316
APENDIX G Experimental Design for phytoremediation of
co-contaminated soil
317
APENDIX H Publications
318
xiv
LIST OF FIGURES
Figure 2.1
Petroleum hydrocarbon structural relationships
Figure 2.2
Main principle of aerobic degradation of hydrocarbons
25
by microorganisms
Figure 2.3
14
Initial steps of bacterial biodegradation pathways for
29
benzene substrates
Figure 2.4
Possible mechanisms of benzene degradation under a
30
anaerobic conditions
Figure 2.5
Possible mechanisms of benzene degradation under
31
aerobic conditions
Figure 2.6
Basic metabolism of n-alkanes degradation
34
Figure 2.7
Factors affecting biodegradation process
35
Figure 2.8
Schematic diagram of soil vapor extraction system
49
Figure 2.9
Air sparging and soil vapor extraction system
56
Figure 2.10
Scheme of different mechanisms of contaminant
74
removal by plants
Figure 2.11
Hypothetical pathway representing the metabolism of
trichoroethylene (TCE) in plant tissues
75
Figure 2.12
Phytoextraction mechanisms
76
Figure 2.13
Phytodegradation mechanisms
77
Figure 2.14
Phytostabilization mechanisms
78
xv
Figure 2.15
Schematic representation of the enzymatic and
microbial activities responsible for the enhanced
80
remediation in rhizosphere zone
Figure 2.16
Plant-degrader interactions potentially involved in
80
rhizodegradation
Figure 2.17
Kinetics of Biodegradation
Figure 2.18
Schematic diagram of an elemental analyser (EA) in
series with IRMS for the analysis of carbon isotope
90
95
ratios
Figure 2.19
Uptake mechanisms on phytoremediation of HMs
Figure 4.1
Biodegradation of diesel fuel in soil contaminated with
5% oil and amended with 5% organic wastes. Bars
98
125
indicates standard error (n = 3)
Figure 4.2
Biodegradation of diesel fuel in soil contaminated with
5% oil and amended with 10% organic wastes. Bars
125
indicate standard error (n = 3)
Figure 4.3
Mean comparison of biodegradation in soil polluted with
129
5% diesel oil amended with 10% organic wastes
Figure 4.4
Biodegradation of diesel fuel in soil contaminated with
10% oil and amended with 5% organic wastes. Bars
130
indicate standard error (n = 3)
Figure 4.5
Biodegradation of diesel fuel in soil contaminated with
130
10% oil and amended with 10% organic wastes. (Bars
indicate standard error, n = 3)
Figure 4.6
Mean comparison of biodegradation in soil polluted with
134
10% diesel oil amended with 10% organic wastes
Figure 4.7
Biodegradation of diesel fuel in soil contaminated with
15% oil and amended with 5% organic wastes. Bars
xvi
135
indicates standard error (n = 3)
Figure 4.8
Biodegradation of diesel fuel in soil contaminated with
15% oil and amended with 10% organic wastes. (Bars
135
indicates standard error, n = 3)
Figure 4.9
Mean comparison of biodegradation in soil polluted with
15% diesel oil amended with 10% organic wastes
139
amended with 10% organic wastes. (Bars indicates
standard error, n = 3)
Figure 4.10
Biodegradation of diesel fuel in soil contaminated with
20% oil and amended with 5% organic wastes. (Bars
140
indicates standard error, n = 3)
Figure 4.11
Biodegradation of diesel fuel in soil contaminated with
20% oil and amended with 10% organic wastes. Bars
141
indicates standard error (n = 3)
Figure 4.12
Mean comparison of biodegradation in soil polluted
with 20% diesel oil amended with 10% organic wastes
Figure 4.13
143
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 5% diesel fuel amended with 5%
148
organic wastes. Bars indicates standard error (n = 3)
Figure 4.14
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 5% diesel fuel amended with
149
10% organic wastes. Bars indicates standard error (n = 3)
Figure 4.15
Total CFU of diesel utilizing bacterial (DUB) in soil
150
contaminated with 5% diesel fuel amended with 5%
organic wastes. Bars indicates standard error (n = 3)
Figure 4.16
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 5% diesel fuel amended with 10%
