Bioremediation of Spent Lubricating Oil-Contaminated Sediments in ...

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BIOREMEDIATION OF SPENT
LUBRICATING OIL-CONTAMINATED
SEDIMENTS IN MANGROVE
MICROCOSM
LEUNG KA KIN
MASTER OF PHILOSOPHY
CITY UNIVERSITY OF HONG KONG
September 2008

CITY UNIVERSITY OF HONG KONG
香港城市大學
Bioremediation of Spent Lubricating Oil-
Contaminated Sediments in Mangrove
Microcosm
以紅樹林微觀實驗系統作
廢潤滑油底泥的生物修復
Submitted to
Department of Biology and Chemistry
生物及化學系
In Partial Fulfillment of the Requirements for the
Degree of Master of Philosophy
哲學碩士學位
by
Leung Ka Kin
梁家健
September 2008
二零零八年九月

Declaration i
Declaration
The research described in this MPhil thesis was conducted under the supervision of
Professor N.F.Y. Tam at the Department of Biology and Chemistry, City University of
Hong Kong. It was an independent work of the author unless otherwise stated and has
not been included in any other thesis or dissertation submitted to this or other institution
for a degree, diploma or any other qualifications. Attention is drawn to the fact that
anyone without the author’s prior consent strictly may not copy, reproduce, transform,
or publish any data derived form the author’s own work in this project.
Leung Ka Kin
September 2008

Abstract ii
Bioremediation of spent lubricating oil-contaminated sediments in mangrove
Abstract
microcosm
submitted by Leung Ka Kin
for the Degree of Master of Philosophy
at City University of Hong Kong (September 2008)
Oil pollution has been recognized as one of the most serious anthropogenic threats
to marine and coastal environments. Coastal wetlands distributed along oil transporting
routes are vulnerable to oil pollution, and these habitats are often contaminated with oil
residues and petroleum hydrocarbons (PH). Bioremediation, the use of biological
processes to remove, destroy or sequester hazardous substances from the environment,
has received increasing attention in recent years for clean-up purposes. The potential of
mangrove wetlands in removing heavy metals, inorganic and organic pollutants from
contaminated sediment have been reported. The present study aims to explore the
feasibility of using mangrove wetlands to remedy sediment contaminated by spent
lubricating oil. A series of microcosm studies were conducted in a greenhouse to
determine the potential of mangrove seedlings of different ages, and the importance of
oil-degrading microorganisms in the bioremediation process.

Abstract iii
The one-year old seedlings of two mangrove species, namely Bruguiera
gymnorrhiza (Bg) and Acanthus ilicifolius (Ai) were planted in sediment contaminated
by spent lubricating oil at a dose of 9.04 ± 1.03 mg oil g -1 sediment fresh weight. The
growth and physiological responses of plants during the four-month experiment were
also investigated. The performance of these two seedlings was compared with the
three-month old A. ilicifolius (3MAi). The results demonstrated that the microcosm
planted with one-year old Ai had the highest removal percentage (average of 44%),
followed by 3MAi (41%), Bg (36%) and the unplanted microcosm (just natural
attenuation) had the lowest removal (only 22%). Not only did the three-month old
seedling have a poorer removal efficiency than the one-year old seedling of the same
species, oxidative stress was found in the roots of the oil treated 3MAi, suggesting that
3MAi was more susceptible to oil pollution and was not suitable for bioremediation.
Growth, measured in terms of leaf number and root biomass, also supported that
one-year old Ai was more resistant to oil toxicity than its younger seedlings, and Bg and
was more advantageous for remedying oil-contaminated sediments. The sediment
properties, including redox potential and microbial count, showed that more oxygen was
consumed in the sediment contaminated by spent lubricating oil. Further, the mangrove

Abstract iv
plants helped increase its oxygen status, leading to more oil degradation by
microorganisms.
The effects of mangrove plants and the importance of oil-degrading
microorganisms on bioremediation of oil-contaminated sediment were further assessed.
A greenhouse microcosm study was conducted to study the four commonly used
bioremediation methods, namely natural attenuation (without plants and without
inoculation of oil-degrading microorganisms), phytoremediation (with one-year old Ai),
biostimulation (with the addition of slow-release-fertilizers as extra nutrients) and
bioaugmentation (with the inoculation of an oil-degrading microbial consortium
enriched from mangrove sediment). A total of nine treatments were prepared to
compare the efficiency of each of the four methods and their various combinations in
removing spent lubricating oil from the contaminated sandy mangrove sediment.
At the end of the four-month treatment, the growth and physiological responses of
Ai in the oil-contaminated sediment was comparable to that in the oil-free control,
indicating that Ai could tolerate the toxicity of spent lubricating oil. With the addition of
nutrients to the contaminated sediments, the root of Ai had the lowest content of

Abstract v
malondialdehyde, an indicator of membrane lipid peroxidation and damage due to free
radicals, suggesting that biostimulation enhanced the plant’s vigor, as well as its
resistance to the reactive oxygen stress caused by oil pollution. This may, in turn,
improve the remediation potential of mangrove plants.
The residual concentrations of total petroleum hydrocarbon (TPH) in the aliphatic
(TPH-F1) and aromatic (TPH-F2) fractions in sediment were measured at the end of the
experiment. The mass balance of TPH-F1 and TPH-F2 showed that the TPH taken up
by the mangrove plant (one-year old Ai) could only account for a very small amount of
its total loss, about 0.4 - 8.4 %, even though Ai could tolerate the oil toxicity. This
indicated that the loss of TPH from the mangrove microcosm was mostly due to
biodegradation by microorganisms in the sediment. The overall bioremediation process
was significantly faster in the microcosm with the inoculation of oil-degrading
consortium (bioaugmentation) than that with biostimulation (with nutrient amendment)
or phytoremediation (with Ai), and >50% of TPH-F1 was removed with
bioaugmentation treatment. The microcosm with the combination of bioaugmentation
and biostimulation achieved more than 80% loss of TPH-F2, irrespective of whether it
was planted or unplanted. These findings further demonstrated that the oil-degrading

Abstract vi
inoculants played a more important role in the degradation of oil, especially the
aromatic PH, than the mangrove plant. The potential of employing the oil-degrading
microbial consortium enriched from mangrove sediment to remedy oil-contaminated
wetland habitats should be further explored.

Acknowledgements vii
Acknowledgements
It is not difficult for me to express my gratitude to my supervisor, Prof Nora Tam.
With her enthusiasm, her inspiration, and her great efforts have helped me throughout
my study period. Her guidance and encouragement have been an incredible learning
experience for me, both academically and personally.
Many thanks to Prof Y S Wong and my qualifying panel, Prof Rudolf Wu and Prof
Lilian Vrijmoed. I thank also the oral examination panel, Prof Kin-chung Ho and Dr
Siu-gin Cheung, who had given me critical comments on the thesis and review my
work.
I am indebted to my many student colleagues for providing a stimulating and fun
environment in which to learn and grow. I am especially grateful to Sidney Chan,
Jianlin Chen, Justin Chu, Qinfeng Gao, Chuling Guo, Xu Han, Dr Tiangang Luan, Ada
Wong, Teresa Wong, Keith Yu, Kelvin Yau, Chunguang Zhang and Hongwei Zhou.
Cyrus Ho and Budd Lau were particularly helpful to my experimental setup and
sampling.
The technicians, Amy Chong, Raymond Chan, Hardy Wong, Eric Shum, Helen Ng
and Benz Chan were particularly helpful in the Department and assisted me in many
different ways.
My love, Wu Yan deserves special mention, for helping me to get through the
difficult times, having discussion on my thesis, and for all the support and caring.
Lastly, and most importantly, I wish to thank God and my parents. To them I
dedicate this thesis.

Table of Content viii
Table of content
Declaration i
Abstract ii
Acknowledgements vii
Table of content viii
List of figures xiv
List of tables xx
Abbreviations xxiv
Chapter 1 Introduction 1
1.1 General introduction 1
1.2 Aim and objectives 5
1.3 Research plan 7
Chapter 2 Literature review 9
2.1 Oil pollution in marine habitats 9
2.1.1 Sources and types 9
2.1.2 Effects of oil pollution 12
2.1.3 Fate of oil in marine environment 12
2.2 Types of petroleum oils 13
2.2.1 Different types of oils 13
2.2.2 Lubricating oil and its composition 17
2.3 Remediation of soil and sediment contaminated with organic
pollutants 18
2.3.1 Chemical and physical methods 20
2.3.2 Natural attenuation 20
2.3.3 Biological methods and case studies 22

Table of Content ix
2.3.3.1 Biostimulation 25
2.3.3.2 Bioaugmentation 25
2.3.3.3 Phytoremediation 30
2.4 Mangrove wetlands 33
2.4.1 Characteristics of mangrove wetlands 33
2.4.2 Oil spills and recovery in mangroves 37
2.4.3 Mangrove wetlands for bioremediation 39
Chapter 3 Materials and methods 41
3.1 Experimental setup 41
3.2 Collection of sediment, spent lubricating oil
and plant materials 41
3.3 Analysis of sediment 43
3.3.1 Determination of the content of total petroleum
hydrocarbons (TPHs) 43
3.3.2 Physiochemical analysis 44
3.3.2.1 Sediment pH 44
3.3.2.2 Texture 44
3.3.2.3 Total organic matter (TOM) 45
3.3.2.4 Total nitrogen (TN) 46
3.3.2.5 Total phosphorus (TP) 46
3.3.2.6 Oxidation-reduction potential (ORP) 47
3.3.2.7 Trace metals content 47
3.3.3 Biological properties 48
3.3.3.1 Populations size of total aerobic
heterotrophs and oil-degrading bacteria 48
3.3.3.2 Sediment dehydrogenase activity 49

Table of Content x
3.4 Analysis of spent lubricating oil 49
3.5 GC-FID analysis 50
3.5.1 Quantification of hydrocarbon concentrations 55
3.5.2 Quality control 56
3.5.2.1 Calibration check 56
3.5.2.2 Accuracy and precision 57
3.6 Analysis of plant growth, physiological parameters
and tissue content 60
3.6.1 Plant growth and biomass 60
3.6.2 Malondialdehyde content (MDA) in plant tissues 60
3.6.3 Total petroleum hydrocarbons in roots 61
3.7 Statistical analyses 61
Chapter 4 Screening study on potential mangrove species for
phytoremediation of oil contaminated sediment 62
4.1 Introduction 62
4.2 Materials and methods 64
4.2.1 Microcosm design 64
4.2.2 Sampling and determination 65
4.2.3 Enrichment of oil-degrading microbial consortia
and their degradation potential 67
4.2.4 Statistical analyses 68
4.3 Results 68
4.3.1 Properties of sediment and lubricating oil used
in this study 68

Table of Content xi
4.3.2 Growth and physiological response of mangrove
plants in oil microcosm 71
4.3.2.1 Leaf number 71
4.3.2.2 Biomass 73
4.3.2.3 MDA content 78
4.3.3 Sediment analyses 81
4.3.3.1 Oxidation-reduction potential (ORP) 81
4.3.3.2 Enumeration of total aerobic heterotrophs 81
4.3.3.3 Petroleum hydrocarbons in sediment and
their removal 85
4.3.4 Enrichment of oil-degrading microbial consortia and
the biodegradation potential of the enriched consortia 92
4.4 Discussion 94
4.4.1 Effects of oil on the growth and physiological
response of mangrove plants and the selection
of mangrove plants for bioremediation 94
4.4.2 Effects of oil contamination on biological and
chemical parameters of sediment 96
4.4.3 Ability of different plant species in phytoremediation 98
4.5 Conclusions 99
Chapter 5 Evaluation of bioremediation methods for the clean-up of
spent lubricating oil in mangrove microcosm 101
5.1 Introduction 101
5.2 Materials and methods 105
5.2.1 Microcosm design 105
5.2.2 Materials 106
5.2.3 Sampling methods 107

Table of Content xii
5.2.4 Statistical analyses 108
5.3 Results 109
5.3.1 Growth and physiology of mangrove plants in oil
contaminated sediment 109
5.3.1.1 Plant growth and biomass 109
5.3.1.2 Malondialdehyde content in root 110
5.3.1.3 Accumulation of petroleum hydrocarbons
in mangrove roots 110
5.3.2 Sediment parameters 117
5.3.2.1 Dehydrogenase activity in bulk sediment 117
5.3.2.2 Comparison of dehydrogenase activity in
bulk and rhizosphere sediment 119
5.3.2.3 Analyses of petroleum hydrocarbons in
sediment 121
5.3.2.4 Mass balance of total petroleum hydrocarbons 126
5.4 Discussion 131
5.4.1 Response of Acanthus ilicifolius planted in oil
contaminated mangrove sediment under different
bioremediation treatments 131
5.4.2 Microbial activity in rhizosphere sediment contaminated
with oil under different bioremediation treatments 132
5.4.3 Evaluation of the effectiveness of different
bioremediation treatments on spent lubricating
oil contaminated mangrove sediment 134
5.5 Conclusions 140

Table of Content xiii
Chapter 6 General discussion and conclusions 141
6.1 Feasibility of using mangrove wetland to clean up
spent lubricating oil 141
6.2 Contributions and significance of the present research 147
6.3 Limitations of the present study and future research 148
6.4 Conclusions 150
Reference 151
Appendix: Conference and Publication 172

List of figures xiv
List of figures
Figure 1.1 The research plan of the present MPhil study 8
Figure 2.1 Summary of petroleum product types, TPH and its analytical
methods with respect to approximate carbon number and boiling
point ranges (adopted from Total Petroleum Hydrocarbon
Criteria Working Group Series, 1998)
Figure 2.2 World map showing mangrove distribution zones. Dark lines
show coastal areas where mangroves occur (Duke, 1992)
Figure 2.3 Major shipping routes between Indian Ocean and Northeast Asia
(adopted from Sien, 1998)
Figure 3.1 Sampling locations of sediment and mangrove plant materials
(MP: Mai Po Nature Reserve; YSO: Yung Shu O; KLH: Kei
Ling Ha Lo Wai)
Figure 3.2 Alkane standards for calibration, eluted according to this
sequence: C16, C18, C20, IS, C22, C24, C26, C28 and C30; elution
ranged from 22.36 to 43.50 min
Figure 3.3 PAHs standards for calibration, eluted according to this
sequence: naphthalene (Nap), acenaphthylene (A), acenaphthene
(Ace), fluorene (F), phenanthrene (Phe), anthracene (Ant),
fluoranthene (Flu), pyrene (Pyr), m-terphenyl (IS),
benzo[a]anthracene (BaA), chrysene (Chr),
benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF),
benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (IP),
dibenzo[ah]anthracene (DA), and benzo[ghi]perylene (BP);
elution ranged from 12.97 to 47.93 min
16
34
35
42
51
52

List of figures xv
Figure 3.4 Chromatography of TPH-F1 of an oiled sediment sample. IS =
internal standard; UCM = Unresolved Complex Mixture; TRP =
Total Resolved Peaks; TPH = Total Petroleum Hydrocarbon.
Integration of the TPH was the area from 1 min before C16 to 1
min after C30 elution range, i.e. from 21.36 to 44.50 min
Figure 3.5 Chromatography of TPH-F2 of an oiled sediment sample. IS =
internal standard; UCM = Unresolved Complex Mixture; TRP =
Total Resolved Peaks; TPH = Total Petroleum Hydrocarbon.
Integration of the TPH was the area from 1 min before
naphthalene to 1 min after benzo[ghi]perylene elution range, i.e.
from 11.97 to 48.93 min
Figure 4.1 Stratified sampling plan of 12 pots in each treatment. The
numbers corresponded to sampling months, and same number
represented triplicates
Figure 4.2 The change of total leaf number in oiled sediment and clean
sediment (the control) pots during the 4-month experiment.
Mean and S.D. of triplicates are shown. (3MAi: three-month old
A. ilicifolius; 1YAi: one-year old Ai; Bg: B. gymnorrhiza)
Figure 4.3 Biomass production of three-month old Acanthus ilicifolius
during the 4-month experiment (Mean and S.D. of three
replicates are shown)
Figure 4.4 Biomass production of one-year old Acanthus ilicifolius during
the 4-month experiment (Mean and S.D. of three replicates are
shown)
Figure 4.5 Biomass production of Bruguiera gymnorrhiza during the
4-month experiment (Mean and S.D. of three replicates are
shown)
53
54
66
72
74
75
76

List of figures xvi
Figure 4.6 MDA content in root of oiled treated and control plants
harvested at days 30 and 120 (Mean and S.D. of nine replicates
are shown; 3MAi: three-month old A. ilicifolius; 1YAi: one-year
old Ai; Bg: one-year old B. gymnorrhiza; *** indicates the
control was significantly different from the oiled treatment at
p≤0.001 according to the independent T-test)
Figure 4.7 MDA content in leaf of oiled treated and control plants
harvested at days 30 and 120 (same legend as in Fig. 4.5; no
significant difference was found between control and oil
treatment for each plant species during two sampling times
according to independent T-test at p≤0.05)
Figure 4.8a Redox potential (standard hydrogen electrode) in 2 cm and 5 cm
deep sediment planted with three-month old A. ilicifolius (3MAi)
and one-year old A. ilicifolius (1YAi) at different sampling times
(Means and S.D. of three replicates are shown)
Figure 4.8b Redox potential (standard hydrogen electrode) in 2 cm and 5 cm
deep sediment planted with one-year old B. gymnorrhiza (Bg)
and the oiled control (OC) at different sampling times (Means
and S.D. of three replicates are shown)
Figure 4.9 Population sizes of total aerobic heterotrophs (measured in terms
of most probable number, MPN) in surface sediment collected at
days 30 and 120 (C: control, O: oiled; 3MAi: three-month old A.
ilicifolius; 1YAi: one-year old Ai; Bg: B. gymnorrhiza; OC:
oiled control; mean and standard deviation of triplicates are
shown)
Figure 4.10 Temporal changes of residual total petroleum hydrocarbons,
aliphatic (F1) and aromatic (F2) fractions, in oiled sediments
planted with three-month old A. ilicifolius. Mean and standard
deviation of three replicates are shown; same letter means no
significant difference at p

List of figures xvii
Figure 4.11 Temporal changes of residual total petroleum hydrocarbons,
aliphatic (F1) and aromatic (F2) fractions, in oiled sediments
planted with one-year old A. ilicifolius. Mean and standard
deviation of three replicates are shown; same letter means no
significant difference at p

List of figures xviii
Figure 5.2 The MDA content in roots of A. ilicifolius under different
treatments at the end of four-month experiment (Mean and
standard deviation of three replicates are shown; Different letters
on top of each bar indicate significant differences among
treatments at p≤0.05, according to one-way ANOVA test).
Control: plant without oil; P: Phytoremediation; F:
Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A
Figure 5.3 The concentrations of the aliphatic fraction of total petroleum
hydrocarbons (TPH-F1) in roots of A. ilicifolius under different
treatments at the end of four-month experiment (Mean and
standard deviation of three replicates are shown; Different letters
on top of each bar indicate significant differences among
treatments at p≤0.05, according to one-way ANOVA test).
Control: plant without oil; P: Phytoremediation; F:
Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A
Figure 5.4 The concentrations of aromatic fraction of total petroleum
hydrocarbons (TPH-F2) in roots of A. ilicifolius under different
treatments at the end of four-month experiment (Mean and
standard deviation of three replicates are shown; No significant
differences among treatments at p≤0.05, according to one-way
ANOVA test). Control: plant without oil; P: Phytoremediation;
F: Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A;
PFA: P+F+A
Figure 5.5 The dehydrogenase activity of bulk sediment in different
treatments at the end of four-month experiment (Mean and
standard deviation of five replicates are shown; Different letters
on top of each bar indicate significant differences among
treatments at p≤0.05, according to one-way ANOVA test).
Control: plant without oil; P: Phytoremediation; F:
Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A; NA: Natural Attenuation
113
114
115
117

List of figures xix
Figure 5.6 The dehydrogenase activity of bulk and rhizosphere sediment in
different planted treatments at the end of four-month experiment
(Mean and standard deviation of five replicates are shown; * and
** indicate the dehydrogenase activity of bulk and rhizosphere
sediments were significantly different according to independent
T-test at 0.05 and 0.01 levels, respectively). Control: plant
without oil; P: Phytoremediation; F: Biostimulation; A:
Bioaugmentation; PF: P+F; PA: P+A; PFA: P+F+A
Figure 5.7 Concentrations of residual petroleum hydrocarbons in the
aliphatic fraction (TPH-F1) and aromatic fraction (TPH-F2).
Mean and standard deviation of three replicates are shown.
Arrows A and B on y-axis indicates the concentrations of
TPH-F1 and TPH-F2 in sediment at day 0, respectively. Control:
plant without oil addition; NA: Natural attenuation; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF:
P+F; PA: P+A; PFA: P+F+A
Figure 5.8 The percentage removal of TPH in F1, F2 and F3 fractions.
Mean and standard deviation of three replicates are shown.
Percentage removal = (Day 0 TPH – Month 4 TPH in sediment)
/ Day 0 TPH x 100%. NA: Natural attenuation; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF:
P+F; PA: P+A; PFA: P+F+A
120
123
125

List of tables xx
List of tables
Table 2.1 Major oil spill accidents reported in the last four decades 10
Table 2.2 Average annual inputs (1990-1999) of petroleum to marine
waters from different sources in thousand tonnes (The National
Academy of Sciences, 2002; nd: no data)
Table 2.3 A classification system derived to estimate the kind of damage
expected from oil spills. Vulnerability increases follow a scale
from 1 to 10 (Gundlach and Hayes, 1978)
Table 2.4 Representative physical parameters for TPH analytical
fractions based on correlation to relative boiling point index
Table 2.5 Typical chemical compounds in petroleum products (Potter
and Simmons, 1998)
Table 2.6 Physical and chemical methods for oil removal (Zhu et al.,
2001)
Table 2.7 Bioremediation treatment technologies (Boopathy, 2000) 23
Table 2.8 Summary of the reported oil bioremediation studies 24
Table 2.9 Literature on biostimulation of hydrocarbons (TPH: total
petroleum hydrocarbons; NA: natural attenuation; F:
biostimulation; FA, biostimulation with inoculants; P:
phytoremediation; PF: biostimulation with plants)
Table 2.10 Literature on bioaugmentation of hydrocarbons (TPH: total
petroleum hydrocarbons; NA: natural attenuation; FA:
bioaugmentation with fertilizer; A: bioaugmentation only)
11
15
17
19
21
27-28
29

List of tables xxi
Table 2.11 Literature on phytoremediation of hydrocarbons (TPH: total
petroleum hydrocarbons; TPAH: total polycyclic aromatic
hydrocarbons; P: phytoremediation, NA: natural attenuation;
PA: phytoremediation with inoculants)
Table 2.12 Six of the thirteen oil spills impacting mangroves compiled by
Lewis (1983)
Table 2.13 Impacts and recovery times for mangroves at eight spills
(adopted from NOAA, 2002)
Table 3.1 Mean recovery (%), standard deviations of the recovery and
percentage relative standard deviation (RSD) of the eight
n-alkanes, in the spiked sediment (n=3)
Table 3.2 Mean recovery (%), standard deviation of the recovery and
percentage relative standard deviation (RSD) of the 16 PAHs
in the spiked sediment (n=3)
Table 4.1 Characteristics of Kei Ling Ha surface sediment (Mean ± S.D.,
n=3)
Table 4.2 Characteristics of spent lubricating oil used in the present
study (Mean ± S.D., n=3)
Table 4.3 Comparison of concentrations of trace metals (µg g -1 dwt) in
clean and oiled sediment (Mean ± S.D., n=3) and the effect
levels. * and ** indicate the two sediment was significantly
different according to the independent T-test at 0.05 and 0.001
levels, respectively
Table 4.4 Results of two-way ANOVA test on leaf number during the
4-month experiment (3MAi: three-month old A. ilicifolius;
1YAi: one-year old Ai; Bg: B. gymnorrhiza)
32
36
38
58
59
69
70
71
73

List of tables xxii
Table 4.5 Results of two-way ANOVA test on dried biomass of root,
stem and leaf during the 4-month experiment (3MAi:
three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: B.
gymnorrhiza)
Table 4.6 Results of two-way ANOVA test on total dried biomass during
the 4-month experiment (3MAi: three-month old A. ilicifolius;
1YAi: one-year old Ai; Bg: B. gymnorrhiza)
Table 4.7 Results of three-way ANOVA test showing the effects of oil
treatments (oiled vs. clean sediment), plant species (Ai vs. Bg)
and two sampling times (Days 30 vs. 120) on the Most
Probable Number (MPN) of total aerobic heterotrophs
Table 4.8 Results of two-way ANOVA test showing the effects of four
treatments (three planted and the oiled control) and sampling
times on the residual concentrations of total petroleum
hydrocarbons (TPH) in sediment
Table 4.9 Residual concentrations of total petroleum hydrocarbons
(TPH-F1, mg g -1 ) in flasks inoculated with six consortia
enriched from rhizosphere or bulk sediment collected from
oiled treated 1YAi microcosms at the end of 5-day degradation
experiment in MSM, the percentage removal and the MPN of
oil-degrading bacteria. Mean and standard deviation of three
replicates are shown
Table 5.1 Root concentration factor (RCF) of TPH-F1 and TPH-F2 in
root grown in oil-contaminated sediment of different
remediation approaches (Mean and standard deviation of three
replicates are shown, different letters in each column represent
significant difference at p

List of tables xxiii
Table 5.2 Results of three-way ANOVA test showing effects of different
treatments on the sediment concentrations of residual
petroleum hydrocarbons in three fractions
Table 5.3 Mass balance of total petroleum hydrocarbons (TPH-F1) in
each microcosm. (Mean and standard deviation of three
replicates are shown, different letters in each column represent
significant difference at p

Abbreviations xxiv
Ai Acanthus ilicifolius
ANOVA Analysis of variance
Bg Bruguiera gymnorrhiza
FIA Flow injector analyser
Abbreviations
GC-FID Gas chromatograph-flame ionization injector
INT 2-p-iodophenyl-3-(p-nitrophenyl)-5-pheny tetrazolium chloride
LCEL Lower chemical exceedance level
LSD Least significant difference
MDA Malondialdehyde
MPN Most probable number
MSM Mineral salt medium
PAH Polycyclic aromatic hydrocarbons
RCF Root concentration factor
ROS Reactive oxygen species
SPSS Statistical package for social science
TKN Total Kjeldahl nitrogen
TN Total nitrogen
TOM Total organic matter
TP Total phosphorous
TPH Total petroleum hydrocarbons
UCEL Upper chemical exceedance level

Chapter 1
Introduction 1
1.1 General introduction
Chapter 1 Introduction
Technological advancement has caused a rapid rise in petroleum consumption
and, as a result, a huge amount of hydrocarbons are being discharged into the
environment, either deliberately or accidentally, every year. There have been many
reported oil spills worldwide. For instance, there was a crude oil spill of 0.04 mega
tonnes into Prince William Sound, Alaska in 1989 (Swannell et al., 1996). In 2002, the
Prestige oil spill occurred 209 km offshore and affected 1,900 km of shore line in
northern and north-western Spain and western France, dumping 63,000 tonnes of fuel
oil (Fernandez-Alvarez et al., 2006). The illegal disposal of spent lubricating oil is
another concern. The leakage of oil from vehicles onto the road, and the subsequent
washing of said oil into the coastal environment is also becoming a significant source of
oil pollution in marine habitats (Ngabe et al., 2000).
Strategies for cleaning up oil spills have been briefly described by Zhu et al.
(2001) in a technical guideline for the bioremediation of marine shorelines and
freshwater wetlands. They are basically classified as 1) physical removal by absorbents,

Chapter 1
Introduction 2
2) uses of chemical dispersants, emulsifiers or solidifiers and 3) natural methods,
including evaporation of the lighter-weight components in the oil, photo-oxidation and
biodegradation by various types of microorganisms. Although conventional methods,
such as physical removal, are the first response option in the United States for cleanup,
bioremediation has emerged as one of the alternative treatment options for oil spills.
Bioremediation strategies include natural attenuation, biostimulation,
bioaugmentation and phytoremediation (Skipper, 1999). Natural attenuation has been
defined as the reliance on natural processes to achieve site-specific remedial objectives
(USEPA, 1999), and it has the fewest adverse ecological impacts. Nitrogen and
phosphorous are major concerns with the field application of bioremediation because of
their limited supply in the natural environment (Oh et al., 2001). The addition of
nutrients to enhance biodegradation is called biostimulation. Soluble inorganics,
slow-release fertilizers, oleophilic fertilizers and crop residues have been applied as
nutrients, and their abilities to optimise and sustain bacterial oil degradation activity
were studied (Oh et al., 2001; Rahman et al., 2003; Barahona et al., 2004; Bento et al.,
2005; Coulon et al., 2005; Kim et al., 2005). Bioaugmentation is the addition of
oil-degrading microorganisms to supplement or augment the existing microbial

Chapter 1
Introduction 3
population. Both stimulation of indigenous oil-degraders through the addition of
nutrients or co-substrates and the supplementation of known oil-degrading microbes
into an existing microbial population to enhance biodegradation are well documented
(Atlas, 1995; Head and Swannell, 1999; Mills et al., 2003). The studies of the
microorganisms with hydrocarbon-degrading ability in contaminated areas, wetlands
and mangroves have also been extensively investigated (Burns et al., 1999; Kato et al.,
2001; Rahman et al., 2002; Yerushalmi et al., 2003).
Phytoremediation, the use of plants to remove, destroy or sequester hazardous
substance from the environment (Glick 2003), has been receiving more attention in the
area of bioremediation in recent years. There are studies using plants to remove
inorganic pollutants, including heavy metals (Weis and Weis, 2004), explosives like
trinitrotoluene (TNT) (Hughes et al., 1997), organic compounds such as atrazine (Singh
et al., 2004), polycyclic aromatic hydrocarbons (PAHs) (Paquin et al., 2002; Muratova
et al., 2003), crude oil (Banks, et al., 2003), a mixture of benzene, toluene and xylene
(BTX) (Suominen et al., 2000) and polychlorinated biphenyls (PCBs) (Chekol et al.,
2004). These studies found that plants, ranging from grasses, leguminous plants and
agricultural crops to freshwater wetland plants or reeds, have the promising ability to

Chapter 1
Introduction 4
remove various types of contaminants. It has been proposed that the enhancement effect
is due to 1) the root exudates provide additional nutrients that increase microbial
activity in the rhizosphere, 2) the adsorption and uptake of contaminants by plants, 3)
the modification of oxygen and water contents around root and 4) the secretion of
enzymes by plants to detoxify contamination.
Mangrove habitats distributed along coastlines of tropical and subtropical
regions are vulnerable to marine pollution due to the low wave energy of tides. It is also
a sink of many organic pollutants such as PCBs and petroleum hydrocarbons, however,
mangrove plants appear to be tolerant to these pollutants. Mangrove sediment and roots
were found to harbour diverse groups of microorganisms, which play essential roles in
pollutant degradation (Al-Sayed et al., 2005). Tam et al. (2002) reported that the
bacteria capable of degrading PAHs were isolated from mangrove sediment, suggesting
some intrinsic PAH-biodegradation potential in mangrove ecosystems. These unique
features of mangrove ecosystems create a suitable environment for removing or
transforming pollutants. However, no attention has been given to the use of mangrove
plants and the indigenous microorganisms in their roots and sediment as
phytoremediation and bioaugmentation agents of petroleum hydrocarbons. Although

Chapter 1
Introduction 5
previous studies have shown that mangrove ecosystems are able to resist the pollutants
and even degrade them, the roles and involvement of plants and microbes in the
remediation process are not clearly known yet. Additionally, no research has been done
to explore the effects of nutrient addition on bioremediation of oil in mangrove
sediment.
1.2 Aim and objectives
The seriousness of spent lubricating oil pollution in marine habitats (Kennish,
1992; Ngabe et al., 2000) and the possibility of bioremediation to remove various types
of oil pollution are the driving force behind this study. The aim of this research is to
explore the feasibility of using mangrove wetlands to remedy sediment which has been
contaminated by spent lubricating oil. A series of microcosm studies were conducted in
a greenhouse to compare the potential of mangrove seedlings of different species and
the importance of oil-degrading microorganisms in the bioremediation process. To
achieve this research aim, the following objectives are identified:
1) To compare the species and age effects of Acanthus ilicifolius and Bruguiera
gymnorrhiza on the remediation of spent lubricating oil;

Chapter 1
Introduction 6
2) To investigate the physiological responses of A. ilicifolius and B. gymnorrhiza
during the remediation process;
3) To examine the degradation potential of spent oil by the bacterial consortia
enriched from the oiled mangrove sediment which was planted with the most
effective species in a culture medium and
4) To evaluate different remediation strategies, including phytoremediation,
biostimulation, bioaugmentation and natural attenuation, individually or in
combination, on the remediation of mangrove sediment contaminated with spent
lubricating oil.
The two mangrove species, Acanthus ilicifolius and Bruguiera gymnorrhiza,
selected in the current study represent woody herb and tree respectively. Although K.
obovata is commonly found in Hong Kong mangrove swamps, this species was not
chose because it was relatively sensitive to oil pollution than A. ilicifolius and B.
gymnorrhiza (Zhang, 2006). B. gymnorrhiza is in the family of Rhizophoraceae to
which Kandelia obovata is also belong.

Chapter 1
Introduction 7
1.3 Research plan
The conceptual framework of the present research is illustrated in Figure 1.1.
The study included two parts. The first part was the screening of mangrove species for
phytoremediation purposes and the isolation of oil-degrading consortium. The second
part further explored the potential of different bioremediation strategies, namely natural
attenuation (NA), phytoremediation (P), biostimulation (F) and bioaugmentation (A),
individually and in various combinations, on sandy mangrove sediment contaminated
with spent lubricating oil using greenhouse microcosms.
The thesis consists of six chapters. Chapter 2 is a literature review and Chapter 3
provides an overview of the materials and methods utilized. Chapters 4 and 5 describe
the results of the two parts of the experiments, and Chapter 6 provides a general
discussion and conclusions.

Chapter 1
Introduction 8
Figure 1.1 The research plan of the present MPhil study.

Chapter 2
Literature review 9
2.1 Oil pollution in marine habitats
2.1.1 Sources and types
Chapter 2 Literature review
Oil pollution has been recognized as one of the most serious anthropogenic
threats to the marine environment. The world production of oil is about three billion
tonnes per year and half of it is transported by sea (Clark, 2001). In the last four decades,
there have been many major oil spills worldwide, and they are summarized in Table 2.1.
For instance, with regard to discharges related to transportation, tanker accidents
accounted for 26.2% of spills and 8.5% of the anthropogenic input is due to oil releases
from fixed installations (e.g. coastal refineries). Though oil spill accidents are perceived
to be the major source of oil pollution in the marine environment, Kennish (1992)
stressed that the amount of oil input from chronic discharges via industrial operations,
municipal sources, urban and river runoff, atmospheric fallout and ocean dumping are
significant and account for 65.2% of oil pollution. This far exceeds the loss from the oil
spill accidents. It is estimated that only 45% of the waste oil is collected throughout the
world, which means that 55% is discarded into the environment (Environmental Oil
Ltd., 2000). The leakage from vehicles onto the roads, and subsequently washed to the
coastal environment, is another important source of oil pollution in marine habitats
(Ngabe et al., 2000).
Petroleum inputs into worldwide marine waters are computed for four major
sources, namely, natural seeps, releases during extraction, transportation and

Chapter 2
Literature review 10
consumption (Table 2.2). The last three are significant sources of anthropogenic
petroleum pollution to the marine environment while the releases from consumption
account for one-third of the total load of petroleum to the sea and represent 85% of
anthropogenic load to North American marine water and 70% worldwide (National
Research Council, 2003).
Table 2.1 Major oil spill accidents reported in the last four decades.
Date of oil
spill
Sep. 16,
1969
Apr. 27,
1986
Mar. 24,
1989
Dec. 3,
1992
Nov. 13,
2002
Location
Buzzards Bay,
West
Falmouth,
MA
Galeta Island,
Panama
Prince
William
Sound, Alaska
La Coruna,
Spain
La Coruna,
Spain
Type of oil /
Name of vessel
No. 2 fuel oil /
Florida
Crude oil /
storage tank
ruptured
Crude oil /
Exxon Valdez
Crude oil /
Aegean Sea
Fuel oil /
Prestige
Amount
of oil
Impact
area
7.0x10 5 L Marsh
1.5x10 7 L Mangrove
42x10 6 L Beach
79,000 t Beach
70,000 t Beach
References
Reddy et
al., 2002
Garrity et
al., 1994
Carls et al.,
2004
Pastor et al.,
2001
Junoy et al.,
2005

Chapter 2
Literature review 11
Table 2.2 Average annual inputs (1990-1999) of petroleum to marine waters from
different sources in thousand tonnes (National Research Council, 2003; nd: no data).
Best estimates Minimum Maximum
Natural seeps 600 200 2000
Extraction 38 20 62
Platforms 0.86 0.29 1.4
Atmospheric deposition 1.3 0.38 2.6
Produced waters 36 19 58
Transportation 150 120 260
Pipeline spills 12 6.1 37
Tank vessel spills 100 93 130
Operational discharges (Cargo washings) 36 18 75
Coastal facility spills 4.9 2.4 15
Atmospheric deposition 0.4 0.2 1
Consumption 480 130 6000
Land-based (River and runoff) 140 6.8 5000
Recreational marine vessel nd nd nd
Spills (Non-tank vessels) 7.1 6.5 8.8
Operational discharges (Vessels ≥ 100 GT) 270 90 810
Operational discharges (Vessels ≤ 100 GT) nd nd nd
Atmospheric deposition 52 23 200
Jettisoned aircraft fuel 7.5 5.0 22
Total (in thousand tonnes) 1300 470 8300

Chapter 2
Literature review 12
2.1.2 Effects of oil pollution
Oil spills have significant short- and long-term impacts on coastal ecosystems.
The most prevalent impacts are due to the physical effects and chemical toxicity of oil,
leading to decreasing primary production, plant die-back and marsh erosion (Ko and
Day, 2004). Damages to ecosystems by oil pollution vary according to the types of
habitats receiving oil. Table 2.3 shows a classification of the vulnerability of different
aquatic habitats. Physical factors such as tidal ranges and waves modify the duration of
the impact of oil spills on habitats (Teruhisa et al., 2003).
2.1.3 Fate of oil in marine environment
An oil spill undergoes several physico-chemical alteration processes such as
evaporation, water-washing or photo-oxidation in addition to biodegradation (Bence et
al., 1996; Sauer et al.; 1998; Prince et al., 2002). These natural processes have been
studied in recent years, especially following oil spillages (Gundlach et al., 1983; Sauer,
1993; Wolfe, 1994; Wang et al., 1998; de Hemptinne et al., 2001). For example,
evaporation and water-washing may lead to strong depletion of low-molecular-weight
constituents (Volkman et al., 1984; Kuo, 1994; MacKay and McAuliffe, 1989; Garrett
et al., 1998; Charrié-Duhaut et al., 2000; Santas and Santas, 2000; Taylor et al., 2001).
Photo-oxidation may also play an important role by the generation of water-soluble
compounds that are more available to biodegrading organisms (Peters and Moldowan,
1993; Albaigés et al., 1985; Maki et al., 2001).

Chapter 2
Literature review 13
One major problem of oil pollution is the persistence of petroleum hydrocarbons
in estuarine and marine sediment. Oil persistence could last for many years if left
untreated. Reddy et al. (2002) studied the sedimentary record from the West Falmouth
oil spill and suggested that petroleum residues continued to persist after 30 years and
are likely to remain indefinitely. Short et al. (2004) showed that the Exxon Valdez oil
spill was the largest reservoir of biologically available PAHs on the impacted beaches
12 years after the spill occurred. Corredor et al. (1990) proposed that the persistence
was due to rapid burial of oil into the reducing depth. Burns et al. (1994) confirmed that
the oil from the Galeta spill was trapped in the anoxic mud substrate of the mangrove
ecosystem for at least 20 years. The findings of Yamamoto et al. (2003) suggested that
it took at least two to three years for the inter-tidal animal community to recover to its
original level after the oil spill.
2.2 Types of petroleum oils
2.2.1 Different types of oils
Crude oil is a complex mixture of hydrocarbon and non-hydrocarbon
compounds varying in chemical composition and physical properties (Kennish, 1997).
Crude oil must be refined before it can be used as different petroleum products with
different boiling ranges of hydrocarbons. Figure 2.1 shows the relationship between the
boiling range and the carbon number for some common petroleum products. Four major
classes of hydrocarbons can be found in oils: 1) straight-chain alkanes, 2) branched-
chain alkanes, 3) cycloalkanes and 4) aromatics. Beside hydrocarbons, sulfur
compounds may amount up to 5% by weight.

Chapter 2
Literature review 14
The leachability and volatility of individual hydrocarbons are determined by
several physical properties of oil, including solubility, vapor pressure and propensity to
bind with soil and organic particles. These properties are summarized in Table 2.4.

Chapter 2
Literature review 15
Table 2.3 A classification system derived to estimate the kind of damage expected from
oil spills. Vulnerability increases follow a scale from 1 to 10 (Gundlach and Hayes,
1978).
Scale Habitats Expected damages and action needed
1 Exposed rocky cliffs
2 Exposed rocky platforms
3 Flat fine sand beaches
4
Medium to coarse-grained
beaches
5 Exposed tidal flats
6
Mixed sand and gravel
beaches
7 Gravel beaches
8 Sheltered rocky coast
9 Sheltered rocky plats
10
Salt marshes and
mangroves
Under high wave energy, oil spill clean-up is usually
unnecessary
Wave action causes a rapid dissipation of oil, generally
within weeks. In most cases clean-up is not necessary
Due to close packing of the sediment, oil penetration is
restricted. Oil usually forms a thin surface layer which can
be efficiently scraped off. Clean-up should concentrate on
the high tide mark, lower beach levels are rapidly cleared of
oil by wave action
Oil forms thick oil-sediment layers and mixes down to 1 m
deep with the sediment. Clean-up damages the beach and
should concentrate on the high water level
Oil does not penetrate in compacted sediment surface, but
biological damage results. Clean-up only if oil
contamination is heavy
Oil penetration and burial occur rapidly, oil persists and has
a long-term impact
Oil penetrates deeply and is buried. Removal of oiled gravel
is likely to cause future erosion of the beach
The lack of wave activity enables oil to adhere to rock
surfaces and tidal pools. Severe biological damage. Cleanup
operations may cause more damage than if the oil is left
untreated
Long-term biological damage. Removal of the oil is nearly
impossible without causing further damage. Clean-up only
if the tidal flat is very heavily oiled
Long-term deleterious effects. Oil may continue to exist for
10 or more years

Chapter 2
Literature review 16
Figure 2.1 Summary of petroleum product types, TPH and its analytical methods with respect to approximate carbon
number and boiling point ranges (adopted from Total Petroleum Hydrocarbon Criteria Working Group Series, 1998).

Chapter 2
Literature review 17
Table 2.4 Representative physical parameters for TPH analytical fractions based on
correlation to relative boiling point index.
Source: Agency for Toxic Substances and Disease Registry, U.S. Department of Health
and Human Services, Public Health Service (1999)
2.2.2 Lubricating oil and its composition
Lubricating oil, a refined petroleum product from crude oil, is used to reduce
friction of engine surfaces. It is a complex mixture of hydrocarbons; its composition,
refining process and the additives used vary with the crude oil source (Table 2.5). Spent

Chapter 2
Literature review 18
lubricating oil or used motor oil consists of 73 - 80% aliphatic compounds, 11 - 15%
monoaromatic, 2 - 5% diaromatic and 4 - 8% polyaromatic and polar fractions
(Vazquezduhalt, 1989). Heavy metal content in the used motor oil is higher than that in
fresh or new motor oil because heavy metals originate from the additives in the fuel and
from motor wear and tear. Other types of compounds present in used motor oil are
polycyclic aromatic hydrocarbons (PAHs), and the concentration increases with motor
operating time (Wong and Wang, 2001).
2.3 Remediation of soil and sediment contaminated with organic pollutants
The remediation of sediment contaminated with organic pollutants is a global
problem that consumes considerable economic resources from industries and
governments alike. Most sediment contaminated with organic pollutants is remedied
using a diverse set of thermal, chemical and physical methods that strip the
contaminants from the sediment (Cunningham et al., 1996). In addition to these
physical and chemical treatment methods, microbial-based remediation has become
more common in the last two decades (USEPA, 1992). This recent system relies on the
stimulation of naturally occurring aerobic populations to degrade the contaminants; this
is mostly accomplished by adding nutrients and increasing oxygen flux within the
contaminated zone. All remediation techniques are done either in place (in situ) or by
removing the contaminated materials and transporting them to other sites for treatment
(ex situ).

Chapter 2
Literature review 19
Table 2.5 Typical chemical compounds in petroleum products (Potter and Simmons,
1998).
Petroleum Products Compound classes
Gasoline 1) High concentrations of BTEXs, monoaromatics and
branched alkanes;
2) Lower concentrations of n-alkanes, alkenes,
cycloalkanes and naphthalenes;
3) Very low concentrations of BTEXs and PAHs.
Kerosene 1) High concentrations of cycloalkanes and n-alkanes;
2) Lower concentrations of monoaromatics and branched
alkanes;
3) Very low concentrations of BTEXs and PAHs.
Diesel (#2) 1) High concentrations of n-alkanes;
2) Lower concentrations of branched alkanes,
cycloalkanes, monoaromatics, naphthalenes and PAHs
3) Very low concentrations of BTEXs.
No. 2 Fuel oil 1) High concentrations of n-alkanes;
2) Lower concentrations of branched alkanes,
cycloalkanes, monoaromatics, naphthalenes and
PAHs;
3) Very low concentrations of BTEXs.
No. 6 Fuel oil 1) High concentrations of n-alkanes, cycloalkanes;
2) Lower concentrations of naphthalenes and PAHs
3) Very low concentrations of BTEXs.
Lubricating & motor oil 1) Low concentrations of barium;
2) High concentrations of cycloalkanes and branched
alkanes;
3) Very low concentrations of BTEXs and PAHs.
Crude oil 1) High concentrations of n-alkanes, and branched
alkanes, cycloalkanes;
2) Lower concentrations of BTEXs, PAHs and
naphthalenes;
3) Very low concentrations of sulfur heterocyclics.