organic wastes. Bars indicates standard error (n = 3)
xvii
150
Figure 4.17
Total CFU of aerobic heterotrophic bacterial (AHB) in
152
soil contaminated with 10% diesel fuel amended with
5% organic wastes. Bars indicates standard error (n = 3)
Figure 4.18
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 10% diesel fuel amended with
152
10% organic wastes. Bars indicates standard error (n = 3)
Figure 4.19
Total CFU of diesel utilizing bacterial (DUB) in soil
154
contaminated with 10% diesel fuel amended with 5%
organic wastes. Bars indicates standard error (n = 3)
Figure 4.20
Total CFU of diesel utilizing bacterial (DUB) in soil
154
contaminated with 10% diesel fuel amended with 10%
organic wastes. Bars indicates standard error (n = 3)
Figure 4.21
Total CFU of aerobic heterotrophic bacterial (AHB) in
156
soil contaminated with 15% diesel fuel amended with 5%
organic wastes. Bars indicates standard error (n = 3)
Figure 4.22
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 15% diesel fuel amended with
156
10% organic wastes. Bars indicates standard error (n = 3)
Figure 4.23
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 15% diesel fuel amended with 5%
157
organic wastes. Bars indicates standard error (n = 3)
Figure 4.24
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 15% diesel fuel amended with
157
10%organic wastes. Bars indicates standard error (n = 3)
Figure 4.25
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil
contaminated with 20% diesel fuel amended with
5% organic wastes. Bars indicates standard error (n = 3)
xviii
160
Figure 4.26
Total CFU of aerobic heterotrophic bacterial (AHB) in
160
soil contaminated with 20% diesel fuel amended with
10% organic wastes. Bars indicates standard error (n = 3)
Figure 4.27
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 20% diesel fuel amended with 5%
161
organic wastes. Bars indicates standard error (n = 3)
Figure 4.28
Total CFU of diesel utilizing bacterial (DUB) in soil
162
contaminated with 20% diesel fuel amended with 10%
organic wastes. Bars indicates standard error (n = 3)
Figure 4.29
Bioavailable fractions in total content of analyzed PAHs
164
in contaminated soil with 5 % diesel fuel and amended
with 10% organic wastes. The same letter represents no
significant difference (p > 0.05)
Figure 4.30
Bioavailable fractions in total content of analyzed PAHs
165
in contaminated soil with 10 % diesel fuel and amended
with 10% organic wastes. The same letter represents no
significant difference (p > 0.05)
Figure 4.31
Bioavailable fractions in total content of analyzed PAHs
166
in contaminated soil with 15% diesel fuel and amended
with 10% organic wastes. The same letter represents no
significant difference (p > 0.05)
Figure 4.32
Bioavailable fractions in total content of analyzed PAHs
167
in contaminated soil with 20% diesel fuel and amended
with 10% organic wastes. The same letter represents no
significant difference (p > 0.05)
Figure 4.33
pH of soil polluted with 5% diesel fuel and amended
168
with 5% organic wastes. Bars indicates standard error (n
= 3)
Figure 4.34
pH of soil polluted with 5% diesel fuel and amended
xix
168
with 10% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.35
pH of soil polluted with 10% diesel fuel and amended
169
with 5% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.36
pH of soil polluted with 10% diesel fuel and amended
170
with 10% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.37
pH of soil polluted with 15% diesel fuel and amended
170
with 5% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.38
pH of soil polluted with 15% diesel fuel and amended
171
with 10% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.39
pH of soil polluted with 20% diesel fuel and amended
171
with 5% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.40
pH of soil polluted with 20% diesel fuel and amended
172