Chapter 2
Literature review 20
2.3.1 Chemical and physical methods
Physical treatment methods for contaminated sediment include thermal
desorption, incineration and soil washing, while chemical treatment consists of
chemical extraction, supercritical fluid oxidation, volatilization, steam extraction,
stabilization and encapsulation (Riser-Robets, 1998). Zhu et al. (2001) described the
commonly used physical and chemical methods and summarized their limitations; this
is presented in Table 2.6. Physical containment and recovery of bulk or free oil are the
primary response options in the U.S. for the clean-up of oil spills in marine and
freshwater shoreline environments. Chemical methods, particularly dispersants, have
been routinely used as a response option in many countries, for example, in the U.K.,
where rough coastal conditions may make mechanical responses problematic (Lessard
and Demarco, 2000). However, chemical methods have not been used extensively in the
U.S. due to the disagreement about toxicity and long term environmental effects
(USEPA, 1999).
2.3.2 Natural attenuation
Intrinsic or natural attenuation, based on several natural processes, includes
biodegradation, abiotic transformation, mechanical dispersion, sorption and dilution to
reduce contaminant concentrations in the environment (Morin, 1997). For natural
attenuation to be a viable approach, the site must have a large, natural supply of
nutrients and oxygen, as well as a group of indigenous microorganisms capable of
degrading contaminants. Even with these factors present, the scale of the pollution

Chapter 2
Literature review 21
would need to be small (Hart, 1996). Further, the natural process takes a long time to
restore the pollution.
Table 2.6 Physical and chemical methods for oil removal (Zhu et al., 2001).
Physical method Description Limitations
Booming and
skimming
Wiping with
absorbents
Mechanical
removal
Washing
Sediment
relocation and
tilling
In-situ burning
Use of booms to contain and
control the movement of
floating oil and use skimmers
to recover it
Use of hydrophobic materials
to wipe up oil from the
contaminated surface
Collection and removal of
oiled surface sediments by
using mechanical equipment
Use of low pressure cold water
flushing or high pressure hot
water flushing
Movement of oiled sediment
from one place to another or
tilling and mixing the
contaminated sediment to
enhance natural cleansing
processes by facilitating the
dispersion of oil into the water
column
Oil on the shoreline is burned
usually when it is on a
combustible substrate
Environmental impact is
minimal if traffic of the cleanup
work force is controlled
Disposal of contaminated waste
For limited amounts of oiled
materials
Should not be considered for
sensitive habitats or where
erosion may result
This method, especially high
pressure or hot water, should not
use for wetlands or other
sensitive areas
Tilling may cause oil penetration
deep into the shoreline sediment.
Oil potentially move to adjacent
water bodies
Significant air pollution and
destruction of plants and animals

Chapter 2
Literature review 22
2.3.3 Biological methods and case studies
Conventional technologies are often expensive and not very effective. According
to the Office of Technology Assessment (OTA, 1990), current mechanical methods
typically recover no more than 10 - 15% of the oil after a major oil spill. Bioremediation
has emerged as one of the most promising secondary treatment options for oil removal
since its successful application after 1989 Exxon Valdez spill (Bragg et al., 1994; Prince
et al., 1994). Bioremediation has been defined by Madsen (1991) as “a managed or
spontaneous process in which biological, especially microbial, catalysis acts on
pollutant compounds, thereby remedying or eliminating environmental contamination.”
The advantages of bioremediation over conventional techniques are that it could be
done on site, is often less expensive, has minimal site disruption and has a higher level
of public acceptance. Nevertheless, bioremediation has its limitations. For example,
some chemicals are not amenable to biodegradation; some metabolites could even be
more toxic than their parent compounds, and the treatability is often site-specific
(Boopathy, 2000). Seven common types of bioremediation are available and
summarized in Table 2.7. They involve actively pumping air or supplementing nutrients
or degraders.
Most of the studies on bioremediation are restricted to laboratory scale
experiments with some field trials. A summary on the literature of oil bioremediation is
shown in Table 2.8. All the laboratory scale experiments have encouraging results,
showing microbes could effectively utilize the oil as their carbon source. Most of the
laboratory studies have focused on the potential of using nutrient amendments to

Chapter 2
Literature review 23
enhance oil biodegradation in salt marsh environments. This is because studies
conducted in other shoreline environments have demonstrated that the microbial
population was rarely a limiting factor, and nutrient addition alone had a greater effect
on oil biodegradation than the addition of microbial products (Lee et al, 1997; Venosa
et al., 1996).
Table 2.7 Bioremediation treatment technologies (Boopathy, 2000).
Treatment Definition
Bioaugmentation
Biostimulation
Addition of bacterial cultures to a contaminated medium;
frequently used in bioreactors and ex situ systems
Stimulation of indigenous microbial populations in soils
and / or ground water; may be done in situ or ex situ
Biofilters Use of microbial stripping columns to treat air emissions
Bioreactors
Bioventing
Composting
Landfarming
Biodegradation in a container or reactor; may be used to
treat liquids or slurries
Method of treating contaminated soils by drawing oxygen
through the soil to stimulate microbial growth and activity
Aerobic, thermophilic treatment process in which
contaminated material is mixed with a bulking agent; can
be done using static piles, aerated piles, or continuously
fed reactors
Solid-phase treatment system for contaminated soils; may
be done in situ or in a constructed soil treatment cell

Chapter 2
Literature review 24
Table 2.8 Summary of the reported oil bioremediation studies.
Oil type Site / Condition
Laboratory scale:
Light Arabian oil Mineral medium
Diesel fuel Mineral medium
Crude oil
Natural seawater
medium
Crude oil Artificial seawater
Field trials:
Bonny Light crude
oil
Experimental oil
spill on shoreline of
Delaware Bay
Remediation type
(efficiency)
Mixed culture from
landfarming degradation
(42% in 28 d)
Bacterial consortium
degradation (90% in 50 d)
Photooxidation and
Natural microbial
degradation
(36% within 8 weeks)
Bacterial and yeast
degradation (10-30% in 5 d)
Biostimulation
(2-fold enhanced rate)
Crude oil Freshwater wetland Biostimulation
(35% in 5 months)
Crude oil
Sub-Antarctic
intertidal sediments
Biostimulation
(90% in first 6 months)
References
Del’Arco and
de França,
1999
Richard and
Vogel, 1999
Dutta and
Harayama,
2000
Zinjarde and
Pant, 2002
Venosa et al.,
1996
Venosa et al.,
2002
Pelletier et al.,
2004

Chapter 2
Literature review 25
2.3.3.1 Biostimulation
Microbial degradation of hazardous compounds requires the presence of
nitrogen and phosphorus. However, nutrients are often limited or deficient in sediment.
Biostimulation is the modification of the environment to enhance the growth of
indigenous microbes, usually by nutrient amendment. Extensive biostimulation studies
on oil degradation have been reported (Table 2.9).
One of the main challenges associated with biostimulation in oil-contaminated
coastal areas is maintaining the optimal nutrient concentrations in contact with the oil.
Oil from offshore spills usually contaminates the inter-tidal zone, where the washout
rate for water-soluble nutrients can be very high, and this can adversely influence the
effectiveness of biostimulation (Zhu et al., 2001). Many attempts have been made in the
design of nutrient delivery systems to overcome the washout problem, a characteristic
of intertidal environments (Prince, 1993). These include oleophilic and slow-release
fertilizer formulations and systems that rely on the subsurface flow of water through the
beach (Wise et al., 1994).
2.3.3.2 Bioaugmentation
The biodegradation of petroleum in the marine environment is carried out
largely by diverse bacterial populations. Generally, in pristine environments, the
hydrocarbon-degrading bacteria comprise < 1% of the total bacterial population (Atlas,
1981). Although hydrocarbon-degrading microorganisms are widespread in nature, they

Chapter 2
Literature review 26
may not be capable of degrading the wide range of potential substrates present in
complex mixtures such as petroleum (Leahy and Colwell, 1990) or where the
indigenous microbial population is low. Bioaugmentation involves the introduction of a
microbial group or a single strain that has the ability to degrade the contaminants. The
seeding of degrading microbes could reduce the lag phase to start the bioremediation
process (Forsyth et al., 1995). Its success lies on the competition between the seeded
microbes and the availability of indigenous degraders. Some researchers have reported
that inoculation had either no positive or only marginal effects on oil biodegradation
rates. Table 2.10 shows the previous research work on bioaugmentation.

Chapter 2 Literature review 27
Table 2.9 Literature on biostimulation of hydrocarbons (TPH: total petroleum hydrocarbons; NA: natural attenuation; F: biostimulation;
FA, biostimulation with inoculants; P: phytoremediation; PF: biostimulation with plants).
Contamination type
Two year aged crude
oil, at 20-50, 70-150,
>200 mg g -1
Arabian light crude oil
(3% v/v)
Diesel contaminated
soil, Long Beach:
C12- C23, 2.8 mg g -1
C23- C40 9.45 mg g -1
Bioremediation system
and time
Phytoremediation with
fertiliser (PF) (1 year)
Biostimulation with inoculants
(FA) of intertidal microcosms
i) 21 days
ii) 91 days
Natural attenuation (NA)
and biostimulation (F)
(12 weeks)
Soil
type
Marsh
soil
Sand
(86 %)
Sand
(61 %)
Nutrient amount Removal of TPH References
N, P and K:
666, 272 and 514 kg ha -1 ,
respectively
Slow release fertiliser
C:N:P=100:10:3,
(NH4)2SO4 and K2HPO4 :
250 and 100 mg kg -1 ,
respectively
PF: 59%
P: 28%
Rate µg C/g sand /day:
i) Aliphatic: 144.4;
Aromatic: 44.4;
ii) Aliphatic: 43.9
Aromatic : 134.4
C12-C23 :
NA: 48.7%, F: 45.8%
C23-C40 :
NA: 45.7%, F: 45.2%
Lin and
Mendelssohn,
1998
Oh et al.,
2001
Bento et al.,
2005
Cont’d

Chapter 2 Literature review 28
Cont’d
Table 2.9 Literature on biostimulation of hydrocarbons (TPH: total petroleum hydrocarbons; NA: natural attenuation; F: biostimulation;
FA, biostimulation with inoculants; P: phytoremediation; PF: biostimulation with plants).
Contamination type
Crude oil at 7.30 mg g -1
Arabian Light crude oil or
diesel at 28.53 and 27.33
mg g -1 , respectively
Diesel at 4 mg g -1
Recently contaminated
and weathered crude oil
at 71 mg g -1
Bioremediation system
and time
Natural attenuation (NA)
and biostimulation (F) (150
d)
Biostimulation (F) with
oleophilic fertilizer (180 d)
Biostimulation (F) with
biosolids addition (56 d)
Biostimulation and
bioaugmentation (FA) of
weathered and recently
contaminated (109 d)
Soil type Nutrient amount Removal of TPH References
Clay
(48%)
Sandy
loam
Clay
(90%)
Sandy
(clay loam)
N, P and K: 850, 85
and 240 ug g -1
respectively
Inipol EAP 22 ®
C:N:P=62:7.4:0.7
NH4NO3, Triple
superphosphate
C:N:P 100:1.25:1
F: 62%
NA: 47%
Total alkane 77-95%
PAHs 80%
96%
Recent contamination:
NA: 2.3%, F: 5.7%
Weathered oil:
NA: 11.5%, F: 14.3%
Chaîneau et
al., 2005
Coulon et al.,
2005
Sarkar et al.,
2005
Trindade et al.,
2005

Chapter 2 Literature review 29
Table 2.10 Literature on bioaugmentation of hydrocarbons (TPH: total petroleum hydrocarbons; NA: natural attenuation; FA:
bioaugmentation with fertilizer; A: bioaugmentation only).
Contamination type Sites of Isolation Amount Strains added Removal of TPH References
Arabian light crude oil
(3% v/v) (FA)
Recently contaminated
and weathered crude oil
at 71 mg g -1 (FA)
Diesel contaminated soil,
Long Beach (A):
C12- C23, 2.8 mg g -1 ;
C23- C40 9.45 mg g -1
.
Crude oil
contaminated
intertidal microcosms
Crude oil
contaminated soil
Diesel contaminated
soil, Long Beach
10 6
cells cm -3 sand
10 8
CFU g −1 soil
2.6 x 10 8
cells ml -1
Pseudomonas sp K12-5,
Moraxella sp K12-7,
Yarrowia lipolytic 180
Nocardia nova,
Rhodotorula glutinis var.
dairenesis
Bacillus cereus,
Bacillus sphaericus,
Bacillus fusiformis,
Bacillus pumilus,
Acinetobacter junii,
Pseudomonas sp.
Rate µg C/g sand /day:
Aliphatics: 144.4%;
Aromatics: 44.4%;
Recent contamination:
NA: 2.3%, FA: 5.7%
Weathered oil:
NA: 11.5%, FA: 14.3%
C12-C23 :
NA: 48.7%, A: 72.7%
C23-C40 :
NA: 45.7%, A: 75.2%
Oh et al.,
2001
Trindade et
al., 2005
Bento et al.,
2005

Chapter 2 Literature review 30
2.3.3.3 Phytoremediation
Phytoremediation, the stimulation of contaminant degradation by the growth
of plants and their associated microorganisms, is emerging as a potentially cost-
effective option for the clean-up of petroleum hydrocarbons in terrestrial
environments (Frick et al., 1999; Banks et al., 2000). Table 2.11 shows that the
phytoremediation works on different types of oil pollution. Phytoremediation has
several advantages: relatively low cost, less disruption to the environment, no need for
disposal sites, high probability of public acceptance and potential versatility to treat a
diverse range of hazardous materials (Cunningham et al., 1993; Salt et al., 1995;
Schnoor et al., 1995).
The mechanisms responsible for oil phytoremediation may include
degradation, containment and the transfer of contaminants from the soil to the
atmosphere (Cunningham et al., 1996). The primary loss mechanism for petroleum
hydrocarbons is the degradation of these compounds by microorganisms in the
rhizosphere of plants (Frick et al., 1999). Phytoremediation is hypothesized to be
particularly effective when used together with nutrient enrichment because
hydrocarbon contamination may result in nutrient deficiencies in the contaminated
sediment. Added fertilizers could increase the rate of oil degradation by indigenous
microorganisms in the rhizosphere and simultaneously stimulate plant biomass
production, thereby increasing the effectiveness of phytoremediation and accelerating
the recovery of the affected wetland plant ecosystem (Zhu et al., 2004).
Extensive studies have been conducted on phytoremediation of petroleum
hydrocarbons in terrestrial environments (Frick et al., 1999). But only limited studies

Chapter 2 Literature review 31
have been carried out on the effectiveness of phytoremediation in enhancing oil
degradation in coastal wetland environments. Lin and Mendelssohn (1998) found that
the application of fertilizers in conjunction with the presence of salt marsh and
brackish marsh transplants significantly enhanced oil degradation in the greenhouse.
In another mesocosm study, Dowty et al. (2001) evaluated the effects of soil organic
matter content, plant species, soil oxygen status and nutrient content on oil
degradation and plant growth responses in fresh marsh environments. The study found
that the amount of oil remaining after 18 months was the lowest in aerated and
fertilized mesocosms containing either Panicum hemitomon or Sagittaria lancifolia,
and a substrate of low organic matter content. Field studies, however, did not
demonstrate such significant effects as in the mesocosm studies. A field trial showed
that the addition of nutrients did not result in significant enhancement of
biodegradation of crude oil, regardless of whether plants were left intact or removed
(Garcia-Blanco and Suidan, 2001). Similar results were also found in the St.
Lawrence River freshwater wetland field study (Garcia-Blanco et al., 2001; Venosa et
al., 2002). On the other hand, results of these field trials suggested that although the
application of fertilizers in conjunction with the presence of wetland plants may not
significantly enhance oil degradation, it could accelerate habitat recovery. There is
evidence to support that nutrient amendments could stimulate vigorous vegetative
growth, and reduce sediment toxicity and oil bioavailability (Lee et al., 2001).

Chapter 2 Literature review 32
Table 2.11 Literature on phytoremediation of hydrocarbons (TPH: total petroleum hydrocarbons; TPAH: total polycyclic aromatic
hydrocarbons; P: phytoremediation, NA: natural attenuation; PA: phytoremediation with inoculants)
Contamination type Plants (Time) Soil type Removal References
Mineral oil in experimental
disposal sites (20×20 m)
(0.25 mg g -1 )
Coal gasification site
(1.50 mg g -1 )
Willow
(1.5 years)
Vetch, mustard and ryegrass
(95 d)
Dredged sediment
Loamy sand
57% planted
15% unplanted
43.4 – 47.0% planted
68.7% unplanted
Vervaeke et al., 2003
Liste and Felgentreu,
2006

Chapter 2 Literature review 33
2.4 Mangrove wetlands
2.4.1 Characteristics of mangrove wetlands
Wetlands are defined as “transitional lands between terrestrial and aquatic
systems where the water table is usually at or near the surface or the land is covered
by shallow water” by the U.S. Fish and Wildlife Service (USFWS, 1979). To be
classified as wetlands, they must have one or more of the following three attributes: (1)
at least periodically the land supports predominantly hydrophytes; (2) the substrate is
predominantly undrained hydric soil and (3) the substrate is non-soil and is saturated
with water or covered by shallow water at some time during the growing season of
each year (USFWS, 1979).
Mangroves can be found in inter-tidal areas of tropical and subtropical
latitudes where they exist in conditions of high salinity, extreme tides, high
temperatures and in muddy, anaerobic soils. Mangroves create unique ecological
environments that host rich assemblages of species. The muddy or sandy sediment of
the mangrove ecosystem is home to a variety of epibenthic, infaunal, and meiofaunal
invertebrates. Since they are surrounded by loose sediment, the submerged roots,
trunks and branches of mangroves are islands of habitats that may attract rich
epifaunal communities including bacteria, fungi, macroalgae and invertebrates
(Kathiresan and Bingham, 2001). These diverse communities are important for the
nitrogen and sulfur cycles (Sherman et al., 1998).
Mangroves have enormous ecological value, for example, they protect and
stabilize coastlines, enrich coastal waters (Pearce, 1996), produce organic carbon and

Chapter 2 Literature review 34
contribute significantly to the global carbon cycle (Alongi et al., 2001; Gonneea et al.,
2004).
Mangroves are well adapted to deal with natural stresses (e.g. temperature,
salinity, anoxia and UV) in inter-tidal regions (Kathiresan and Bingham, 2001).
Because of their proximity to population centers, mangroves are sensitive to human
activities and are threatened by developments. Also, the distribution of mangroves
worldwide overlaps the regions of active oil production and transportation (Figs. 2.2
& 2.3). Mangrove habitats are therefore easily susceptible to oil contamination. Some
oil spill accidents affecting mangroves are summarized in Table 2.12.
Figure 2.2 World map showing mangrove distribution zones. Dark lines show coastal
areas where mangroves occur (Duke, 1992).

Chapter 2 Literature review 35
Figure 2.3 Major shipping routes between Indian Ocean and Northeast Asia (adopted
from Sien, 1998).

Chapter 2 Literature review 36
Table 2.12 Six of the thirteen oil spills impacting mangroves compiled by Lewis
(1983).
Name of vessels
and date of the
spill
Argea Prima, 16,
Jul 1962
Whitewater,
13 Dec, 1968
Santa Augusta,
1971
Zoe Colocotroni,
18 Mar, 1973
St. Peter,
Feb, 1976
Howard Star,
5 Oct, 1978
Location Type of oil
Guanica,
Puerto Rico
Galeta Island,
Panama
St. Croix, U.
S. Virgin
Islands
Cabo Rojo,
Puerto Rico
Colombia &
Ecuador
Tampa,
Florida
Amount of oil
spilled
Mangrove species
affected
Crude 10,000 tons Not reported
Diesel oil and
Bunker C
20,000 barrels
Rhizophora mangle
Avicennia sp.
Crude 12.5 million litres Rhizophora mangle
Venezuelan
crude
Crude
20% diesel
80% Bunker C
37,000 barrels
243,442 barrels
carried; quantity
spilled unknown
40,000 gallons
Rhizophora mangle
Avicennia
germinans
Rhizophora sp.
Avicennia sp.
Rhizophora mangle
Avicennia
germinans
Laguncularia
racemosa

Chapter 2 Literature review 37
2.4.2 Oil spills and recovery in mangroves
The most significant damage from oil accidents occurs when the oil is driven
by wind and tides onto the shore. An oil spill vulnerability index has been worked out
by Gundlach (1978). Mangroves were ranked as the most vulnerable habitat (Table
2.3). This may be due to the high organic carbon content and anoxic conditions in the
sediment of mangroves that hinder microbial degradation, as well as the low energy
environment that reduces flushing and water washing. The oil trapped in sediment
continues to persist and is likely to remain indefinitely. The oil residue from the spill
can exert toxic effect for many years and prevent re-colonization. The National
Oceanic and Atmospheric Administration’s (NOAA, 2002) Environmental Sensitivity
Indices, commonly used as a tool for spill contingency planning around the world,
rank mangrove forests as the most sensitive tropical, coastal habitat.
Oil from spills and from petroleum production has permeated many
mangroves. In a report written by the NOAA entitled ‘Oil Spills in Mangroves:
Planning and Response Considerations’, some recent cases of oil spills impacts and
recovery times are summarized (Table 2.12). In most of the studies, mangroves were
re-grown in the oil-impacted areas, but tree height, area of open canopy and other
parameters remained different from controls. Grant et al. (1993) showed that the oiled
sediment, or the oil remaining in the sediment, inhibited new establishment and
decreased the survival of mangrove seedlings for several years.
Oil deposited onto mangroves from oil slicks is brought in by tides and waves.
Mangrove trees have specialized root structures, prop roots and pneumatophores, for

Chapter 2 Literature review 38
breathing under waterlogged sediment. Oil coating on the lenticels of these roots is
one of the contributing factors for plant deaths. Apart from this, long-term and sub-
lethal effects could arise from the chemical toxicity inherent in oil and its persistent
residues. The extent of mangrove damages from oil pollution varies, depending on the
type of oil, and the magnitude and frequency of spilling. For example, fresh oil causes
more leaf loss in Avicennia seedlings than that of aged oil (Grant et al., 1993).
Table 2.13 Impacts and recovery times for mangroves at eight spills (adopted from
NOAA, 2002).
Spill location Oil type Mangrove impacts
Era, Australia, 1992
Santa Augusta, US
Virgin Islands, 1971
Zoe Colocotronis,
Puerto Rico, 1973
Bahia las Minas,
Panama, 1986
Roosevelt Roads,
NAS, Puerto Rico,
1986, 1999
Tampa Bay, 1993
Bunker
fuel
Avicennia marina
75-100 ha impacted
Crude Rhizophora mangle
Venezuela
crude
Crude
Jet Fuel -5
No. 6 &
No. 2 fuel
Rhizophora mangle
Avicennia nitita
Rhizophora mangle
Avicennia germinans
Lagunicularia racemosa
Pelliciera rhizophorae
Lagunicularia racemosa
6 ha killed (1986)
31 acres impacted (1999)
Avicennia germinans
Rhizophora mangle
Lagunicularia racemosa
5.5 acres oiled
Mangrove
recovery
> 4 yr.
> 7 yr. (little to no
recolonisation)
> 6 yr. (mangrove
fringe)
> 5 yr. (fringing
mangroves)
> 6 yr. (recovery
underway)
> 1 yr.
> 1.5 yr.
> 2 yr.
References
Wardop et al.,
1997
Lewis, 1979
Nadeau and
Nergquist,
1977
Gilfillan et al.,
1981
Garrity et al.,
1994
Duke et al.,
1997
Ballou and
Lewis, 1989
Wilkinson et
al., 2001
Levings et al.,
1995, 1997

Chapter 2 Literature review 39
2.4.3 Mangrove wetlands for bioremediation
Over the last decade, the petroleum industry has shown interests in using
constructed wetlands to manage and process wastewater and storm water at a variety
of installations, including refineries, pumping stations and oil and gas wells (Knight et
al., 1999).
Mangroves, as described in Section 2.4.2, are known coastal ecosystems
which are regularly polluted by accidental oil spills (Getter et al., 1981). The anoxic
characteristics of the sediment just few centimeters below the surface inhibits the
biodegradation of organic pollutants, which leads to elevated concentrations of PAHs
in mangrove sediments (Tam et al., 2008). Wetland plants provide many positive
attributes for remediating contaminants. The rhizosphere of wetland plants provides
an enriched zone for microbes to degrade pollutants (Macek et al., 2000; Meharg and
Cairney, 2000). It is believed that wetland plants are capable of oxidizing the
rhizosphere through the release of oxygen from leaves to roots to soils. Plants can also
stimulate growth and metabolism of soil microbes by providing root exudates of
carbon, enzymes and nutrients which can result in more than a 100-fold increases in
microbial counts (Macek et al., 2000). Tam and Wong (1995, 1996) and Tam et al.
(2002) found that mangrove sediments could trap wastewater-borne nutrients, heavy
metals and toxic organic pollutants that could be processed by bacterial communities
and mangrove plants. PAH-degrading bacteria, which have been isolated from
mangrove sediment, have shown promising degrading ability (Tam et al., 2002).
Microcosms using Kandelia candel seedlings for pyrene remediation revealed that
89% of pyrene in surface sediment was removed after six months (Ke et al., 2003). In

Chapter 2 Literature review 40
addition to this example, no other work has been reported on the use of mangrove
species as phytoremediation agents for organic pollutants. In fact, the potential use of
mangrove plants to decontaminate sediment is worthy of further investigation (Burn et
al., 1999; Ramsay et al., 2000).

Chapter 3
Materials and methods 41
3.1 Experimental set-up
Chapter 3 Materials and methods
Two experiments employed pot seedlings nurtured in a greenhouse at City
University of Hong Kong for four months were carried out during September to
December 2004, and July to November 2005, respectively. The first experiment was to
screen different mangrove plant species for phytoremediation purposes. One-year old
Bruguiera gymnorrhiza and Acanthus ilicifolius of three-month and one-year old were
used. The second experiment focused on the one-year old Acanthus ilicifolius. Both
experiments had similar experimental set-up, except the mangrove species or sediment
varied according to the objectives of the experiment. Details of the designs of each
experiment were discussed in individual chapter.
3.2 Collection of sediment, spent lubricating oil and plant materials
Sediment was collected from two mangrove swamps, Kei Ling Ha Lo Wai, Sai
Kung and Mai Po Nature Reserve in Hong Kong SAR during low tide periods. The
surface debris was spaded away, and sediment on the surface to 5 cm was collected and
put into plastic trays. The sediment was then mixed before use.
Spent lubricating oil was collected from a local garage in one batch and stored in
a plastic barrel.

Chapter 3
Materials and methods 42
Mature propagules of Bruguiera gymnorrhiza were collected from Yung Shu O
mangrove swamp in Sai Kung, and seeds of Acanthus ilicifolius were harvested from
Mai Po Nature Reserve (Fig. 3.1). B. gymnorrhiza was germinated by inserting one-
fourth of the propagule into a tray of wet sediment. A. ilicifolius was germinated by
taking the seeds out of the fruits and placed on a tray of wet sediment. Germination was
taken place in City University’s greenhouse. Each of the germinated seedlings, around
one month after planting, was transplanted into a plastic bag (6 cm wide x 10 cm height)
and kept outside of the greenhouse for one month with frequent irrigation of tap water
prior to the experiment.
Figure 3.1 Sampling locations of sediment and mangrove plant materials (MP: Mai Po
Nature Reserve; YSO: Yung Shu O; KLH: Kei Ling Ha Lo Wai).

Chapter 3
Materials and methods 43
3.3 Analysis of sediment
Sediment was treated in different ways before analyses. Some sediment was
freeze-dried for hydrocarbons content, some were air-dried for physiochemical
properties including nutrient and metal content, and the remaining fresh sediment was
used for microbial assays.
3.3.1 Determination of the content of total petroleum hydrocarbons (TPHs)
Five to ten grams of freeze-dried sediment were weighed and put into a 100 ml
pre-ashed conical flask. Around one gram activated copper was added to remove
sulphur. 60 ml of 1:1 n-hexane and dichloromethane mixture was added to the sediment
sample. The sample was then ultra-sonicated for 30 minutes (50-60 Hz, 105 W,
ultrasonic bath, Branson 3210, USA). The ultrasonication was repeated twice with
additional 50 ml dichloromethane for each time. The extracts were collected and filtered
through Whatman No. 541 filter paper. The extracts were concentrated to around 0.5 ml
by a rotary evaporator (Heidolph, Laborator 4000) of 24 °C.
The cleanup and fractionation of the extracts were done by a self-packed silica
gel column. About 3 g of activated silica gel (heated in 550 °C for at least 2 hours) was
packed into a solvent-rinsed 10 ml pipette of a diameter of 0.6 cm and a length of 29 cm
with the bottom outflow plugged with glass wool. 1 cm of sodium sulfate was added on
the top of the silica gel to absorb water. After tapping the column to firmly pack the
silica gel, 20 ml of n-hexane was added to condition the column. The concentrated

Chapter 3
Materials and methods 44
extract was applied to the column with an additional 3 ml n-hexane for complete
transfer. All the solvent eluted up to this stage was discarded. 12 ml of n-hexane was
used to elute the aliphatic fraction (F1) and another 12 ml of 1:1 benzene : n-hexane was
used to elute the aromatic fraction (F2). All fractions were concentrated to less than 1
ml by nitrogen gas blow down. 100 µl of the internal standard (1000 mg ml -1 ), 5-α-
androstane was spiked to the F1 fraction for quantitative analysis using gas
chromatography as described below. M-terphenyl was used as an internal standard for
the F2 fraction. All fractions were made up to 1 ml in volumetric flasks and stored in
brown GC-vials before GC injection.
3.3.2 Physiochemical analysis
3.3.2.1 Sediment pH
The pH value of sediment was measured in 1:1 sediment to water (w/v) ratio.
Ten grams of air-dried sediment in 10 ml deionized water were stirred vigorously to
form a thin paste and stood for an hour before pH measurement. The pH was measured
using standard pH meter (Thermo Orion 9103, USA).
3.3.2.2 Texture
Fifty grams of air-dried sediment were placed in a 200 ml medical flat bottle
with 100 ml deionized water and 25 ml sodium hexametaphosphate (10 % w/v), and
shaken on a horizontal shaker for 24 hours. The entire suspension from the medical flat
bottle was sieved through a 63 µm sieve. The suspension was then transferred into a 1 L

Chapter 3
Materials and methods 45
measuring cylinder and made up to 1 L by deionized water. The sediment particles on
the sieve, >63 µm, were transferred to a crucible. The suspension in the measuring
cylinder was mixed thoroughly by inversion. An aliquot of 20 ml of suspension at a
depth about 15 cm representing particles of size

Chapter 3
Materials and methods 46
3.3.2.4 Total nitrogen (TN)
Sediment samples were digested using Kjeldahl acid digestion method (Page et
al., 1982). Total Kjeldahl Nitrogen (TKN) in the digest was measured using Flow
Injector Analyser (FIA) colorimetry (Lachat QuikChem 8000, USA). This was done by
putting 0.5 g air-dried sediment into a 50 ml digestion tube with 5 ml concentrated
sulphuric acid and a copper Kjeldahl catalyst tablet (consisted of 1.5 g K2SO4 and 0.125
g CuSO4 . 5H2O) and few anti-bumping granules. The tubes were heated in a block
digestor at 160 °C for an hour to remove water vapor. The temperature was raised to
390 °C for 3 hours, until the sample was clear. After cooling, the digested samples were
filtered through Whatman No. 42 filter paper and diluted to 50 ml in a volumetric flask
with double distilled deionized (3D) water. The digests were stored in 4 °C before FIA
measurement. Four grams of air-dried sediment were shaken with 16 ml KCl (2M) for
an hour on a horizontal shaker. The extract was filtered through Whatman No. 42 filter
paper and then measured by FIA. Total Nitrogen in the sediment was obtained by
adding the concentration of nitrate and nitrite and TKN, and expressed in term of oven-
dried weight.
3.3.2.5 Total phosphorus (TP)
Sediment samples were digested using the same Kjeldahl acid method as
described above and measured by FIA colorimetry (Lachat QuickChem Method 8000,
USA). Two grams of air dried sediment were shaken with 0.5 M NaHCO3 in 1:10 (w/v)

Chapter 3
Materials and methods 47
ration for 30 mins on a horizontal shaker. The extract was then filtered through
Whatman No. 42 filter paper and measured by FIA.
3.3.2.6 Oxidation-reduction Potential (ORP)
ORP of the sediment at 2 cm and 5 cm deep was measured monthly during low
tide using the Redox Platinum electrode (Ag/AgCl Saturated KCl) (TPS, IJ 64,
Australia). The values were referred to the standard hydrogen electrode according to the
instruction manual of the electrode.
3.3.2.7 Trace metals content
The concentrations of total heavy metals including Pb, Cd, Zn, Cu, Ni and Cr
and extractable ions (K, Na, Ca and Mg) in sediment were determined according to the
standard methods described by Page (1982). In brief, one gram of air-dried sediment
was digested with 10 ml of concentrated nitric acid at 190 °C block digestor for 4 hours.
Extractable metals were shaken with 1 M ammonium acetate (1:20 w/v) for 15 minutes
on a horizontal shaker. The filtered extracts were kept in the refrigerator at 4 °C until
analysis for heavy metals and ions by ICP-AES (Inductively Coupled Plasma–Atomic
Emission Spectrometry, Plasma 1000, Perkin Elmer, USA) and atomic absorption
spectrophotometer (Shimadzu, AA-6501S, Japan), respectively.

Chapter 3
Materials and methods 48
3.3.3 Biological properties
3.3.3.1 Population size of total aerobic heterotrophs and oil-degrading bacteria
The number of total aerobic heterotrophs was estimated by the Most Probable
Number (MPN) method. Ten grams of fresh surface sediment collected during low tides
were shaken in a medical flat bottle containing 90 ml of sterile Ringer’s solution on a
horizontal shaker for 15 minutes. A series of 10-fold dilution was made for the
suspension. Standard nutrient agar purchased from Oxoid (CM0001) was prepared and
sterilised. About 10 ml agar was poured onto each agar plate. Every nutrient agar plate
was divided into half and each half representing a 10-fold serial dilution factor was
further divided into five divisions (five replicates). A drop of 10 µl of the diluted culture
was inoculated onto each division of the nutrient agar plate as a spot. The plates were
incubated for at least two days at 28 °C. The bacterial colony appeared on each division
was recorded as positive growth. The total number of positive growth was counted and
the bacterial population size was then computed from a standard five-tube MPN table
following the method described in the Standard Methods for the Examination of Water
and Wastewater (1980).
The population size of the oil-degrading bacteria was enumerated using the
same MPN method as for the total aerobic heterotophs except that nutrient agar was
replaced by mineral salt medium (MSM)-agar coated with oil. MSM had the following
composition (mg l -1 ): (NH4)2SO4, 1000; K2HPO4, 800; KH2PO4, 200; MgSO4.7H2O,
200; CaCl2.2H2O, 100; trace elements made up of FeSO4.7H2O, 12; MnSO4.H2O, 3;
ZnSO4.7H2O, 3; CoCl2.6H2O, 1; (NH4)6Mo7O24.7H2O, 1. The oiled MSM-agar was

Chapter 3
Materials and methods 49
prepared by dissolving MSM in agar, 20 µl of spent lubricating oil was then dropped on
the MSM-agar plate and the inoculated plate was incubated for two weeks. The colony
appeared on the oil spot at the end of incubation was counted as positive.
3.3.3.2 Sediment dehydrogenase activity
Dehydrogenase activity was estimated according to Alef and Nannipieri (1995).
One gram of fresh sediment (S) was weighed and put into a test tube containing 1 M
Tris(hydroxymethyl) aminomethane buffer (pH 7) and 9.88 mM of 2-(p-iodophenyl)-3-
(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Two sub-samples (S) were tested
with one autoclaved control (C). They were then incubated at 40 ºC for 2 hrs and
extracted with N,N-dimethylformamide and ethanol (1:1 v/v). The absorbance was
measured at 464 nm. The calibration curve was made with iodonitrotetrazolium chloride
(INF) in four concentrations (0, 100, 200 and 500 µg INF). The dehydrogenase activity
was expressed as µg INF g -1 dwt 2h -1 and calculated as follow:
3.4 Analysis of spent lubricating oil
µg INF g -1 dwt 2h -1 = (S - C) / dwt sediment
The solvent extractable, aliphatic total petroleum hydrocarbons (TPH-F1) and
aromatic total petroleum hydrocarbon (TPH-F2) in spent lubricating oil were analysed
according to the method described by Wang et al. (1994 a, b) with minor modifications.
In short, around 0.7 g of the spent lubricating oil was dissolved in n-hexane and made
up to 10 ml. 0.2 ml of the dissolved waste lubricating oil was applied to the self-packed

Chapter 3
Materials and methods 50
silica gel column, same as that of the sediment extraction. Results were expressed in
weight (mg) of TPH per weight (g) of spent lubricating oil.
3.5 GC-FID Analysis
Quantitative analysis of F1 and F2 fractions were done by GC-FID (Hewlett-
Packard 5890 installed with a flame ionization detector). The capillary column was Rtx-
5 fused silica column of 30 m long, 0.32 mm internal diameter and 0.25 µm film
thickness (Restek, Bellefonte, PA). The column flow was adjusted to a flow rate of 1-
1.5 ml /min. The injector and detector temperatures were kept at 290 °C and 300 °C,
respectively. The oven temperature program was as follow: held at 50 °C for 2 min,
increased to 300 °C at 6 °C / min and a final held at 300 °C for 16 min. Sample extracts
(1 µl) were injected in a splitless mode with 1 min purge off. Helium gas was used as
carrier gas and total petroleum hydrocarbons of aliphatic and aromatic fractions (TPH),
unresolved complex mixture (UCM) and total resolved peaks (TRP) were quantified
based on a five-point calibration curve plotting peak area of standard (ASt) / peak area of
internal standard (AIS) against concentration of standard (CS) / concentration of internal
standard (CIS) (refer to section 3.5.1). The integration of TPH, UCM and TRP were
made between elution range from 1 min before to 1 min after the standards, i.e. 1 min
before n-C16 to 1 min after n-C30 for F1 (Fig. 3.2); and the integration for F2 was 1 min
before naphthalene to 1 min after benzo[ghi]perylene (Fig. 3.3). The TPH area was the
sum of the resolved peaks, and the unresolved hydrocarbons (or UCM) appeared as a
hump between the lower baseline and the base of the resolved peaks subtracting the
internal standards (Figs. 3.4 and 3.5).

Chapter 3
Materials and methods 51
Figure 3.2 Alkane standards for calibration, eluted according to this sequence: C16, C18,
C20, IS, C22, C24, C26, C28 and C30; elution ranged from 22.36 to 43.50 min.

Chapter 3
Materials and methods 52
Figure 3.3 PAHs standards for calibration, eluted according to this sequence:
naphthalene (Nap), acenaphthylene (A), acenaphthene (Ace), fluorene (F), phenanthrene
(Phe), anthracene (Ant), fluoranthene (Flu), pyrene (Pyr), m-terphenyl (IS),
benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF),
benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (IP),
dibenzo[ah]anthracene (DA), and benzo[ghi]perylene (BP); elution ranged from 12.97
to 47.93 min.

Chapter 3
Materials and methods 53
Figure 3.4 Chromatography of TPH-F1 of an oiled sediment sample. IS = internal
standard; UCM = Unresolved Complex Mixture; TRP = Total Resolved Peaks; TPH =
Total Petroleum Hydrocarbon. Integration of the TPH was the area from 1 min before
TRP
UCM
C16 to 1 min after C30 elution range, i.e. from 21.36 to 44.50 min.
IS
TPH-F1

Chapter 3
Materials and methods 54
Figure 3.5 Chromatography of TPH-F2 of an oiled sediment sample. IS = internal
standard; UCM = Unresolved Complex Mixture; TRP = Total Resolved Peaks; TPH =
Total Petroleum Hydrocarbon. Integration of the TPH was the area from 1 min before
naphthalene to 1 min after benzo[ghi]perylene elution range, i.e. from 11.97 to 48.93
min.
IS
TRP
UCM
TPH-F2

Chapter 3
Materials and methods 55
3.5.1 Quantification of hydrocarbon concentrations
Five point calibration curves were set up by plotting AS / AIS against CS / CIS and
the resulting linear regression lines were presented as follows:
AS
AIS =
m CS
CIS
where AIS = area of internal standard
AS
= area of the analyte
(3.1)
m = slope of the regression line (response factor)
CIS
CS
= concentration of internal standard
= concentration of standard
The mean response factor (MRF), that is the mean of the slope of the regression
equation 3.1 of the eight even carbon number n-alkanes from n-C16 to n-C30 or 16
priority PAHs listed by USEPA, was calculated and used as the response factor in the
equation 3.2. The concentrations of TPH or UCM in the extracts were calculated by the
following equation 3.2:
where AIS
AA
CA =
AA x CIS
AIS x MRF
= area of internal standard
= area of the TPH or UCM
MRF = mean response factor
CA
CIS
= concentration of TPH or UCM
= concentration of internal standard
(3.2)

Chapter 3
Materials and methods 56
The concentrations of the TPH or UCM in the extract calculated by the equation 3.2
were converted to concentrations in sediment (weight of TPH or UCM / weight of the
freeze-dried sediment used for extraction).
3.5.2 Quality control
The quality control measures were carried out following the “Method for the
determination of extractable petroleum hydrocarbons” published by Massachusetts
Department of Environmental Protection in 1998.
3.5.2.1 Calibration check
Calibration curves were determined before each batch of sample analysis by
GC-FID. To further ensure the response factors were similar throughout each batch of
sample injection, a mixture of standards and internal standards of each fraction (F1 and
F2) were injected after 20 sample injections. The response factors were then compared.
The relative percent difference (RPD), as shown in equation 3.3, must be within ±25%.
RPD = (RF1 – RF2) / (RF1 + RF2) / 2 * 100% (3.3)
where RF1 = response factor from calibration curves before sample injection
RF2 = response factor from calibration check during sample injection

Chapter 3
Materials and methods 57
3.5.2.2 Accuracy and precision
The accuracy of the eight-even-number carbons (n-C16 to n-C30) and 16 PAHs
was tested. Extractions were done with three replicates of 5 gram freeze-dried Kei Ling
Ha Lo Wai sediment spiked with the standards (with the expected concentration of 20
mg g -1 ). The recoveries for n-alkanes (n-C16 to n-C30) were between 70 – 97 % (Table
3.1) and that for PAHs were 23 – 99 % (Table 3.2). These results were comparable with
those reported by previous workers (Zheng et al., 2002; Ke et al., 2005) and fulfilled the
acceptance criteria suggested by Burns et al., (1997), indicating that the analytical
procedures were reproducible and acceptable.
The precision in terms of the relative standard deviation (RSD, equation 3.4) for
n-alkanes and PAHs were found to be within the acceptable range of 25 % required by
Massachusetts Department of Environmental Protection (1998), except naphthalene,
acenaphthylene and acenaphthene. For these three low molecular weight PAHs, the
RSD varied from 34-56%.
RSD = standard deviation of the recovery / mean of the recovery x 100% (3.4)

Chapter 3
Materials and methods 58
Table 3.1 Mean recovery (%), standard deviations of the recovery and percentage
relative standard deviation (RSD) of the eight n-alkanes, in the spiked sediment (n=3).
n-alkanes Mean recovery (%) Standard Deviation RSD (%)
n-C16 78.28 0.0117 7.48
n-C18 96.83 0.0132 6.82
n-C20 67.05 0.0081 6.04
n-C22 68.69 0.0143 10.43
n-C24 75.21 0.0180 11.94
n-C26 78.28 0.0029 1.86
n-C28 79.75 0.0054 3.36
n-C30 83.00 0.0155 9.32

Chapter 3
Materials and methods 59
Table 3.2 Mean recovery (%), standard deviation of the recovery and percentage
relative standard deviation (RSD) of the 16 PAHs in the spiked sediment (n=3).
PAHs Mean recovery (%)
Standard
Deviation
RSD (%)
Naphthalene 23.29 0.0262 56.34
Acenaphthylene 49.07 0.0336 34.27
Acenaphthene 43.01 0.0385 44.75
Fluorene 63.17 0.0206 16.29
Phenanthrene 66.60 0.0249 18.70
Anthracene 62.37 0.0298 23.90
Fluoranthene 71.54 0.0133 9.29
Pyrene 66.66 0.0231 17.32
Benzo(a)anthracene 70.26 0.0067 4.79
Chrysene 74.57 0.0076 5.09
Benzo(b)fluranthene 64.27 0.0085 6.60
Benzo(k)fluoranthene 85.90 0.0063 3.67
Benzo(a)pyrene 75.91 0.0063 4.15
Indeno(1,2,3,cd)pyrene 74.74 0.0089 5.94
Dibenzo(ah)anthracene 99.36 0.0077 3.86
Benzo(ghi)perylene 62.91 0.0056 4.44

Chapter 3
Materials and methods 60
3.6 Analysis of plant growth, physiological parameters and tissue content
3.6.1 Plant growth and biomass
The growth of mangrove plant was determined in terms of leaf number, stem
height, and biomass every month. Leaves unfolded completely were counted. Stem
height of A. ilicifolius was the length between the base of the most distal pair of leaves
and the sediment, while that of B. gymnorrhiza was the length between the tip of the
propagule where the stem emerged and the bottom of the most distal opened pair of
leaves. Plant sample were collected monthly and each plant was separated into leaf,
stem and root portions, weighed and dried for 2 days until constant weight at 70 °C.
Dried biomass of each portion was then weighed.
3.6.2 Malondialdehyde content (MDA) in plant tissues
0.5 g of fresh plant sample was ground and homogenized in 5 ml of 5 %
trichloroacetic acid (TCA) and centrifuged at 3,000 g for 10 min. 2 ml of the
supernatant were reacted with 2 ml 0.67 % thiobarbituric acid (TBA) in boiling water
for 30 min. The absorbance at 450, 532 and 600 nm was measured and the
malondialdehyde (MDA) content was calculated using the following equation:
MDA content = 6.45 (A532 – A600) – 0.56 (A450)
The MDA content was expressed in µmol g -1 fresh weight.