with 10% organic wastes. Bars indicates standard error
(n = 3)
Figure 4.41
Seed germination toxicity index (%) A) amended soil
175
with 5% organic wastes B) amended soil with 10%
organic wastes
Figure 4.42
Correlation between oil degradation in 20% pollution
182
and CO2 evolution, A) Soil amended with SC, B) Soil
amended with PS, C) Soil amended with TL, D)
Unamended control soil
Figure 4.43
Correlation between oil degradation in 15% pollution
and CO2 evolution, A) Soil amended with SC, B) Soil
xx
183
amended with PS, C) Soil amended with TL, D)
Unamended control soil
Figure 4.44
Correlation between oil degradation in 10% pollution
184
and CO2 evolution, A) Soil amended with SC, B) Soil
amended with PS, C) Soil amended with TL, D)
Unamended control soil
Figure 4.45
Correlation between oil degradation in 5% pollution and
185
CO2 evolution, A) Soil amended with SC, B) Soil
amended with PS, C) Soil amended with TL, D)
Unamended control soil
Figure 4.46
Dehydrogenase activity (DHA) in soil polluted with 5%
187
diesel fuel, (A) soil amended with 5% organic wastes,
(B) soil amended with 10% organic wastes
Figure 4.47
Dehydrogenase activity (DHA) in soil polluted with 10%
188
diesel fuel, (A) soil amended with 5% organic wastes,
(B) soil amended with 10% organic wastes
Figure 4.48
Dehydrogenase activity (DHA) in soil polluted with 15%
188
diesel fuel, (A) soil amended with 5% organic wastes,
(B) soil amended with 10% organic wastes
Figure 4.49
Dehydrogenase activity (DHA) in soil polluted with 20%
189
diesel fuel, (A) soil amended with 5% organic wastes,
(B) soil amended with 10% organic wastes
Figure 4.50
Biodegradation
efficiency
obtained
through
the
195
respirometric data in soil polluted with5% diesel oil and
amended with A) 10% organic wastes B) 5% organic
wastes. Bars indicates standard error (n = 3)
Figure 4.51
Biodegradation
efficiency
obtained
through
the
respirometric data in soil polluted with10% diesel oil and
amended with A) 10% organic wastes B) 5% organic
xxi
195
wastes. Bars indicates standard error (n = 3)
Figure 4.52
Biodegradation
efficiency
obtained
through
the
196
respirometric data in soil polluted with 15% diesel oil
and amended with A) 10% organic wastes B) 5% organic
wastes. Bars indicates standard error (n = 3)
Figure 4.53
Biodegradation
efficiency
obtained
through
the
196
respirometric data in soil polluted with 20% diesel oil
and amended with A) 10% organic wastes B) 5% organic
wastes. Bars indicates standard error (n = 3)
Figure 4.54
Biodegradation of diesel fuel in soil contaminated with
197
5% oil and amended with 10% organic wastes. (Bars
indicates standard error n = 3)
Figure 4.55
Biodegradation of diesel fuel in soil contaminated with
198
10% oil and amended with 10% organic wastes. (Bars
indicate standard error n = 3)
Figure 4.56
Biodegradation of diesel fuel in soil contaminated with
199
15% oil and amended with 10% organic wastes. (Bars
indicates standard error n = 3)
Figure 4.57
Biodegradation of diesel fuel in soil contaminated with
200
20% oil and amended with 10% organic wastes. (Bars
indicates standard error n = 3)
Figure 4.58
Chromatogram of residual diesel fuel in contaminated
soil with 5% oil
a) amended with 5% SC (T1)
203
b)
amended with 5% SC (T 365) c) unamended control soil
(T 365)
Figure 4.59
Chromatogram of residual diesel fuel in contaminated
soil with 5% oil a) amended with 10% SC (T1) b)
amended with 10% SC (T 365) c) unamended control
soil (T 365)
xxii
204
Figure 4.60
Chromatogram of residual diesel fuel in contaminated
206
soil with 10% oil a) amended with 5% SC (T1) b)
amended with 5% SC (T 365) c) unamended control soil
(T 365)
Figure 4.61
Chromatogram of residual diesel fuel in contaminated
207
soil with 10% oil a) amended with 10% SC (T1) b)
amended with 10% SC (T 365) c) unamended control
soil (T 365)
Figure 4.62
Chromatogram of residual diesel fuel in contaminated
209
soil with 15% oil a) amended with 5% SC (T1) b)
amended with 5% SC (T 365) c) unamended control soil
(T 365)
Figure 4.63
Chromatogram of residual diesel fuel in contaminated
210
soil with 15% oil a) amended with 10% SC (T1) b)
amended with 10% SC (T 365) c) unamended control
soil (T 365)
Figure 4.64
Chromatogram of residual diesel fuel in contaminated
211
soil with 20% oil a) amended with 5% SC (T1) b)
amended with 5% SC (T 365) c) unamended control soil
(T 365)
Figure 4.65
Chromatogram of residual diesel fuel in contaminated
212
soil with 20% oil a) amended with 10% SC (T1) b)
amended with 10% SC (T 365) c) unamended control
soil (T 365)
Figure 4.66
Biodegradation of diesel fuel in soil contaminated with
214
1% oil. Bars indicates standard error (n = 3)
Figure 4.67
Biodegradation of diesel fuel in soil contaminated with
214
2.5% oil. Bars indicates standard error (n = 3)
Figure 4.68
Biodegradation of diesel fuel in soil contaminated with
xxiii
216
5% oil. Bars indicates standard error (n = 3)
Figure 4.69
Total CFU of aerobic heterotrophic bacterial (AHB) in
217
soil contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.70
Total CFU of aerobic heterotrophic bacterial (AHB) in
217
soil contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.71
Total CFU of diesel utilizing bacterial (DUB) in soil
218
contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.72
Total CFU of diesel utilizing bacterial (DUB) in soil
219
contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.73
pH of soil contaminated with 1% diesel fuel planted with
220
D. reflexa. Bars indicates standard error (n = 3)
Figure 4.74
pH of soil contaminated with 2.5% diesel fuel planted
220
with D. reflexa. Bars indicates standard error (n = 3)
Figure 4.75
pH of soil contaminated with 5% diesel fuel planted with
221
D. reflexa. Bars indicates standard error (n = 3)
Figure 4.76
Biodegradation of diesel fuel in soil contaminated with
227
1% oil. Bars indicates standard error (n = 3)
Figure 4.77
Biodegradation of diesel fuel in soil contaminated with
227
2.5% oil. Bars indicates standard error (n = 3)
Figure 4.78
Biodegradation of diesel fuel in soil contaminated with
228
5% oil. Bars indicates standard error (n = 3)
Figure 4.79
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
xxiv
230
Figure 4.80
Total CFU of aerobic heterotrophic bacterial (AHB) in
230
soil contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.81
Total CFU of diesel utilizing bacterial (DUB) in soil
232
contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.82
Total CFU of diesel utilizing bacterial (DUB) in soil
232
contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.83
pH of soil contaminated with 1% diesel fuel planted with
233
P.polystachyus. Bars indicates standard error (n = 3)
Figure 4.84
pH of soil contaminated with 2.5% diesel fuel planted
234
with P.polystachyus. Bars indicates standard error (n = 3)
Figure 4.85
Biodegradation of diesel fuel in soil contaminated with
239
1% oil. Bars indicates standard error (n = 3)
Figure 4.86
Biodegradation of diesel fuel in soil contaminated with
239
2.5% oil. Bars indicates standard error (n = 3)
Figure 4.87
Biodegradation of diesel fuel in soil contaminated with
240
5% oil. Bars indicates standard error (n = 3)
Figure 4.88
Total CFU of aerobic heterotrophic bacterial (AHB) in
242
soil contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.89
Total CFU of aerobic heterotrophic bacterial (AHB) in
242
soil contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.90
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
xxv
244
Figure 4.91
Total CFU of diesel utilizing bacterial (DUB) in soil
244
contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.92
pH of soil contaminated with 1% diesel fuel planted with
245
P.polystachyus. Bars indicates standard error (n = 3)
Figure 4.93
pH of soil contaminated with 2.5% diesel fuel planted
246