Chapter 3
Materials and methods 61
3.6.3 Total petroleum hydrocarbons in roots
0.5 gram of freeze-dried root was used to extract the petroleum hydrocarbons in
roots and analysed in the same way as that for sediment. The hydrocarbon content was
expressed in term of freeze-dried weight.
3.7 Statistical analyses
The effects of each factor, including different species of mangrove plants,
bioremediation treatments and sediment types on the concentration of residual
petroleum hydrocarbons in sediment and root, plant growth and physiological
parameters, and microbial count and activity were done by one-way analysis of variance
(ANOVA) test. Two-way ANOVA was employed to test the effects of times and types
of plants in the removal of petroleum hydrocarbon in the preliminary screening. The
sources of variations of different bioremediation methods were compared by three-way
ANOVA. If ANOVA results were significant (p ≤ 0.05), least significant difference
(LSD) multiple comparison would be used to determine the difference among
treatments. All statistical analyses were performed with the software, Statistical Package
for Social Sciences (SPSS 11.0 for Windows, SPSS Inc., USA), except three-way
ANOVA which was done by SigmaStat. 3.0.1 for Window (SPSS Inc., USA).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 62
4.1 Introduction
Chapter 4 Screening study on potential mangrove species
for phytoremediation of oil contaminated sediment
Phytoremediation, the use of plants to remove, destroy or sequester hazardous
substances from the environment, has drawn more and more attention in bioremediation
research in recent years (Glick, 2003). Extensive studies on phytoremediation have been
focused on terrestrial and annual wetland plants (Frick et al. 1999a, b). Plants have been
used to remove inorganic pollutants, including heavy metals (Weis and Weis, 2004),
explosives like trinitrotoluene (TNT) (Hughes et al., 1997), organic compounds such as
atrazine (Singh et al., 2004), polycyclic aromatic hydrocarbons (PAHs) (Paquin et al.,
2002; Muratova et al., 2003), crude oil (Banks et al., 2003), benzene, toluene, p-xylene
(BTX) (Suominen et al., 2000) and polychlorinated biphenyls (PCBs) (Chekol et al.,
2004). Grasses, leguminous plants, agricultural crops and freshwater wetland plants and
reeds have shown promising abilities in removing environmental contaminants. It has
been proposed that the enhancement effects of plants are due to the following reasons: 1)
the root exudates provide additional nutrients that increase microbial activity in
rhizosphere, 2) the adsorption and uptake of contaminants by plants, 3) the modification
of oxygen and water contents around roots and 4) the secretion of enzymes by plants to
detoxify contamination (Anderson et al., 1993; Cunningham et al., 1993; Salt et al.,
1995; Schnoor et al., 1995).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 63
Mangrove habitats are vulnerable to marine pollution due to the low wave
energy of tides. The mangrove sediment is a sink of many organic pollutants such as
PCBs and petroleum hydrocarbons; but mangrove plants appeared to be tolerant to these
pollutants. It has been reported that mangrove plants in a mangrove swamp in Hong
Kong SAR were able to recover from an accidental oil spill which occurred nearby,
with new leaves and new buds produced only a few months after the spill (Tam et al.,
2005). Tam et al. (2002) also reported that the bacteria capable of degrading PAHs
could be isolated from mangrove sediment, suggesting that the sediment had some
intrinsic PAH-biodegradation potential. However, research on the use of mangrove
plants and their associated microorganisms as phyto- or bio-remediation measures for
toxic organic pollutants is still limited. The roles and involvement of plants in the
remediation process are relatively unknown. For any plant to be considered for
phytoremediation purposes, the plant must be able to grow and resist the pollutants.
However, the tolerance of different plant species, including mangroves, should be
different.
Growth of plants can be reflected by changes of leaf number and biomass. The
leaf function to derive solar energy for a plant’s metabolism via photosynthesis is an
important growth factor. A plant’s roots are in direct contact with the contaminated
sediment, and act as the plant’s first barrier and defensive measure. The root biomass
and concentration of contaminants in roots are also significant stress indicators. It is
important for a plant to have healthy roots in phytoremediation because the process
relies on roots to stimulate biodegradation, adsorption and uptake of the pollutant. The
physiological response of plants under stress could also be assessed by the extent of

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 64
lipid peroxidation. Malondialdehyde (MDA), the product of lipid and protein
peroxidation, is commonly used for measuring plants under stress.
The aims of the present study are: 1) explore the potential of mangrove
ecosystems to remove petroleum hydrocarbons in spent lubricating oil; 2) compare the
ability of two mangrove species, namely Acanthus ilicifolius and Bruguiera
gymnorrhiza, in the remediation of spent lubricating oil; 3) examine the temporal
change of petroleum hydrocarbons during the phytoremediation process; 4) understand
the growth and physiological response of mangrove plants, and the changes in microbial
activity in sediment during the remediation process and 5) enrich oil-degrading
microbial consortia and test their biodegradation ability for bioaugmentation purposes.
4.2 Materials and methods
4.2.1 Microcosm design
A total of 48 plastic pots, each with a diameter of 12 cm and height of 11.5 cm,
containing about 1.8 kg fresh weight sediment, was prepared to be individual mangrove
microcosms. The sediment was first evenly mixed with 100 ml of spent lubricating oil,
and then allowed to stand for one day to let the volatile organic compounds in the oil
evaporate before adding sediment to the pot. The 48 pots were divided into four oil-
treatment groups, each had 12 replicates. The four groups were: 1) three seedlings of
three-month old A. ilicifolius were planted (3MAi) in each pot; 2) two seedlings of one-
year old A. ilicifolius were planted (1YAi); 3) one seedling of one-year old B.
gymnorrhiza (Bg) were planted and 4) oiled control without plants (OC). The number of

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 65
seedlings among three planted groups was different because the sizes of the seedlings
were different, with more seedlings being planted if they were smaller in size. In
addition, three parallel, oil-free plant controls (the sediment did not have any oil added
to it and was considered to be ‘clean’ sediment), 12 pots for each plant group and a total
of 36 pots, were set up to monitor plant growth under conditions without oil, and were
used for comparing the effects of oil on different mangrove species and ages.
The pots were arranged into groups of 12 and then placed in a large tank. The
pots were subjected to one tidal cycle a day, with 12-hour high and low tides. During
the high tide period, the artificial seawater (prepared by dissolving the artificial salts
purchased from Instant Ocean, USA in deionised water to achieve a salinity of 15 ‰)
was pumped in to submerge the pots in a depth of seawater approximately 3 cm above
the sediment surface. At the end of 12-hour high tide, the seawater was drained back to
the storage tank. The tank and the pots were then left dried and exposed to air for 12
hours, similar to the low tide condition. The seawater was reused for about one month
before renewal, the salinity was checked and deionised water was added to compensate
for any evaporation loss. The concentration of residual petroleum hydrocarbons in the
used seawater was also determined. This experiment lasted for four months, from
September to December, 2004. Triplicate pots from each treatment and the control were
retrieved monthly and four samplings were carried out during the experiment.
4.2.2 Sampling and determination
To minimize the possible positional effects of pots inside the tank, stratified

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 66
sampling was employed to select the triplicates. The sampling plan was illustrated in
Figure 4.1, which enable at least one sample from each row was collected at each
months.
Figure 4.1 Stratified sampling plan of 12 pots in each treatment. The numbers
corresponded to sampling months, and same number represented triplicates.
During each sampling, before the pots were retrieved, the oxidation and
reduction potential of the sediment, at 2 cm and 5 cm depths, for each pot, were
measured one hour after low tide using the redox potential electrode as described in
Section 3.3.2.6. The plants were harvested and the roots were carefully separately from
the sediment. The sediment adhering to the root was collected as the rhizosphere root
and the rest was called bulk sediment. The fresh bulk sediment was used for the
enumeration of the total aerobic heterotrophs and oil-degrading (or oil-resisting)
bacteria, using the method described in Section 3.3.3.1. The remaining bulk sediment
was divided into two portions, one was air-dried and the other was freeze-dried. The
plant growth, in terms of leaf number and biomass, was measured according to Section
3.6. The MDA content in the fresh mangrove root and leaf collected at the end of first
and the fourth months was analyzed (Section 3.6.2).
1 3 4
3 1 2
4 2 1
2 4 3

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 67
4.2.3 Enrichment of oil-degrading microbial consortia and their degradation
potential
Both the bulk and rhizosphere sediment from the oiled treated 1YAi were
collected at the end of the 4-month experiment and used to enrich oil-degrading
microbial consortia. Around 10 g of fresh sediment was transferred to a medical flat
bottle containing 90 ml of sterilized mineral salt medium (MSM) and shaken on a
horizontal shaker for 15 minutes. 10 ml of the sediment slurry was then transferred to a
conical flask containing 90 ml sterilized MSM (at salinity of 15 ‰ using NaCl to adjust
the salinity) and 1% (v/v) of spent lubricating oil. The flask was shaken at room
temperature (20 o C ± 2 o C), in the dark, at 150 rpm for the oil-degrading microbes to
grow. After one week of cultivation, the first enriched consortium was sub-cultured by
transferring it to another flask containing 90 ml sterilized MSM with oil. This
subculture procedure was repeated twice to obtain the third enriched consortium.
An aliquot of 5 ml of the third enriched consortium was inoculated into a 100 ml
conical flask containing 45 ml MSM and 1% (v/v) spent lubricating oil. Another flask
with the same concentration of MSM and oil, but without any microbial inoculum, was
prepared and used as the abiotic control. The flasks were shaken on an orbital shaker at
150 rpm at room temperature (20 o C ± 2 o C) in the dark. The concentrations of residual
petroleum hydrocarbons in the medium were determined at the end of the 5-day
incubation, as described in Section 3.3.1. The most probable number (MPN) of the oil-
degrading bacteria at Day 0 and Day 5 were measured using the same method as
described in Section 3.3.3.1.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 68
4.2.4 Statistical analyses
A parametric two-way analysis of variance (ANOVA) was used to test any
significant differences among sampling times (first to fourth month) and the four oil-
treatments (3MAi, 1YAi, Bg and OC) for each parameter. If ANOVA results were
significant (p ≤ 0.05), the least significant difference (LSD) multiple comparison would
be used to determine where the difference among treatments was. An independent T-test
was employed to compare any difference of root MDA content between the two
sampling times. The T-test was also used to compare the concentrations of pollutants in
oiled and clean sediment. All statistical analyses were performed with the software,
Statistical Package for Social Sciences (SPSS 11.0 for Windows, SPSS Inc., USA).
4.3 Results
4.3.1 Properties of sediment and lubricating oil used in this study
The general characteristics of the sediment collected from Kei Ling Ha (KLH),
Sai Kung (Fig. 3.1) are summarised in Table 4.1. The sediment had a very high
percentage of sand-sized particles and could be classified as sandy soil, according to the
standard soil triangle. The total organic matter content was around 2.5% and was
comparable to that in other mangrove swamps in Hong Kong (Guo et al., 2005). The
nutrient contents were also similar to the Sai Keng sediment, a typical Hong Kong
mangrove stand adjacent to KLH, as reported by Tam and Wong (1998).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 69
Table 4.1 Characteristics of Kei Ling Ha surface sediment (Mean ± S.D., n=3).
Properties Values
Texture
Sand % 83.93 ± 0.44
Silt % 5.64 ± 0.80
Clay % 10.34 ± 0.34
Total Organic Matter % dwt 2.49 ± 0.07
pH (1:1 DI water) 7.5 ± 0.1
TKN µg g -1 dwt 193.99 ± 10.31
TP µg g -1 dwt 186.24 ± 6.32
Sodium (Na) µg g -1 dwt 133.17 ± 0.95
Potassium (K) µg g -1 dwt 20.14 ± 0.64
Magnesium (Mg) µg g -1 dwt 36.35 ± 0.65
Calcium (Ca) µg g -1 dwt 29.03 ± 5.99
Table 4.2 shows that the spent lubricating oil contained around 350 mg g -1 of
total aliphatic hydrocarbon (TPH-F1) (around 69.4% of total petroleum hydrocarbons);
while 153 ± 7 mg g -1 (30.6%) were aromatic hydrocarbons (TPH-F2). The sediment
spiked with oil at the beginning of the experiment had a concentration of total aliphatic
hydrocarbon (TPH-F1) of 8.50 ± 0.60 mg g -1 freeze-dried sediment, the total aromatic
fraction (TPH-F2) was 0.54 ± 0.43 mg g -1 and the total petroleum hydrocarbon (TPH-F3)
was 9.04 ± 1.03 mg g -1 . The heavy metals in the oiled sediment were summarized in
Table 4.3. Higher concentrations of lead and zinc were observed in oiled sediment,
more so than in the uncontaminated sediment. However, none of the trace metals fell
above the Lower Chemical Exceedance Level (LCEL), as suggested by the

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 70
Development Bureau of HKSAR. The oiled sediment fell under the Category L of the
marine sediment classification scheme used in Hong Kong. The scheme divides
sediment into three classes: (1) Category L (low contamination), sediment with all
contaminant levels not exceeding the lower chemical exceedance level (LCEL); (2)
Category M (moderate contamination), sediment with any one or more contaminant
levels exceeding the LCEL and none exceeding the upper chemical exceedance level
(UCEL); and (3) Category H (high contamination), sediment with any one or more
contaminant levels exceeding the UCEL. The LCEL and UCEL for each contaminant
(e.g. metals) are derived from data on natural background level in the marine sediments
of Hong Kong and from a literature review of the potential adverse
ecological/toxicological effects of the contaminant on the marine biota (Lau-Wong et al.,
1993).
Table 4.2 Characteristics of spent lubricating oil used in the present study (Mean ± S.D.,
n=3).
Properties Values
Spent lubricating oil
Density (g ml -1 ) 0.83
Total Aliphatic Hydrocarbon (F1) mg g -1 fresh wt oil 347 ± 116
Total Aromatic Hydrocarbon (F2) mg g -1 fresh wt oil 153 ± 7
Total Petroleum Hydrocarbon (F3) mg g -1 fresh wt oil 500 ± 123
Oiled sediment (Day 0)
Total Aliphatic Hydrocarbon (F1) mg g -1 freeze-dried wt 8.50 ± 0.60
Total Aromatic Hydrocarbon (F2) mg g -1 freeze-dried wt 0.54 ± 0.43
Total Petroleum Hydrocarbon (F3) mg g -1 freeze-dried wt 9.04 ± 1.03

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 71
Table 4.3 Comparison of concentrations of trace metals (µg g -1 dwt) in clean and oiled
sediment (Mean ± S.D., n=3) and the effect levels. * and ** indicate the two sediment
was significantly different according to the independent T-test at 0.05 and 0.001 levels,
respectively.
Values
Metals Clean sediment Oiled sediment LCEL – UCEL #
Lead (Pd) * 14.44 ± 0.67 18.08 ± 0.45 75 - 110
Cadmium (Cd) 0.51 ± 0.13 0.69 ± 0.03 1.5 - 4
Zinc (Zn) ** 38.61 ± 2.39 78.88 ± 2.28 200 - 270
Copper (Cu) 11.19 ± 8.90 7.93 ± 0.31 65 - 110
Nickel (Ni) 5.60 ± 0.22 4.34 ± 1.37 40
Chromium (Cr) 4.67 ± 0.26 4.93 ± 0.73 80 - 160
# LCEL: lower chemical exceedance level, biological effects would be observed above this
level; UCEL: upper chemical exceedance level, adverse biological effects would be observed
above this level, adopted from WBTC (W) No. 34/2002 Management of Dredged / Excavated
Sediment (http://www.devb-wb.gov.hk).
4.3.2 Growth and physiological response of mangrove plants in oil microcosm
4.3.2.1 Leaf number
Oil treatment did not have any significant effect on the leaf number of A.
ilicifolius, irrespective to its age (Fig. 4.2 & Table 4.4). However, B. gymnorrhiza in
oiled sediment had significantly lower leaf number than the respective control pots with
clean sediment during the experiment. In both oiled and clean sediment, the effect of
sampling time was significant, but the temporal changes varied among all planted

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 72
groups. There was increment of leaf number in A. ilicifolius of both ages but the number
of leaves declined with experimental time in B. gymnorrhiza (Fig. 4.1 and Table 4.4).
Leaf no. / pot
Leaf no. / pot
Leaf no. / pot
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
Control
Oiled
3MAi
1YAi
Bg
0 20 40 60 80 100 120 140
Days
Figure 4.2 The change of total leaf number in oiled sediment and clean sediment (the
control) pots during the 4-month experiment. Mean and S.D. of triplicates are shown.
(3MAi: three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: B. gymnorrhiza).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 73
Table 4.4 Results of two-way ANOVA test on leaf number during the 4-month
experiment (3MAi: three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: B.
gymnorrhiza).
Variations 3MAi 1YAi Bg
Between
treatment
Between time
Interaction
4.3.2.2 Biomass
F1,24 = 0.136
p = 0.716
F5,24 = 6.969
p < 0.001
F5,24 = 0.278
p = 0.921
F1,24 = 0.313
p = 0.581
F5,24 = 6.136
p = 0.001
F5,24 = 1.711
p = 0.170
F1,24 = 5.325
p = 0.030
F5,24 = 3.702
p = 0.013
F5,24 = 2.144
p = 0.095
The root biomass of all oiled treated groups was comparable to that of the
controls with clean sediment (Figs. 4.3, 4.4 & 4.5). These results suggest that oil in the
sediment did not have any adverse effect on plant growth for both mangrove species,
even the roots of 3MAi were not damaged by oiled sediment; however, significant
difference was found in stem biomass of 3MAi and Bg as well as leaf biomass of both
Ai treatments (Table 4.5). The significant changes with sampling time was observed in
the root and total biomass of 1YAi (Tables 4.5 & 4.6) and stem biomass of Ai and leaf
biomass of 1YAi and Bg (Table 4.5). The plants grew very slowly during the 4-month
experimental period (September to December 2004), as this is between autumn and
winter time when plants are normally under slow growth and have leaf shedding. The
arrested plant growth may affect the response of plants to the toxic effect of oil.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 74
Average oven dried biomass (g / pot)
Average oven dried biomass (g / pot)
0.4
0.3
0.2
0.1
0.0
0.8
0.6
0.4
0.2
0.0
3MAi Leaf
30 60 90 120
3MAi Root
30 60 90 120
Days
1.0
0.8
0.6
0.4
0.2
0.0
3MAi Stem
30 60 90 120
3MAi Total
30 60 90 120
Figure 4.3 Biomass production of three-month old Acanthus ilicifolius during the 4-
month experiment (Mean and S.D. of three replicates are shown).
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Days
Control
Oiled

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 75
Average oven dried biomass (g / pot)
Average oven dried biomass (g / pot)
0.5
0.4
0.3
0.2
0.1
0.0
10
8
6
4
2
0
1YAi Leaf
30 60 90 120
1YAi Root
30 60 90 120
Days
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
12
10
8
6
4
2
0
1YAi Stem
30 60 90 120
1YAi Total
30 60 90 120
Days
Control
Oiled
Figure 4.4 Biomass production of one-year old Acanthus ilicifolius during the 4-month
experiment (Mean and S.D. of three replicates are shown).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 76
Average oven dried biomass (g / pot)
Average oven dried biomass (g / pot)
7
6
5
4
3
2
1
0
12
10
8
6
4
2
0
Bg Leaf
30 60 90 120
Bg Root
30 60 90 120
Days
2.5
2.0
1.5
1.0
0.5
0.0
16
14
12
10
8
6
4
2
0
Bg Stem
30 60 90 120
Bg Total
30 60 90 120
Days
Control
Oiled
Figure 4.5 Biomass production of Bruguiera gymnorrhiza during the 4-month
experiment (Mean and S.D. of three replicates are shown).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 77
Table 4.5 Results of two-way ANOVA test on dried biomass of root, stem and leaf
during the 4-month experiment (3MAi: three-month old A. ilicifolius; 1YAi: one-year
old Ai; Bg: B. gymnorrhiza).
Variations 3MAi 1YAi Bg
Root
Between
treatment
Between time
Interaction
Stem
Between
treatment
Between time
Interaction
Leaf
Between
treatment
Between time
Interaction
F1,16 = 0.181
p = 0.676
F3,16 = 0.778
p = 0.523
F3,16 = 1.602
p = 0.228
F1,16 = 9.880
p = 0.006
F3,16 = 6.858
p = 0.003
F3,16 = 31.703
p > 0.001
F1,16 = 11.771
p = 0.003
F3,16 = 1.069
p = 0.390
F3,16 = 10.426
p > 0.001
F1,15 = 0.433
p = 0.520
F3,15 = 5.274
p = 0.011
F3,15 = 3.623
p = 0.038
F1,16 = 0.175
p = 0.681
F3,16 = 13.484
p > 0.001
F3,16 = 1.075
p = 0.388
F1,15 = 70.880
p > 0.001
F3,15 = 6.622
p = 0.004
F3,15 = 7.718
p = 0.002
F1,16 = 0.016
p = 0.901
F3,16 = 1.650
p = 0.218
F3,16 = 0.394
p = 0.759
F1,16 = 6.313
p = 0.023
F3,16 = 1.869
p = 0.176
F3,16 = 0.859
p = 0.482
F1,16 = 0.139
p = 0.716
F3,16 = 8.066
p = 0.003
F3,16 = 13.456
p > 0.001

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 78
Table 4.6 Results of two-way ANOVA test on total dried biomass during the 4-month
experiment (3MAi: three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: B.
gymnorrhiza).
Variations 3MAi 1YAi Bg
Between
treatment
Between time
Interaction
4.3.2.3 MDA content
F1,16 = 2.624
p = 0.125
F3,16 = 1.143
p = 0.362
F3,16 = 16.693
p < 0.001
F1,16 = 0.012
p = 0.915
F3,16 = 3.495
p = 0.040
F3,16 = 2.705
p = 0.080
F1,16 = 3.552
p = 0.078
F3,16 = 0.681
p = 0.576
F3,16 = 1.111
p = 0.374
The MDA contents of the roots in the first and the last months of the experiment
are shown in Figure 4.6. A significantly higher MDA content was found in root of oiled
3MAi than its control in both of the sampling times (1 st and 4 th month), suggesting the
roots might have suffered from oxidative stress, even though reductions in growth and
root biomass were not statistically detected (Fig. 4.3). The leaf MDA content of 3MAi
did not show any significant difference between the oiled treatment and the control
group (Fig. 4.7). For the one-year old seedlings, irrespective to whether they were Ai or
Bg, the MDA content in the root and leaf of the oiled treated seedlings were comparable
to that in control. There was also no change in MDA content with sampling time for all
plant groups.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 79
MDA content (umol / g fwt)
10
8
6
4
2
0
***
3MAi
***
Day 30 Day 120
10
8
6
4
2
0
MDA content in root
1YAi
Day 30 Day 120
10
8
6
4
2
0
1YBg
Day 30 Day 120
Figure 4.6 MDA content in root of oiled treated and control plants harvested at days 30 and 120 (Mean and S.D. of nine replicates are
shown; 3MAi: three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: one-year old B. gymnorrhiza; *** indicates the control was
significantly different from the oiled treatment at p≤0.001 according to the independent T-test).
Control
Oil

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 80
MDA content (umol / g fwt)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
3MAi
Day 30 Day 120
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
MDA content in leaf
1YAi
Day 30 Day 120
10
8
6
4
2
0
1YBg
Day 30 Day 120
Figure 4.7 MDA content in leaf of oiled treated and control plants harvested at days 30 and 120 (same legend as in Fig. 4.5; no significant
difference was found between control and oil treatment for each plant species during two sampling times according to independent T-test at
p≤0.05).
Control
Oil

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 81
4.3.3 Sediment analyses
4.3.3.1 Oxidation-reduction potential (ORP)
Figures 4.8a and b shows that in all planted groups, the oiled sediment at both
depths had significantly lower redox potential than their respective control (clean
sediment), indicating the presence of hydrocarbons in oil would deplete oxygen, causing
a reduction in the redox potential of the sediment. The redox potential in oiled sediment
dropped gradually over the duration of the experiment, but a similar decrease was not
found in the control. The decrease with time was most obvious in the oiled sediment
without any vegetation (OC), and the OC sediment had more negative redox potential
values than the planted groups, at both depths. The data reflected that mangrove plants
could translocate oxygen from their aerial parts to the roots, and release oxygen to the
root zone sediment.
4.3.3.2 Enumeration of total aerobic heterotrophs
The population sizes of the total aerobic heterotrophs (TAH) in sediment
collected at Days 30 and 120 are shown in Figure 4.8. Generally, oil treated sediment
had significantly higher population sizes than the clean sediment of the control (Table
4.7). In all treatments, lower numbers of TAHs were found in sediment collected at
Day 120 than that at Day 30. The number of TAHs did not show any significant
difference between two plant species (Ai and Bg). Similarly, the ages of Ai (three-
month old and one-year old seedlings) also had no significant effect on TAH numbers
in either the oiled treated or control groups.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 82
Redox potential / mV
Redox potential / mV
300
200
100
0
-100
-200
3MAi (2cm depth)
-300
0 20 40 60 80 100 120 140
400
300
200
100
0
-100
-200
Days
1YAi (2cm depth)
-300
20 40 60 80 100 120 140
Days
Redox potential / mV
Redox potential / mV
300
200
100
0
-100
-200
3MAi (5cm depth)
Days
Control
Oiled
-300
20 40 60 80 100 120 140
400
300
200
100
0
-100
-200
1YAi (5cm depth)
-300
20 40 60 80 100 120 140
Figure 4.8a Redox potential (standard hydrogen electrode) in 2 cm and 5 cm deep
sediment planted with three-month old A. ilicifolius (3MAi) and one-year old A.
ilicifolius (1YAi) at different sampling times (Means and S.D. of three replicates are
shown).
Days

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 83
Redox potential / mV
Redox potential / mV
400
300
200
100
0
-100
200
100
0
-100
-200
1YBg (2cm depth)
-200
20 40 60 80 100 120 140
Days
OC (2cm depth)
-300
20 40 60 80 100 120 140
Days
Redox potential / mV
Redox potential / mV
400
300
200
100
0
-100
200
100
0
-100
-200
1YBg (5cm depth)
-200
20 40 60 80 100 120 140
Days
OC (5cm depth)
-300
20 40 60 80 100 120 140
Days
Control
Oiled
Figure 4.8b Redox potential (standard hydrogen electrode) in 2 cm and 5 cm deep
sediment planted with one-year old B. gymnorrhiza (Bg) and the oiled control (OC) at
different sampling times (Means and S.D. of three replicates are shown).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 84
Log MPN / g dwt
10
8
6
4
2
0
Day 30
Day 120
O3MAi C3MAi O1YAi C1YAi OBg CBg OC
Figure 4.9 Population sizes of total aerobic heterotrophs (measured in terms of most
probable number, MPN) in surface sediment collected at days 30 and 120 (C: control, O:
oiled; 3MAi: three-month old A. ilicifolius; 1YAi: one-year old Ai; Bg: B. gymnorrhiza;
OC: oiled control; mean and standard deviation of triplicates are shown).

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 85
Table 4.7 Results of three-way ANOVA test showing the effects of oil treatments (oiled
vs. clean sediment), plant species (Ai vs. Bg) and two sampling times (Days 30 vs. 120)
on the Most Probable Number (MPN) of total aerobic heterotrophs.
Variations Values
Oil F1,41 = 21.166, p

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 86
of aromatic (TPH-F2) fraction fluctuated with experimental time, and there was no
significant difference between Days 0 to 120 in all planted and OC groups. In terms of
total petroleum hydrocarbon (TPH), the two-way ANOVA results showed that there
was no significant difference among four treatments but residual hydrocarbon
concentrations dropped with sampling times (Table 4.8).
At the end of the 4-month experiment, total losses of oil (TPH-F3) were 41%,
44%, 36% and 22% in 3MAi, 1YAi, Bg and OC treatments, respectively (Fig. 4.14).
Although A. ilicifolius had more oil removal than the treatment planted with B.
gymnorrhiza in term of removal percentage, no statistical difference could be found.
The removal in the oiled control without any plants (similar to natural attenuation) was
the lowest. The one-year old A. ilicifolius also showed a higher removal percentage and
a faster rate than its younger counterpart, suggesting the larger the plant, the better the
removal.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 87
TPH / mg g -1
10
8
6
4
2
0
a
AC
ab
3M Ai
ab
B B
0 20 40 60 80 100 120 140
Days
Figure 4.10 Temporal changes of residual total petroleum hydrocarbons, aliphatic (F1)
and aromatic (F2) fractions, in oiled sediments planted with three-month old A.
ilicifolius. Mean and standard deviation of three replicates are shown; same letter means
no significant difference at p

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 88
TPH mg g -1
10
8
6
4
2
0
a
AB
bc
A
1Y Ai
b
AB
0 20 40 60 80 100 120 140
Days
Figure 4.11 Temporal changes of residual total petroleum hydrocarbons, aliphatic (F1)
and aromatic (F2) fractions, in oiled sediments planted with one-year old A. ilicifolius.
Mean and standard deviation of three replicates are shown; same letter means no
significant difference at p

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 89
TPH mg g -1
12
10
8
6
4
2
0
a
a
A
ab
ab
B
Bg
bc bc
0 20 40 60 80 100 120 140
Days
Figure 4.12 Temporal changes of residual total petroleum hydrocarbons, aliphatic (F1)
and aromatic (F2) fractions, in oiled sediments planted with B. gymnorrhiza. Mean and
standard deviation of three replicates are shown; same letter means no significant
AB
difference at p

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 90
TPH mg g -1
OC
10 a ab
F1
F2
8
6
4
2
0
A AB
bc
0 20 40 60 80 100 120 140
Days
Figure 4.13 Temporal changes of residual total petroleum hydrocarbons, aliphatic (F1)
and aromatic (F2) fractions, in the sediment of oiled control. Mean and standard
deviation of three replicates are shown; same letter means no significant difference at
p

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 91
Table 4.8 Results of two-way ANOVA test showing the effects of four treatments (three
planted and the oiled control) and sampling times on the residual concentrations of total
petroleum hydrocarbons (TPH) in sediment.
TPH-F3 mg g -1
Variations TPH-F1 TPH-F2 TPH-F3
Between treatments
Between days
Interaction
12
10
8
6
4
2
0
F3,49 = 1.802
p = 0.166
F3,49 = 11.338
p < 0.001
F9,49 = 1.074
p = 0.407
40.9 43.6
F3,49 = 0.369
p = 0.776
F3,49 = 18.095
p < 0.001
F9,49 = 1.594
p = 0.157
35.7
22.2
Day 0 3MAi 1YAi Bg OC
F3,49 = 1.950
p = 0.141
F3,49 = 9.462
p < 0.001
F9,49 = 1.266
p = 0.292
Figure 4.14 The residual total petroleum hydrocarbons (TPH-F3) in sediment at Day
120 in different treatments. The number on top of each bar was the percentage loss of
TPH-F3 compared with that at day 0 which was calculated by the formula: (Day 0 TPH-
F3 – Day 120 TPH-F3) / Day 0 TPH-F3.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 92
4.3.4 Enrichment of oil-degrading microbial consortia and the biodegradation
potential of the enriched consortia
The degradation potential of the consortia enriched from 1YAi microcosm was
studied for five days. The TPH-F1 remained in the flask and the percentage removals
are shown in Table 4.9. The consortia enriched from the bulk sediment had higher
removal percentages than that from rhizosphere sediment. The degradation potential
was not related to the increase in MPN of the oil-degrading bacteria. These results
suggested that each of the enriched consortia might have different a microbial
composition and degradability, even under similar environmental conditions. Based on
the degradation potential, B2 consortium had the highest percentages of TPH removal,
and it was therefore selected for the bioaugmentation experiment in the following
chapter.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 93
Table 4.9 Residual concentrations of total petroleum hydrocarbons (TPH-F1, mg g -1 ) in
flasks inoculated with six consortia enriched from rhizosphere or bulk sediment
collected from oiled treated 1YAi microcosms at the end of 5-day degradation
experiment in MSM, the percentage removal and the MPN of oil-degrading bacteria.
Mean and standard deviation of three replicates are shown.
Sediment and
Enriched culture
Rhizosphere
sediment
Bulk
sediment
R1
Residual
TPH conc
18.40
TPH %
removal
25.28
MPN at
Day 0
4.38
MPN at
Day 5
4.69
% increase
in MPN
7.08
R2 19.87 19.34 4.54 5.54 22.01
R3 22.81 7.41 4.73 5.96 26.02
B1 13.60 44.80 5.26 5.54 5.50
B2 12.10 50.86 5.20 5.96 14.60
B3 15.02 39.02 4.96 6.20 24.99
R1, R2 and R3 are the three replicates of rhizosphere sediment enriched consortia; B1, B2 and
B3 are the three replicates of the bulk sediment enriched consortia. The residual TPH-F1 in
control flasks without any inoculum was 24.63 ± 3.26 mg g -1 . % removal = TPH-F1 of Day 5
control – Day 5 inoculated flask / Day 5 control x 100%. % increase was the increase of MPN /
ml of the medium after 5 days. Only TPH-F1 was done but this result could represent at least
80% of the total TPH.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 94
4.4 Discussion
4.4.1 Effects of oil on the growth and physiological response of mangrove plants
and the selection of mangrove plants for bioremediation
The concentrations of petroleum hydrocarbons in the oiled contaminated
sediment (Table 4.2) were similar to that in Yi O mangrove sediment, which was
polluted by more than 60,000 gallons of crude oil due to the fuel oil spill that occurred
in the Pearl River Estuary on 14 November 2000 (Ke et al., 2002; Tam et al., 2005).
These studies reported that oil concentration (TPH-F3) in sediment seven months after
the oil spill accident was about 1.8 to 12.3 mg g -1 . In another study on the total
petroleum hydrocarbons in Hong Kong marine sediment, concentrations ranging from
5.9 to 1996 µg TPH g -1 freeze-dried weight were reported, and these values were
derived from surface sediment samples and represented a long-term input from near
shore water activities (Zheng and Richardson, 1999). It is obvious that the mangrove
and coastal sediment is susceptible to oil pollution and the concentration of oil used in
the present study could represent realistic levels in the contaminated environment. In
addition to hydrocarbons, heavy metals are also present in spent lubricating oil,
probably due to the wear and tear of the machines. Elevated concentrations of lead and
zinc were found in the present study (Table 4.2). Similarly, Al-Arfaj and Alam (1993)
reported that higher than baseline traces of metals concentrations (including lead,
cadmium, chromium, copper, nickel and zinc) were observed in oiled site samples after
the 1991 Gulf War oil spill in the Abu Ali area. Although elevated petroleum
hydrocarbons and heavy metals were found in mangrove sediment spiked with spent
lubricating oil, their contamination levels were not extremely high and did not exceed
the LECL.

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 95
In the present study, Bruguiera gymnorrhiza (Bg) in oiled treatment showed a
significant reduction of leaf number compared to the control with clean sediment. In
contrast to Bg, one-year old Acanthus ilicifolius were not affected by the addition of
spent lubricating oil and various growth parameters, including leaf number, biomass of
leaf, stem and root and MDA content in leaf and root, did not show any significant
difference between the oiled treatment and the control. Acanthus ilicifolius was better
adapted to contaminated sediment than Bruguiera gymnorrhiza. These results were
similar to those reported by Zhang et al. (2007) who found that the early growth of B.
gymnorrhiza, including height, leaf number and biomass, was reduced by spent
lubricating oil treatment, and the content of free radicals, malondialdehyde (MDA) and
the activity of superoxide dismutase (an anti oxidant enzyme) were increased with oil
addition. In an outdoor experiment carried out by Ye and Tam (2007), a decrease in leaf
number, biomass, chlorophyll and carotenoid contents, nitrate reductase, peroxidase and
superoxide dismutase activities, and increases in malonaldehyde contents in the leaves
of two mangrove species, Aegiceras corniculatum and Avicennia marina, were under 5
L m -2 spent lubricating oil concentration, which was equivalent of an addition of 127 ml
of oil.
Studies on other plants have also suggested poor plant growth in hydrocarbons
contaminated soils, for example summer vetch (Vicia sativa L.) showed 52.6%
reduction in dried root yields after 95 days of treatment in soil contaminated with
petroleum hydrocarbons at a concentration of 1517 mg TPHs kg -1 (Liste and Felgentreu,
2006). Nevertheless, Lin and Mendelssohn (1996) showed that Spartina alterniflora
(salt marsh plant) was not affected by Louisiana crude oil in a high dosage of 24 L m -2
and that Sagittaria lancifolia (freshwater marsh plant) exhibited even significantly

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 96
higher total biomass compared to the controls, without oil addition. Escalante-Espinosa
et al. (2005) found that Cyperus laxus Lam., a predominant native species growing in
weathered contaminated sites in tropical swamps, were healthy (no outward signs of
phytotoxicity) at 5 g of TPH kg -1 of dry perlite.
When the three-month old seedling was compared with one-year old A.
ilicifolius in the present study, the physiological responses revealed that older seedlings
were more resistant to oil toxicity. The MDA content in roots of younger Acanthus
ilicifolius (three-month) was found to be affected with the addition of lubricating oil but
not in the one-year old seedling. Peralta-Videa et al. (2004) also demonstrated that the
tolerance of alfalfa plants to Cd, Cu and Zn was positively correlated with the age of the
plants. The effect of growth stage or the ages of plant in the bioremediation of toxic
organic contaminants has seldom been reported.
4.4.2 Effects of oil contamination on biological and chemical parameters of
sediment
The redox potential of sediment reflects the oxidization and reduction conditions.
Aerobic sediment usually has a redox range of +350 to +700 mV, and anaerobic
sediment is in the range of -250 to +350 mV (DeLaune et al., 1997). Moreover, redox
potential is also a sensitive measurement of the degree of reduction and the types of
process that will take place in wetland sediment, for example, denitrification occurs at a
redox potential of approximately 250 mV and sulphates are reduced to sulphides at a
redox potential between –100 and –200 mV (Mitsch and Gosselink, 2000). In addition,
redox potential reduction is often associated with carbon oxidation in oil-contaminated

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 97
soil (LaRiviere et al., 2003). Bioremediation depends on the coupling of electron
acceptors with the degradation of petroleum (Cozzarelli et al., 1994). In the present
study, the redox potentials of the surface sediment (2 cm) in the planted groups were
around +200 to +300 mV during the 4-month period in the clean sediment control
(without oil) while that of oiled treatments became negative values and dropped to -100
to -200 mV with more negative values in the oiled treatment without plants (the OC
group in Fig. 4.8b). The presence of wetland plants, including mangrove, is generally
believed to have the ability to transfer oxygen from their aerial part to the roots and
release to the surrounding, thus alter the redox condition of the sediment in the root
zone (the root and sediment interface). These results suggest that the addition of oil
reduced the redox potential of the sediment, changed it from aerobic to anaerobic, but
the reduction was less in planted groups. The biotransformation of hydrocarbons is
mainly through oxidation, which may use oxygen within the sediment. In marine
wetlands, sulphate reduction is usually the most important process when oxygen is
limited since seawater contains an abundant supply of sulphate (Mitsch and Gosselink,
2000).
Although the addition of oil decreased the redox potential, more aerobic
heterotrophs were found in the surface sediment of the oil treatments than that in the
control (clean sediment without oil) (Fig. 4.9 & Table 4.7). This indicated that the
population size of total aerobic heterotrophs was significantly stimulated by the addition
of lubricating oil. A similar finding was reported by Liste and Felgentreu (2006) in that
the total count of bacteria was boosted at the beginning of oil dosing. After the initial
increases, the number of total aerobic heterotrophs decreased towards the end of 4-
month experiment. There are two possible explanations: 1) the amount of residual

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 98
hydrocarbons in sediment was lower at the end than that at the beginning of the
experiment indicating that some hydrocarbons might have been used as carbon sources
by the heterotrophs, and the depletion of hydrocarbons caused a decrease in bacterial
number or 2) the reduction of redox potential in sediment also led to a decrease of
aerobic bacteria towards the end of the experiment. In a study of the redox dynamics
during the recovery of an oil-impacted estuarine wetland, LaRiviere et al. (2003)
reported that the reduction of redox potential was due to the biological oxidation of the
crude oil components by alkane- and PAH-degraders.
Microbial population in vegetated soil is generally believed to be higher than
that in non-vegetated soil (Anderson et al., 1993). The difference in bacterial count
usually found between planted and unplanted treatments could be explained by the
rhizosphere effect which modifies the number and composition of microbial populations
(Escalante-Espinosa et al., 2005); the plant exudates could stimulate microbial activity.
However, the differences between the planted and non-vegetated oil-contaminated
sediment in the present study were insignificant (Fig. 4.9). Nevertheless, the MPN of
the total aerobic heterotrophic bacteria in the surface sediment was similar to that
reported by Euliss et al. (2008), that the MPN values in the sediment collected from a
petroleum contaminated site planted with arrowhead (Sagitaria latifolia) and sedge
(Carex stricta) in Indiana Harbour were at also around 10 7 g -1 dwt.
4.4.3 Ability of different plant species in phytoremediation
Performance of different plant species in phytoremediation is important in
deciding which plant species should be used to remove the contaminant, and is often the

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 99
first step when conducting research. The plant remediation was found to vary with both
species composition and varieties (Liste and Alexander, 2000; Dowty et al., 2001; Liste
and Prutz, 2006). In the present study, A. ilicifolius had slightly higher removal
percentages of TPH than B. gymnorrhiza, and one-year old Ai also performed slightly
better than the three-month old Ai. At the end of 4-month experiment, the removal
percentages of the planted groups ranged from 36 to 44 %, which were slightly higher
than that of the non-vegetated oiled sediment (22% removal, probably due to natural
attenuation). However, the results were statistically insignificant due to large standard
deviations (Fig. 4.14 & Table 4.8). The slight differences between mangrove species
and ages may be attributed to differences in root morphology and root biomass, leading
to variations in the amounts of root exudates or oxygen release to the rhizosphere. When
the radical oxygen release (ROL) pattern was examined by the methylene blue staining
method, oxygen was found to have been released from the root surface of the entire
lateral root of A. ilicifolius, while B. gymnorrhiza released oxygen only at the root tip.
Moreover the root of B. gymnorrhiza was covered by thick outer layer which might also
limit the oxygen release (unpublished data).
4.5 Conclusions
The present experiment, based on growth and physiological responses, showed
that one-year old A. ilicifolius was less susceptible to contamination of spent lubricating
oil than B. gymnorrhiza of the same age, and was also less affected than the three-month
old seedling. The addition of spent lubricating oil changed the sediment properties, for
instance, redox potential was reduced while the microbial count such as total aerobic
heterotroph was increased. Although mangrove plants could increase the oxygen status

Chapter 4
Screening study on potential mangrove species for phytoremediation of oil contaminated sediment 100
in sediment, microbial number in planted sediment was comparable to that in unplanted
ones. In terms of the remediation of the mangrove sediment contaminated with spent
lubricating oil, microcosms planted with one-year old Acanthus ilicifolius had the
highest removal percentages of total petroleum hydrocarbons (TPH), followed by the
three-month old seedling of the same species, and then by Bruguiera gymnorrhiza. The
lowest removal percentage was recorded in the unplanted oiled control that had 22%
removal by natural attenuation at the end of 4-month experiment. The study also
demonstrated that the sediment contained microorganisms capable of degrading TPH,
but the ability varied among the sediments. The most effective microbial consortium
(B2) was enriched from the bulk sediment of the contaminated microcosm planted with
one-year old A. ilicifolius, and this consortium will be used for the research on
bioaugmentation in the next chapter.

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 101
5.1 Introduction
Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm
Bioremediation is an economical and environmental alternative tool for managing
environments contaminated by oil, hydrocarbons and similar organic pollutants. The
success of bioremediation depends on the presence of microorganisms that are able to
degrade pollutants in contaminated environments. It also relies on the potential of
establishing and maintaining conditions favorable to the degraders. Natural
environments such as marine and estuarine sediment have been reported as sources of
microorganisms and they harboured a diverse group of indigenous microbes with oil or
hydrocarbon degrading ability, their diversity and abundance were reported to be
increased with degree of contamination (Daane et al., 2001; Ke et al., 2002; Ke et al.,
2003; Guo et al., 2005). A large number of microbial consortia or pure bacterial strains
with PAH-degrading ability have been isolated from diverse environments, including
polluted and pristine soils, sediment and water bodies (Leys et al., 2004; Miller et al.,
2004; Zhou et al., 2006,). These reports suggested that indigenous microorganisms had
the ability to remedy contaminants and the process is known as natural attenuation.