with P.polystachyus. Bars indicates standard error (n = 3)
Figure 4.94
Biodegradation of diesel fuel in soil contaminated with
248
1% oil. Bars indicates standard error (n = 3)
Figure 4.95
Biodegradation of diesel fuel in soil contaminated with
248
2.5% oil. Bars indicates standard error (n = 3)
Figure 4.96
Biodegradation of diesel fuel in soil contaminated with
249
5% oil. Bars indicates standard error (n = 3)
Figure 4.97
Total CFU of aerobic heterotrophic bacterial (AHB) in
soil contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
251
Figure 4.98
Total CFU of aerobic heterotrophic bacterial (AHB) in
251
soil contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.99
Total CFU of diesel utilizing bacterial (DUB) in soil
252
contaminated with 1% diesel fuel. Bars indicates
standard error (n = 3)
Figure 4.100
Total CFU of diesel utilizing bacterial (DUB) in soil
contaminated with 2.5% diesel fuel. Bars indicates
standard error (n = 3)
252
Figure 4.101
pH of soil contaminated with 1% diesel fuel planted with
253
P.polystachyus. Bars indicates standard error (n = 3)
Figure 4.102
pH of soil contaminated with 2.5% diesel fuel planted
with P.polystachyus. Bars indicates standard error (n = 3)
xxvi
254
LIST OF TABLES
Table 1.1
Annual number of oil spills
Table 2.1
Chemical
Structures
of
9
Various
Categories
of
15
Hydrocarbons
Table 2.2
Relation of carbon number and boiling point ranges
17
Table 2.3
Selected diesel fuel hydrocarbons and some chemical
17
properties of them
Table 2.4
Microorganisms reported to utilize petroleum fractions
23
for growth
Table 2.5
Factors affecting the biodegradation process
44
Table 2.6
Treatment techniques reviewed
46
Table 2.7
Chemical and physical techniques for oil remove
47
Table 2.8
Summary of advantages and disadvantages of in situ and
ex situ on bioremediation processes
Table 2.9
Literature on bioremediation techniques for treating
60
65
contamination
Table 2.10
Data cost for bioremediation projects
67
Table 2.11
Advantages and disadvantages of phytoremediation
70
technology
Table 2.12
Table 2.13
Phytoremediation overview
Plants used for phytoremediation of petroleum
82
86
hydrocarbon
Table 2.14
Stable isotope fraction ratios
xxvii
93
Table 3.1
Dry matter and chemical composition of tea leaf
101
Table 3.2
Physico-chemical composition of soybean cake waste
103
Table 3.3
Chemical composition of potato peel wastes
104
Table 3.4
Experimental Design of phytoremediation
118
Table 3.5
Experimental Design
121
Table 4.1
Soil and organic wastes characterization
123
Table 4.2
Net percentage (%) loss of TPH in soil amended with
137
5% diesel fuel and 5% organic waste amendments
Table 4.3
Net loss percentage (%) of TPH in soil amended with
128
5% diesel fuel and 10% organic waste amendments
Table 4.4
Analysis of variance for biodegradation of 5% diesel
128
fuel amended with 10% organic wastes
Table 4.5
Net (%) loss of TPH in soil amended with 10% diesel
132
fuel with 5% organic waste amendment
Table 4.6
Net (%) loss of TPH in soil amended with 10% diesel
133
fuel with 10% organic waste amendment
Table 4.7
Analysis of variance for biodegradation of 10% diesel
133
fuel amended with 10% organic wastes
Table 4.8
Net percentage (%) loss of TPH in soil amended with
137
15% diesel fuel and 10% organic waste amendments
during bioremediation
Table 4.9
Net percentage (%) loss of TPH in soil amended with
15% diesel fuel and 5% organic waste amendments
xxviii
137
Table 4.10
Analysis of variance for biodegradation of 15% diesel
138
fuel amended with 10% organic wastes
Table 4.11
Net percentage (%) loss of TPH in soil amended with
142
20% diesel fuel and 10% organic waste amendments
Table 4.12
Net percentage (%) loss of TPH in soil amended with
142
20% diesel fuel and 5% organic waste amendments
Table 4.13
Analysis of variance for biodegradation of 20% diesel
143
fuel amended with 10% organic wastes
Table 4.14
kinetic model and half- life of diesel fuel degradation