Chapter 5 Evaluation of bioremediation methods for the
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However, natural attenuation is usually a slow process and takes a long time to remove
contaminants.
In addition to natural attenuation, two remediation approaches, namely
bioaugmentation and biostimulation, have been studied in recent years to enhance
bioremediation rates of toxic organic pollutants such as hydrocarbons in oil. In the
former method, microorganisms with known and desirable degrading ability will be
inoculated or added to the contaminated sediment to act as supplement to the existing
microbial community and to increase the rate of biodegradation. The inoculated
degraders could be isolated from the same contaminated sediment or from different
sources. Bioaugmentation approach provides readily available degrading microbes to
the contaminated environment so a rapid bioremediation rate can be achieved. Rahman
et al. (2003) found that the inoculation of a bacterial consortium had significant positive
effects on the bioremediation of n-alkane in petroleum sludge. Dave et al. (1994) also
showed that 70% of oil in contaminated soil was remedied using oil degrading cultures.
However, the efficacy of bioaugmentation is debatable and contradictory results have
been published. Tam and Wong (2008) reported that mangrove sediment had sufficient
indigenous microorganisms capable of naturally remedying PAH contamination and the

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 103
inoculation of PAH-degrading bacterial strains isolated from mangrove sediment or
planting with Aegiceras corniculatum were not better than natural attenuation.
The biostimulation approach aims to stimulate the activity and abundance of the
indigenous degrading microorganisms by the addition of nutrients, growth factors
and/or co-substrates or by the alternation of environmental conditions, such as aeration
(Zhu et al., 2001). It has been reported that although natural soils or sediment contained
the microbes necessary for degrading organic pollutants, supplementation of inorganic
nutrients would speed up the degradation process (Guo et al., 2005). It is because the
organic contaminant such as oil would increase the carbon content in sediment and
widen the C:N ratio that became unfavorable to microbial activity (Choi et al., 2002).
Lee et al. (2008) found that the bioremediation rate of heavy mineral oil in
contaminated soil was significantly accelerated when the soil was amended with
compost. However, the results of amendment are not always positive, and some
researches have reported adverse or no beneficial effects of amendments. Schaefer and
Juliane (2007) found that the additives including coffee grounds and horticultural and
brewery waste had no enhancement effect on the biodegradation of total petroleum
hydrocarbon degradation and explained that microorganisms might prefer more

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 104
available additives such as available nutrients and simple carbons over less degradable
hydrocarbons. Marja et al. (2005) also reported that compost extract did not enhance
biodegradation of diesel oil.
Another approach to enhance bioremediation of contaminated sediment is
phytoremediation that relies on plants to remove pollutants (Refer to Chapter 4). In
recent years, some researchers have tried to combine the bioaugmentation approach
with biostimulation, and/or the combination of bioaugmentation and phytoremediation,
and reported that the combined effects were better than single approach and natural
attenuation. Trindade et al. (2005) showed that the biodegradation efficiency with a
combination of bioaugmentation and biostimulation treatments was approximately
twice as that using natural attenuation.
The effectiveness of different bioremediation treatment methods depends on the
characteristics of the contaminated environment and must be evaluated case by case.
The present study therefore aims to compare the performance of different
bioremediation techniques, namely bioaugmentation, biostimulation, phytoremediation
and natural attenuation in remediation of spent lubricating oil contaminated mangrove

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 105
sediment. One-year old Acanthus ilicifolius was selected as it was the best species for
oil remediation as shown in Chapter 4. The oil-degrading consortium enriched, isolated
and proved to have oil-degrading ability in previous experiments (Chapter 4) was used
as the inoculum for bioaugmentation approach. Biostimulation was applied by adding
the commercially available slow-release fertilizer with a C:N:P ratio of 100:11:11. The
interactive effects of the aforementioned methods were also studied aiming to identify
the most efficient and appropriate approach for remediation of mangrove habitats
contaminated and impacted by lubricating oil.
5.2 Materials and methods
5.2.1 Microcosm design
A total of 24 microcosms, each contained sandy mangrove sediment contaminated
with 100 ml of spent lubricating oil, were set up as described in Section 4.2.1 (Chapter 4)
in July 2005. The 24 microcosms were randomly divided into eight treatments, each
with three replicates, and placed in the same greenhouse as in Chapter 4. The treatments
were: (i) unplanted (NA, natural attenuation); (ii) planted with one-year old A. ilicifolius
(P, phytoremediation); (iii) unplanted but with inoculation of the enriched oil-degrading

Chapter 5 Evaluation of bioremediation methods for the
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consortium (A, bioaugmentation); (iv) unplanted but with the addition of slow-releasing
fertilizer (F, biostimulation); (v) planted with inoculation of the consortium (PA,
combination of phytoremediation and bioaugmentation); (vi) planted with the addition
of fertilizer (PF, combination of phytoremediation and biostimulation); (vii) unplanted
but with the inoculum and fertilizer (FA, combination of biostimulation and
bioaugmentation); and (viii) planted with inoculum and fertilizer (PFA, combination of
three approaches). Three microcosms with clean sediment (i.e. without oil
contamination) and planted with one-year old A. ilicifolius were prepared as the control
to monitor plant growth (C).
5.2.2 Materials
Acanthus ilicifolius, mangrove sediment and spent lubricating oil were cultivated
or collected as described in Chapter 3, the Materials and methods chapter. One-year old
A. ilicifolius was selected because of the highest removal found in the screening
experiment (Chapter 4). For the treatments with plants (P, PA, PF, PFA and C), two
seedlings of A. ilicifolius were planted in each pot. For the biostimulation treatments (F,
PF, FA and PFA), the slow-release fertilizer was purchased from a local horticulture

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 107
shop (Hi Control) with a C:N:P ratio of 100:11:11 was added to each pot at an amount
of 5.0 g at the beginning of the experiment to provide a continuous supply of balanced
nutrients. The oil-degrading consortium isolated from the previous screening
experiment proving to have the best removal of Total Petroleum Hydrocarbon (TPH) in
liquid culture (Chapter 4) was used as the inoculum. For the bioaugmentation treatments
(A, PA, FA and PFA), the consortium was inoculated to each pot at the beginning of the
experiment at an inoculum size of 10 5 MPN oil-resisting bacteria g -1 sediment. The
population size of total aerobic bacteria in the inoculum was 10 6 MPN g -1 sediment.
5.2.3 Sampling methods
At the end of the four-month experiment, the microcosms were harvested and
the materials in each pot were separated into plant and sediment. The MDA content in
roots and biomass of different plant parts, leaf, stem and root, were freshly tested and
weighted. Roots were freeze-dried for TPH analysis. The microbial activity in
rhizosphere and bulk sediment was determined using fresh sediment surrounding the
root and surface sediment (at a depth of 0-2 cm), respectively. The remaining portion of
the sediment sample was either air-dried or freeze-dried after homogenizing, depending

Chapter 5 Evaluation of bioremediation methods for the
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on the parameters interested. The parameters to be measured and the procedures were
the same as that described in Chapter 3.
5.2.4 Statistical analyses
Mean and standard deviation values of three replicates for each treatment
were calculated. One-way analysis of variance (ANOVA) was used to compare the
effects of the addition of oil, fertilizer amendment and bacterial inoculum on the growth
of mangrove plant which was measured in terms of dried biomass and malondialdehyde
(MDA) content in root. Three-way ANOVA was employed to test the effects of plant,
bacterial inoculum and fertilizer amendment on the concentrations of residual petroleum
hydrocarbons in three fractions (TPH-F1, TPH-F2 & TPH-F3). If ANOVA results were
significant (p ≤ 0.05), the least significant difference (LSD) multiple comparison would
be used to determine where the difference among treatments was. All statistical analyses
were performed with the software, Statistical Package for Social Sciences (SPSS 11.0
for Windows, SPSS Inc., USA).

Chapter 5 Evaluation of bioremediation methods for the
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5.3 Results
5.3.1 Growth and physiology of mangrove plants in oil-contaminated sediment
5.3.1.1 Plant growth and biomass
The dried biomass of leaf, stem and root of mangrove plants after four-month
exposure to sediment contaminated with spent lubricating oil were shown in Figure 5.1.
The plant growth in spent oil-contaminated sediment was not significantly different
from that in the control, except stem biomass, suggesting that Acanthus ilicifolius
seedlings could tolerate the oil toxicity, a pre-requisite for a plant species used for
phytoremediation. As for the oil-contaminated microcosms, the one without any
amendment, just with plants, the biomass was generally lower than those in the
microcosms with inoculum or fertilizer or both. The microcosms with the addition of
both oil-degradation bacteria and fertilizer (PFA) had higher dried biomass than those
without any addition (P) and with just single treatment (PF or PA). The addition of
either fertilizer (PF) or oil-degrading bacteria (PA) did not have any significant
enhancement effect on plant growth. The root biomass contributed significantly more to
total plant biomass than stem and leaf in all treatments, indicating the importance of
roots in phytoremediation.

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 110
5.3.1.2 Malondialdehyde content in root
The MDA content in roots of A. ilicifolius under different treatments after
four-month oil exposure was in the range of 0.35 to 3.55 µmol g -1 FW (Fig. 5.2). The
root MDA content in mangrove seedlings harvested from the oil-contaminated sediment
was comparable to that in the control (without oil) except the one with the fertilizer
amendment (PF), indicating that there was no significant stress imposed by the addition
of spent lubricating oil to mangrove roots. The PF treatment had the lowest root MDA
content, suggesting that the addition of nutrients may increase plant vigor and make
them more resistance to the reactive oxygen species (ROS) stress due to oil pollution.
These plant data suggested that the mangrove species, A. ilicifolius, was able to
withstand the contamination of spent lubricating oil in mangrove sediment and this
species could be used to for phytoremediation of spent lubricating oil.
5.3.1.3 Accumulation of petroleum hydrocarbons in mangrove roots
The concentrations of the two fractions of petroleum hydrocarbons, TPH-F1 and
TPH-F2 in roots of A. ilicifolius under different treatments after four-month oil

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 111
exposure were shown in Figures 5.3 and 5.4, respectively. The plants grown in
oil-contaminated sediment had significantly higher concentrations of aliphatic
hydrocarbons (TPH-F1) in roots that those in the control (oil-free sediment). Among
those grown in contaminated sediment, the root TPH-F1 concentrations from plants
collected from microcosms with PFA treatments, the combination of all possible
remediation approaches, were significantly lower than that from other treatments (Fig.
5.3). In terms of TPH-F2 concentrations in roots, there was no significant difference
among all treatments, and the oil-treated plants had comparable values as those in the
control (Fig. 5.4). The relative concentrations of TPH-F1 were significantly higher than
that of TPH-F2 in roots for all plants harvested from the oil-contaminated sediment.

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 112
Dried biomass / plant (g)
14
12
10
8
6
4
2
0
Leaf
Control
P
PF
PA
PFA
Stem Root Total
Figure 5.1 Biomass of leaf, stem and root of A. ilicifolius under different treatments at
the end of four-month experiment. Mean and standard deviation of three replicates are
shown. Different letters on top of each bar indicate significant difference among
treatments at p≤0.05 according to one-way ANOVA. Control: plant without oil; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A.
ab a ab ab
b c
ab a a
bc
ab
a
ab
ab
b
ab
a
ab
ab
b

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 113
MDA (umol g -1 FW)
4
3
2
1
0
Control P PF PA PFA
Figure 5.2 The MDA content in roots of A. ilicifolius under different treatments at the
end of four-month experiment (Mean and standard deviation of three replicates are
shown; Different letters on top of each bar indicate significant differences among
treatments at p≤0.05, according to one-way ANOVA test). Control: plant without oil; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A.
a
a
b
a
a

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 114
mg TPH-F1 /g root freeze dried wgt
8
6
4
2
0
a
b
Control P PF PA PFA
Figure 5.3 The concentrations of the aliphatic fraction of total petroleum hydrocarbons
(TPH-F1) in roots of A. ilicifolius under different treatments at the end of four-month
experiment (Mean and standard deviation of three replicates are shown; Different letters
on top of each bar indicate significant differences among treatments at p≤0.05,
according to one-way ANOVA test). Control: plant without oil; P: Phytoremediation; F:
Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA: P+F+A.
bc
bc
ac

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 115
mg TPH-F2 /g root freeze dried wgt
4
3
2
1
0
Control P PF PA PFA
Figure 5.4 The concentrations of aromatic fraction of total petroleum hydrocarbons
(TPH-F2) in roots of A. ilicifolius under different treatments at the end of four-month
experiment (Mean and standard deviation of three replicates are shown; No significant
differences among treatments at p≤0.05, according to one-way ANOVA test). Control:
plant without oil; P: Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF: P+F;
PA: P+A; PFA: P+F+A.

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 116
The root concentration factor (RCF) was calculated by dividing the concentration
of TPH in roots by the concentration of TPH in sediment. The RCF of TPH-F1 was
comparable among all treatments but the PFA treatment had significantly higher RCF of
TPH-F2 (Table 5.1). These results suggested that mangrove plants could enhance the
uptake of aromatic hydrocarbons more than that of aliphatic hydrocarbons. The plants
with better growth and higher vigor such as those collected from PFA treatments
appeared to have more accumulation of aromatic hydrocarbons but the opposite was
found for the aliphatic hydrocarbons uptake.
Table 5.1 Root concentration factor (RCF) of TPH-F1 and TPH-F2 in root grown in
oil-contaminated sediment of different remediation approaches (Mean and standard
deviation of three replicates are shown, different letters in each column represent
significant difference at p

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 117
5.3.2 Sediment parameters
5.3.2.1 Dehydrogenase activity in bulk sediment
In general, the dehydrogenase activity in unplanted treatments with both fertilizer
and inoculum (FA treatment) was significantly higher than that in treatments with single
bioremediation method (either biostimulation or bioaugmentation, F or A treatment)
(Fig. 5.5). Dehydrogenase activity in the control (oil-free sediment) was at around 15.1
µg INF g -1 dwt 2h -1 , comparable to that in unplanted treatment with bioaugmentation (A
treatment) (19.08 µg INF g -1 dwt 2h -1 ). The highest dehydrogenase activity was found in
the planted microcosms supplemented with both fertilizer and oil-degrading consortium
(PFA treatment) with an average value of 255.4 µg INF g -1 dwt 2h -1 (Fig. 5.5).

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 118
µg INF g -1 dwt 2h -1
400
300
200
100
0
bc
NA FA F A Control P PF PA PFA
Figure 5.5 The dehydrogenase activity of bulk sediment in different treatments at the
end of four-month experiment (Mean and standard deviation of five replicates are
shown; Different letters on top of each bar indicate significant differences among
treatments at p≤0.05, according to one-way ANOVA test). Control: plant without oil; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A; NA: Natural Attenuation.
c
ab
a a
c
ab
ab
c

Chapter 5 Evaluation of bioremediation methods for the
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5.3.2.2 Comparison of dehydrogenase activity in bulk and rhizosphere sediment
For the control (oil-free sediment), the dehydrogenase activity in bulk sediment
was comparable to that in rhizosphere sediment (Fig. 5.6). For the microcosms
contaminated with spent lubricating oil, the dehydrogenase activity in bulk sediment
was significantly lower than that in rhizosphere sediment (Fig. 5.6). For example, the
value of dehydrogenase activity in rhizosphere sediment was approximately 379.4 µg
INF g -1 dwt 2h -1 in the planted treatments with fertilizer (PF), while the respective value
in the bulk sediment was around 52.8 µg INF g -1 dwt 2h -1 , which was almost seven
times lower than that in rhizosphere sediment. However, there was no significant
difference in dehydrogenase activity between the bulk and rhizosphere sediment in the
planted microcosms supplemented with both fertilizer and inoculum (PFA).

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 120
µg INF g -1 dwt 2h -1
800
600
400
200
0
*
Bulk
Rhizosphere
Control P PF PA PFA
Figure 5.6 The dehydrogenase activity of bulk and rhizosphere sediment in different
planted treatments at the end of four-month experiment (Mean and standard deviation of
five replicates are shown; * and ** indicate the dehydrogenase activity of bulk and
rhizosphere sediments were significantly different according to independent T-test at
0.05 and 0.01 levels, respectively). Control: plant without oil; P: Phytoremediation; F:
Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA: P+F+A.
**
*

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 121
5.3.2.3 Analyses of petroleum hydrocarbons in sediment
The petroleum hydrocarbon content in sediment is an important parameter to
measure the effectiveness of bioremediation. The petroleum hydrocarbons in the
aliphatic fraction (TPH-F1) contributed more than 80% of total petroleum hydrocarbons
(TPH-F3) in the contamination, so their removal is vital to the evaluation. Figure 5.7
compared the residual concentrations of TPH-F1 and TPH-F2 among treatments. The
contaminated sediment without any amendment (NA), just natural attenuation, had the
highest residual concentration. The inoculation of oil-degrading bacterial consortium
alone (A) and bioaugmentation with A. ilicifolius plantation (PA) showed the lowest
residual concentrations. The planting of seedlings alone (P) had little effect on the
removal according to three-way ANOVA test (Table 5.2). There was also no significant
difference between planted and unplanted microcosms. Not only the presence of A.
ilicifolius had little enhancement effect, fertilization also did not have any positive
effect on bioremediation of residual TPH in sediment (Table 5.2). The combination of
fertilizer amendment with the inoculation of enriched bacterial consortium (FA) showed
reduction of biodegradation compared to bioaugmentation alone (A). Similarly,
combined the biostimulation approach with phytoremediation and bioaugmentation
(PFA) did not enhance the oil removal in the microcosms. However, inoculation of

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 122
oil-degrading bacterial consortium (A or PA) significantly reduced the residual TPH-F1
concentrations in sediment and improved the removal of petroleum hydrocarbons. This
suggested that bioaugmentation (A) strategy could promote the removal of aliphatic
hydrocarbons better than phytoremediation (P) or biostimulation (F) or their
combinations.
The aromatic hydrocarbons (TPH-F2) did not follow the same pattern as that of
TPH-F1. Different bioremediation approaches had no significant effects on their
removal and there was no significant difference among all treatments according to
three-way ANOVA test (Table 5.2). When comparing the residual concentrations of
TPH-F2 in all treatments with the initial contamination (at Day 0), significant removal
was observed in all treatments with the inoculation of the enriched oil-degrading
bacterial consortium, i.e. FA, PFA, PA and A (F8, 18=3.363, p=0.016). The percentage
removal of total petroleum hydrocarbons among different treatments were shown in
Figure 5.8. The inoculation of oil-degrading bacteria could enhance the oil removal with
56.4 % and 53.8 % removal in bioaugmentation (A) and in microcosm received a
combination of phytoremediation and bioaugmentation treatments (PA), respectively,
while natural attenuation could only remove 17.6% of TPH-F3 over four months.

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 123
TPH mg g -1 freeze dw
A 8
B
10
6
4
2
0
NA P PF F A FA PA PFA
unfertilised fertilised
unplanted planted
uninoculated inoculated
Figure 5.7 Concentrations of residual petroleum hydrocarbons in the aliphatic fraction
(TPH-F1) and aromatic fraction (TPH-F2). Mean and standard deviation of three
replicates are shown. Arrows A and B on y-axis indicates the concentrations of TPH-F1
and TPH-F2 in sediment at day 0, respectively. Control: plant without oil addition; NA:
Natural attenuation; P: Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF:
P+F; PA: P+A; PFA: P+F+A.
F2
F1

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 124
Table 5.2 Results of three-way ANOVA test showing effects of different treatments on
the sediment concentrations of residual petroleum hydrocarbons in three fractions.
Sources of
TPH-F1 TPH-F2 TPH-F3
variations df F p F p F p
Planted (P) 1 0.005 0.946 0.051 0.824 0.00003 0.996
Inoculated (A) 1 17.859

Chapter 5 Evaluation of bioremediation methods for the
clean-up of spent lubricating oil in mangrove microcosm 125
% removal
% removal
% removal
100
80
60
40
20
0
120
100
80
60
40
20
0
80
60
40
20
0
NA P PF F A FA PA PFA
NA P PF F A FA PA PFA
unfertilised fertilised unplanted planted
uninoculated inoculated
F1
F2
NA P PF F A FA PA PFA
Figure 5.8 The percentage removal of TPH in F1, F2 and F3 fractions. Mean and
standard deviation of three replicates are shown. Percentage removal = (Day 0 TPH –
Month 4 TPH in sediment) / Day 0 TPH x 100%. NA: Natural attenuation; P:
Phytoremediation; F: Biostimulation; A: Bioaugmentation; PF: P+F; PA: P+A; PFA:
P+F+A.
F3

Chapter 5 Evaluation of bioremediation methods for the
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5.3.2.4 Mass balance of total petroleum hydrocarbons
To further investigate the effects of various bioremediation strategies on the
removal of spent lubricating oil in mangrove sediment, the mass balances of the two
fractions of total petroleum hydrocarbons, TPH-F1 and TPH-F2, were calculated
(Tables 5.3 & 5.4). The accumulation of hydrocarbons in mangrove roots accounted for
0.4 to 1.0% of the total output of TPH-F1 and 0.5 to 8.4% of that of TPH-F2. When the
four planted treatments (P, PF, PA and PFA) were compared, no significant difference
was found in TPH-F1 and TPH-F2 concentrations in roots as well as their contribution
to total output of petroleum hydrocarbons. The accumulation in sediment was the
largest sink for the aliphatic and aromatic petroleum hydrocarbons, and accounted for
more than 98% of TPH-F1 and 90% of the TPH-F2 in the planted treatments (Tables
5.3 & 5.4).
In terms of the aliphatic hydrocarbons, the amounts of TPH-F1 accumulated in
sediment varied from treatments to treatments. The microcosms with the inoculation of
oil-degrading consortium, bioaugmentation approach such as A and PA treatments had
the lowest amount of TPH-F1 remained in the sediment (Table 5.3). The microcosms
without any enhancement approach, just natural attenuation (NA treatments) had the

Chapter 5 Evaluation of bioremediation methods for the
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highest residual amount in sediment. The phytoremediation strategy with the planting of
A. ilicifolius had little enhancement effect on the removal of TPH-F1. These results
suggested that the microbial degradation appeared to be more significant in removing
aliphatic hydrocarbons in mangrove sediment. The total losses of the aliphatic
petroleum hydrocarbons (Input – Output) varied among treatments, with the highest
percentages of losses observed in microcosms with bioaugmentation, A and PA,
followed by those with the addition of fertilizers, F, PFA and FA. The microcosms with
mangrove plants (P and PF) had lower percentages of losses of TPH-F1 and the least
loss of TPH-F1, only 10%, was found in the microcosms with just natural attenuation.
These findings further proved that bioaugmentation and its combination with
phytoremediation was the best strategy to remedy spent oil contaminated mangrove
sediment and natural attenuation was a very slow process, explaining why spent oil or
similar organic pollutants would accumulate in sediment without any remediation.
The trend for the aromatic fraction, TPH-F2 was slightly different from that for the
TPH-F1. The lowest amounts of TPH-F2 remained in sediment were found in the
microcosms with biostimulation and bioaugmentation (FA and PFA treatments although
statistically there was no significant difference among treatments, probably due to large

Chapter 5 Evaluation of bioremediation methods for the
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variations among replicates in the same treatments. In terms of overall losses of TPH-F2,
more than 80% of the aromatic hydrocarbons were lost in the two bioaugmentation
treatments at the end of the four-month experiment (Table 5.4).