145
amended with 5% organic wastes
Table 4.15
Kinetic model and half- life of diesel fuel degradation
146
amended with 10% organic wastes
Table 4.16
Seed germination toxicity test (%)
173
Table 4.17
CO2 Produced (mg/100g) in soil polluted with 20%
176
diesel fuel
Table 4.18
CO2 Produced (mg/100g) in soil polluted with 15%
177
diesel fuel
Table 4.19
CO2 Produced (mg/100g) in soil polluted with 10%
178
diesel fuel
Table 4.20
CO2 Produced (mg/100g) in soil polluted with 5% diesel
180
fuel
Table 4.21
Matrix
of
correlation
coefficients
between
CO2
evolution, Dehydrogenase activity and TPH degradation
Table 4.22
Isotopic Composition (δ13C) of carbon dioxide in
biodegradation experiments (‰)
xxix
191
193
Table 4.23
Dry mass of Dracaena plant parts at the end of
224
experiment (270 days)
Table 4.24
Dry mass of Podocarpus plant parts at the end of
236
experiment (270 days)
Table 4.25
Heavy metal concentrations of diesel fuel, soil
255
contaminated with 2.5% oil and unpolluted soil before
remediation
Table 4.26
Residual Zn concentration in soil remediated with
Dracaena and polluted with 80 ppm Zn after 6 months
Table 4.27
Residual Pb concentration in soil remediated with
Dracaena and polluted with 60 ppm Pb after 6 months
Table 4.28
257
Pb contents in D. reflexa in soil contaminated with 60
258
ppm Pb
Table 4.30
256
Zn contents with D. reflexa in soil contaminated with 80
ppm Zn
Table 4.29
256
Bioconcentration and translocation factors of Zinc in
260
Dracaena remediated soil
Table 4.31
Bioconcentration and translocation factors of Pb in
261
Dracaena remediated soil
Table 4.32
Rate of constant metals uptake by Dracaena
262
Table 4.33
Residual Zn concentration in soil remediated with
263
Podocarpus and polluted with 80 ppm Zn after 6 months
xxx
Table 4.34
Residual Pb concentration in soil remediated with
264
Podocarpus and polluted with 60 ppm Pb after 6 months
Table 4.35
Zn contents with P. polystachyus in soil contaminated
265
with 80 ppm Zn
Table 4.36
Pb contents in P. polystachyus in soil contaminated with
266
60 ppm Pb
Table 4.37
Bioconcentration and translocation factors of Zinc in
267
Podocarpus remediated soil
Table 4.38
Bioconcentration and translocation factors of Lead in
268
Podocarpus remediated soil
Table 4.39
Rate of constant metals uptake in remediate soil with
269
Podocarpus
Table 4.40
Percentage of diesel fuel biodegradation by microbial
isolates
xxxi
271
LIST OF PLATES
Plate 2.1
Dracaena reflexa
87
Plate 2.2
Podocarpus polystachyus
88
Plate 3.1
Tea leaf used in this study
102
Plate 3.2
Soy cake used for study
103
Plate 3.3
Potato skin used for study
105
Plate 3.4
Gas chromatography
109
Plate 3.5
Biolog microstation machine
111
Plate 3.6
Isotope Ratio Mass Spectrometer
115
Plate 4.1
D. reflexa, A: Dracaena amended with TL, B:
225
Dracaena amended with SC, C: Dracaena amended
with PS, D: Control Plant, E: Phytotoxicity effect of oil
on Dracaena
Plate 4.2
P.polystachyus, A: Polystachyus amended with TL, B:
Polystachyus amended with SC, C: Polystachyus
amended with PS, D: Control Plant, E: Phytotoxicity
effect of oil on Polystachyus
xxxii
238
LIST OF SYMBOLS AND ABBREVIATIONS
AHB
Aerobic Heterotrophic Bacteria
BCF
Bioconcentartion Factor
CF–IRMS
Continuous Flow Isotope Ratio Mass Spectrometer
CFUs
Colony Forming Units
DUB
Diesel Fuel Utilizing Bacteria
EIA
Energy Information Administration
EPA
Environmental Protection Agency
GC-MSD
Gas Chromatography/ Mass Spectrometry Detector
HPCD
Hydroxypropyl[B]Cyclodextrin
ICP- OES
Inductively Coupled Plasma-Optical Emission Spectroscopy
INTF
Iodonitrotetrazolium Formazan
MSM
Mineral Salt Medium
NRC
National Research Council
NPL
National Priorities List
PS
Potato Skin
PAH
Polyaromatic Hydrocarbon
TL
Tea Leaf
TPH
Total Petroleum Hydrocarbon
TF
Translocation Factor
SC
Soybean Cake
USITC
United States International Trade Commission
VOCs
Volatile Organic Compounds
xxxiii