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 129
Table 5.3 Mass balance of total petroleum hydrocarbons (TPH-F1) in each microcosm. (Mean and standard deviation of three replicates are
shown, different letters in each column represent significant difference at p

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 130
Table 5.4 Mass balance of total petroleum hydrocarbons (TPH-F2) in each microcosm. (Mean and standard deviation of three replicates are
shown, no significant difference was found among treatments at p

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 131
5.4 Discussion
5.4.1 Response of Acanthus ilicifolius planted in oil contaminated mangrove
sediment under different bioremediation treatments
The comparison of physiological responses of Acanthus ilicifolius grown in
contaminated mangrove sediment remedied with different strategies could suggest if
interactions occurred between strategies such as biostimulation and/or bioaugmentation
to the plant, whether they give positive stimulation on plant growth and reduce the
oxidative stress due to the contaminant. The malondialdehyde (MDA) content in root
could be used as an indicator of plant resistance and its acclimation to lubricating oil.
The present study found that A. ilicifolius planted in contaminated sediment with
biostimulation strategy, that is the addition of fertilizer (PF), produced less MDA than
other treatments, indicating the oil had less effect on plant growth. Lee et al. (2001) also
reported that nutrient amendments could stimulate vigorous vegetative growth and
reduce sediment toxicity and oil bioavailability. Similarly, Dowty et al. (2001) showed
that Sagittaria lancifolia displayed a short-term response of increased productivity
while Panicum hemitomon had the highest biomass production and photosynthetic rates
at the end of the 18-month experiment when slow-release fertilizer was supplemented to
fresh marsh environment. It is often suggested that nutrient addition would benefit plant

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 132
growth and increase plant’s vigor or resistance to environmental stresses, especially for
the mangrove plant as mangrove habitat is often nutrient deficient (Feller, 1995).
5.4.2 Microbial activity in rhizosphere sediment contaminated with oil under
different bioremediation treatments
Biological activity in soil accounts for most of the transformation of organic
contaminants and it is always important to examine microbial activity during
bioremediation (Marin et al., 2005; Lee et al., 2008). Dehydrogenase activity is a
common indicator reflecting biological activity in soil (Kraigher et al., 2006). In the
present study, the dehydrogenase activities in rhizosphere sediment was generally
higher than that in bulk sediment (Fig. 5.4), indicating the roots of mangrove plants
stimulated more biological activity in contaminated sediment. Kaimi et al. (2006) also
found that the number of aerobic bacteria and dehydrogenase activity in rhizosphere
soil of the diesel-contaminated environment were higher than in that in the root-free
soil and these microbial parameters were closely correlated with the growth of roots.
Mangroves are known to be able to oxidize the rhizosphere by translocating oxygen
through arenchyma tissues and lenticels on above-ground root structures to
below-ground roots, which then leaks into the rhizosphere and changes the Eh and pH

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 133
condition, thus the rhizosphere environment is often more aerobic than the bulk
sediment (Gleason et al., 2003). Plants may also secrete a number of enzymes to
degrade organic contaminants in soil, including phenol oxidizing enzymes, laccases,
peroxidases, and dehydrogenase (Salt et al., 1998; Wenzel et al., 1999). The root
exudates secreted by roots containing different types of carboxylic acids, strong
organic acids, alcohols, carbohydrates and proteins may serve as carbon and nutrient
sources for the growth of soil microorganisms (Alkorta and Garbisu, 2001; Yoshitomi
and Shann, 2001; Rentz et al., 2005). These explained why the rhizosphere soil or
sediment would support more biological activity and higher values of dehydrogenase
activity in the present study.
The dehydrogenase activities in sediment varied among different bioremediation
treatments. Oil contamination with just natural attenuation had comparable
dehydrogenase activity as the control (without oil contamination) but the activity was
significantly enhanced under phytoremediation treatment (Fig. 5.3). Not only plants, a
combination of biostimulation and bioaugmentation (FA) also stimulated the activity
as the biological activity is often enhanced by nutrients and more bacteria able to resist
to oil contamination,

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 134
5.4.3 Evaluation of the effectiveness of different bioremediation treatments on
spent lubricating oil contaminated mangrove sediment
The removal of petroleum hydrocarbons was most efficient under the
bioaugmentation treatment with the inoculation of oil-degrading microbial consortium
while phytoremediation (planting of mangrove seedlings) and biostimulation (addition
of slow-release fertilizer) alone did not show significant benefit. The interaction among
three treatment methods further showed that bioaugmentation and biostimulation (FA)
and combining these two together with phytoremediation (PFA) were more significant
than other combinations (Tables 5.3 & 5.4). In terms of the amount of hydrocarbons
dissipation, bioaugmentation (A) and planting together augmented with bacteria (PA)
showed the highest removal percentage (Figs. 5.7 & 5.8). The addition of fertilizer
alone did not enhance the oil removal which might suggest that nutrient was not the
limiting factor in the bioremediation of lubricating oil in contaminated sediment. The
present results revealed that bioaugmentation could speed up the remediation process
in mangrove microcosms.
The rationale for the bioaugmentation approach was based on the belief that
indigenous microbial population in contaminated soil or sediment might not be capable

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 135
of degrading the wide range of substrates that are complex and in mixture such as
petroleum. It might also take a long time for the indigenous microbes to adapt to the
contamination even if they could resist and degrade the contaminants. The inoculation
of oil-degrading bacteria could then reduce the lag phase before the bioremediation
begins (Zhu et al., 2001). One key factor to make bioaugmentation success in natural
environment or real field situation is that the inoculated microbes must be able to
survive in the environment and effectively live with the indigenous microbial
population. It is generally agreed that if the inoculum is enriched or isolated from the
same environment, it would be easier to compete with the indigenous microbes. Many
researchers and companies have isolated bacteria with degrading ability and applied
them back to the field (Kim et al., 2005; Liste and Prutz, 2006). Nevertheless,
commercial bacterial inoculums have been used to restore oil pollution, such as the
USEPA has compiled a list of bioremediation agents (USEPA, 2000) and product
schedule as part of the National Oil and Hazardous Substances Pollution Contingency
Plan (NCP) (Zhu et al., 2001). Different sources and types of the inoculum would vary
the efficacy of the bioaugmentation treatments. In the present study, the removal
percentages of the aliphatic and aromatic fractions of petroleum hydrocarbons were
comparable to and even better than those in literatures on similar experiments as

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 136
summarized in Tables 5.4, 5.5 and 5.6. The oil-degrading consortium was enriched
from the same mangrove sediment previously contaminated with the same type of
spent lubricating oil. Therefore, it should be easy for the inoculum to adapt and start
the biodegradation process once added.

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 137
Table 5.5 Results of biostimulation experiments in the present study and those in published in literatures.
Contamination
type
Two year aged crude oil, at 20-50,
70-150, >200 mg/g
Diesel contaminated soil, Long
Beach at i) C12- C23, 2.80 mg/g;
ii) C23- C40, 9.45 mg/g
Crude oil at 7.30 mg/g
Recently contaminated and
weathered crude oil at
71 mg/g
Spent lubricating oil
9.21 mg/g
Bioremediation system
and Time
Phytoremediation with
fertilizer (PF)(1 year)
Natural attenuation (NA),
Biostimulation (F)
(12 weeks)
Natural attenuation (NA),
Biostimulation (F) (150 d)
Biostimulation (F)
Bioaugmentation (A) of
weathered and recently
contaminated (109 d)
Biostimulation (F)
(4 months)
Soil
type
Marsh soil
Sand
(61 %)
Clay
(48%)
Sandy
(clay loam)
Sandy
(84%)
Nutrient
amount
N, P and K: 666, 272 and 514
kg ha -1 , respectively
(NH4)2SO4 and K2HPO4 : 250
and 100 mg kg -1 , respectively
N, P and K:
850, 85 and 240
µg g -1 respectively
C:N:P 100:1.25:1
Slow- release fertilizer
C:N:P 100:11:11
Removal of TPH % References
PF: 59%
P: 28%
i) C12-C23 : NA: 48.7%, F: 45.8%
ii) C23-C40 : NA: 45.7%, F: 45.2%
NA: 47%
F: 62%
Recent contamination:
NA: 2.3%, FA: 5.7
Weathered oil:
NA: 11.5%, FA:14..3%
NA: 17.6% F: 44.1%
FA: 41.3% PF: 21.6%
Lin and
Mendelssohn,
1998
Bento et al.,
2005
Chaîneau et al.,
2005
Trindade et al.,
2005
Present study

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 138
Table 5.6 Results of bioaugmentation experiments in the present study and those in published literatures.
Contamination level Sites of isolation Amount Strains added Removal % References
Recently contaminated and
weathered crude oil at 71 mg/g
Diesel:
i) C12- C23, 2.80 mg/g;
ii) C23- C40, 9.45 mg/g
Spent lubricating oil 9.41 mg/g
Crude oil contaminated
soil
Diesel contaminated soil,
Long Beach
Experimental spill
microcosm
10 8
CFU g −1 soil
2.6 x 10 8
cells ml -1
1x10 6 MPN/ g
sediment
Nocardia nova and
Rhodotorula glutinis var. dairenesis
Bacillus cereus, Bacillus sphaericus,
Bacillus fusiformis, Bacillus pumilus,
Acinetobacter junii and Pseudomonas sp.
Consortium and speciation not identified
Recent contamination:
FA: 5.7
Weathered oil:
FA:14..3%
i) C12-C23: 72.7%
ii) C23-C40: 75.2%
FA: 41.3%
A: 56.4%
Trindade et al.,
2005
Bento et al.,
2005
Present study

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 139
Table 5.7 Results of phytoremediation experiments in the present study and those in published literatures.
Contamination type (concentration) Plants (Time) Soil type Removal % References
Mineral oil in experimental disposal (20× sites
20 m) (0.25 mg/g)
Willow (1.5 years) Dredged sediment
Coal gasification site (1.50 mg/g) Vetch, mustard and ryegrass (95 d) Loamy sand
Spent lubricating oil mangrove sediment
(9.41 mg/g)
Mangrove, Acanthus ilicifolius
(4 months)
Sandy
57% planted
15% unplanted
43.4 – 47.0% planted
68.7% unplanted
30.2% planted
17.6% unplanted
Vervaeke et al., 2003
Liste and Felgentreu, 2006
Present study

Chapter 5
Evaluation of different bioremediation methods of spent lubricating oil in mangrove microcosm 140
5.5 Conclusions
The present study suggests that the loss of TPH from spent lubricating oil
contaminated mangrove sediment was mostly due to biodegradation by sediment
microbes, particularly the bioaugmentation. The inoculation of oil-degrading
consortium (A) isolated from the same habitat alone was effective in removing
TPH-F1 (more than 50% loss) than planting of mangroves alone or amendment of
slow-releasing fertilizer. To enhance the degradation efficiency, a combination
approach with adding the oil-degrading microbial consortium together with fertilizer or
with plants (FA and PFA treatments) achieved more than 80% losses of TPH-F2.
Although planting of mangrove enhanced the dehydrogenase activity in sediment,
especially the rhizosphere sediment, the percentage loss of TPH in each microcosm
due to plant uptake was very small, about 0.4 - 8.4 % in both aliphatic (TPH-F1) and
aromatic (TPH-F2) fractions. These results revealed that mangrove sediment naturally
contained oil-degrading microbes but they might not be in a large amount or in very
active mode. If they were enriched and used as the inoculum, they would work
together with the indigenous microbial population and significantly enhanced the
remediation process and improved the restoration of the oil-contaminated mangrove
habitat.

Chapter 6
General discussion and conclusions 141
Chapter 6 General discussion and conclusions
6.1 Feasibility of using mangrove wetland to clean up spent lubricating oil
The clean up of oil spills in coastal marshes and similar environments remains a
problematic issue worldwide because wetlands can be extremely sensitive to
disturbance. It has been suggested that physical and chemical methods for cleaning up
can induce secondary impacts, for example foot traffic and equipment on
oil-contaminated sediment surface can have significant adverse effects on recovery by
trampling vegetation, accelerating erosion and burying oil into anaerobic sediment
where it may persist for decades (Getter et al., 1984). It is then suggested to allow the
wetland to recover on its own, using no clean up techniques to avoid such adverse
impacts (Mendelssohn et al., 1993). However, natural processes to remove oil
contamination may be very slow and ineffective, it has been reported that residual oil
was still present at a large amount several decades after the oil spill accident (Duke et
al., 1998). Therefore, there is a need to develop less destructive clean-up methods, such
as bioremediation and phytoremediation. Most of the phytoremediation works were
conducted on either freshwater or terrestrial plants. There is a research crack for

Chapter 6
General discussion and conclusions 142
contamination in marine and coastal environments.
Mangroves distributed along inter-tidal coastlines are subject to various
environmental stresses, in particular salinity, periodic flooding and nutrient deficiency,
in addition to anthropogenic pollution. Many freshwater plants or salinity sensitive
species are unable to survive in this habitat. Mangroves in contrast have developed a
series of physiological and morphological adaptations that have allowed them to
successfully grow under high salinity condition, up to 35 ppt (Ye et al., 2005). Salt
exclusion, salt secretion and tolerance of high salt concentration within plant tissues are
the main strategies. Moreover, mangrove plants were found to grow well in high tide
position where only receiving tidal flushing twice a month (Fabre, 1999). Therefore, oil
pollution if happened in marine or coastal environments, mangroves could be employed
for in-situ bioremediation without considering other problems such as salinity and tidal
flush. Another advantage of using mangrove for phytoremediation is that mangroves
play a very important role in soil formation, shoreline protection, and stabilization. The
extensive, aboveground root structures (prop roots, drop roots, and pneumatophores) of
mangrove plants act as a sieve, reducing current velocities and shear, and enhancing

Chapter 6
General discussion and conclusions 143
sedimentation and sediment retention (Carlton, 1974; Augustinus, 1995). Sediment is
stabilized by mangroves which then reduce the risk of erosion and oil dispersion.
Mangroves, commonly found in areas with the most shipping and industrial
activities are subject to oil spill. Duke et al. (1998) summarized that around 5,000 tons
of different kinds of oils have been spilled in the vicinity of mangrove habitats in
Australia since 1970, resulting at least 220 hectares of mangroves oiled and around 13
hectares of mangrove plants killed. Difference species of mangrove would have
different degree of endurance to oil pollution, for instance, Levings and Garrity (1995)
reported that 20% of Rhizophora mangle were totally defoliated and appeared dead
while no death was observed in Avicennia germinans and Laguncularia racemosa in a
heavily oiled site. The present study also found that one-year old Acanthus ilicifolius
was less susceptible to contamination of spent lubricating oil than Bruguiera
gymnorrhiza of the same age, and was also less affected than the three-month old
seedling. Also higher percentage removal of oil was found in microcosms planted with
one-year old A. ilicifolius and this treatment gave the highest removal percentages of
total petroleum hydrocarbons (TPH). The lowest removal percentage was recorded in
the unplanted oiled control and only 22% removal was obtained by natural attenuation

Chapter 6
General discussion and conclusions 144
at the end of 4-month experiment. The results of phytoremediation could be considered
effective in term of the removal percentage in the present study (Table 5.6). It is
generally accepted that the introduction of a vegetative cover accelerates the
disappearance of persistent organic pollutants in the root zone (Merkl et al., 2005; Rentz
et al., 2005). Nevertheless, literatures reported the use of mangroves for
phytoremediation of lubricating oil is still limited.
Mangrove ecosystem has been proven to remedy different kinds of pollutants by its
plants, sediment and associated microorganisms. Ke et al. (2003) found the
microorganisms in the pyrene-contaminated mangrove sediment had been acclimated
and developed capability to degrade PAHs. The study on the remediation of
hydrocarbon-contaminated soil also reported that the number of the PAH-degraders in
the rhizosphere of alfalfa could be seven times higher than that in the plant-free polluted
soil but could be four times lower in the rhizosphere of reed (Muratova et al., 2003).
Not only affecting the population size of the PAH-degraders, the present study showed
that the dehydrogenase activity of rhizosphere sediment was higher than that of bulk
sediment, suggesting the presence of plant was advantageous to the remediation process.
The effect of roots, generally known as the rhizosphere effect, is probably species

Chapter 6
General discussion and conclusions 145
specific, explaining why phytoremediation could be beneficial, had no effect or even
hindered the degradation and removal of contaminants.
Mangrove habitats are often nutrient deficient due to tidal flushing. Burns et al.
(2000) found that the addition of fertilizer to the salt marshes stimulated the degradation
of the lighter Gippsland oil. The potential application of resource-ratio theory in
hydrocarbon biodegradation was discussed (Smith et al., 1998; Head and Swannell,
1999), suggesting that manipulation of the N:P ratio might result in the enrichment of
different microbial populations, and the optimal N:P ratio could be different for
degradation of different compounds. However, in some habitats particularly the marine
shoreline, maintaining a certain nutrient ratio is almost impossible under the dynamic
washout. The use of slow release fertilizer is one of the approaches to provide
continuous sources of nutrients to oil contaminated sediment. The present study found
that the performance of biostimulation treatment alone was not better than
bioaugmentation, but the nutrients encouraged the degradation rate of the enriched
degrading consortium (bioaugmentation and biostimulation treatment FA). This result
suggests that a suitable nutrient ratio could be critical. Oil-degrading microorganisms
are ubiquitous in the environment, and their population size can be increased by many

Chapter 6
General discussion and conclusions 146
folds after being exposed to oil contamination (Pritchard and Costa, 1991). The present
experiment employed the indigenous bacteria consortium as bioaugmentation agent
posed positive effects on the removal efficiency. This suggests that oil-degrading
bacteria were naturally contained in mangrove sediment but not in substantial amount
for effective natural attenuation, thus enriched inoculum could provide active and
considerable amount of microbes to improve the bioremediation of oil contamination.
Oil can be persistent once they entered the environment and weathered.
Weathering referred to the result of biological, chemical and physical processes that
could affect the types of hydrocarbons that remain in a soil (Loehr et al., 2001). Those
processes enhanced the sorption of hydrophobic organic contaminants (HOCs) to the
soil matrix, decreasing the rate and extent of biodegradation (Bosma et al., 1997).
Generally, only the fraction of HOCs dissolved in the aqueous phase is available for
microbiological degradation while the sorbed fraction has a low bioavailability (Allan et
al., 1997). Moreover, a weathered oil-contaminated soil normally contains a recalcitrant
fraction of compounds composed, basically, of high molecular weight hydrocarbons
(higher than C25 compounds), which could not be degraded by indigenous
microorganisms. In contrast, a recently oil-contaminated soil contains a higher amount

Chapter 6
General discussion and conclusions 147
of saturated and aliphatic compounds, which are the most susceptible to the microbial
degradation (Trindade et al.,2005). However, pollutant compounds in a recently
contaminated soil are potentially more toxic to the native microorganisms, leading to
longer adaptation time (lag phase) before degrading the pollutant and even to an
inhibition of the biodegradation process. Many studies on the long-term fate of oil
accidents showed that there was still petroleum hydrocarbons remained in sediment
even after 30 years (Peacock et al., 2007). The present research reported the toxicity of
freshly contaminated oil sediment and the possibility of removing the oil using different
bioremediation strategies. The study on weathered oil might be interesting especially in
mangrove sediment which could have different matrices, texture, anoxic condition and
importance to marine ecosystems. More in-depth research is needed.
6.2 Contributions and significance of the present research
� This is the first systematic study on the feasibility of various bioremediation
strategies for in-situ clean-up of mangrove microcosms contaminated by spent
lubricating oil. The results demonstrate that bioaugmentation (addition of enriched
oil-degrading consortium) is a very effective strategy to remove oil contamination
compared with natural attenuation (intrinsic biodegradation) in microcosm.

Chapter 6
General discussion and conclusions 148
� This study represents a pioneer work on using mangrove microcosm to restore
spent lubricating oil contamination sediment. Acanthus ilicifolius was able to grow
in the oiled sediment, and no obvious reduction of growth and physiological
response were observed. A. ilicifolius was first found to enhance microbial activity
in rhizosphere and increase redox potential of oil contaminated sediment. The
present work also showed the interaction of different bioremediation methods and
it is not necessary to have synergetic effects.
� The significance of plant absorption and microbial degradation of TPH was first
elucidated in the present study, suggesting that most TPH removal was due to
microbial processes in the sediment.
6.3 Limitations of the present study and future research
� The experiment was conducted in greenhouse microcosm environment, thus
results might be different from that in field environment, where constant wave and
tide are flushed in different magnitudes and times with other uncontrollable
factors. Pilot field trail may deem necessary.
� Previous studies reported that the sampling time, if most in-situ bioremediation
study, were few months to years (Zhu et al., 2004). The present sampling period

Chapter 6
General discussion and conclusions 149
of four-month might be too long to clearly differentiate the degradation trend of
different bioremediation strategies. More frequent samplings are therefore
suggested to have a better picture of the experiment. On the other hand, it may be
interesting to study the bioremediation of weathered oil in mangrove environment
which may require a long term work.
� The present study tested only one regime of fertilizer, inoculum concentration,
plant species and oil concentration. Dowty et al. (2001) conducted an assessment
by altering the variables and reported bioremediation was effective in which
combination of regime and in what concentration.
� The diversity of the enriched oil-degrading microbial consortium and the
biodegradation ability of each isolate were yet to identify for a better
understanding of the specificity of the microbes in mangrove sediment.
� This study was conducted in autumn to winter period when plant growth is slow
and the results may be different if plants are more active, such as in summer time.
The seasonal effect on the growth of mangrove and its potential to remedy oil
should be conducted.
� Current research focused on two mangrove species only, work on other
oil-tolerant species and multi-species system may show a comprehensive picture

Chapter 6
General discussion and conclusions 150
of phytoremediation using mangroves.
� The roles of rhizosphere and root exudates of mangroves are not clear but should
be understood to optimize the oil remediation process.
� Spent lubricating oil was studied owing to its chronic input to the marine
environment, results may not be applied to other types of oil pollution, such as
fuel oil. Future focus could be on crude oil which has been reported in many spill
accidents.
6.4 Conclusions
Among various bioremediation strategies tested, either alone or in different
combinations, bioaugmentation of oil-degrading enriched consortium together with the
indigenous oil-degrading bacteria in mangrove sediment was found to be the most
suitable method in degradation and removal of total aliphatic hydrocarbons (TPH) in
mangrove microcosms contaminated by spent lubricating oil. Biostimulation (with the
addition of slow-release fertilizers) and phytoremediation (with the planting of tolerant
mangrove species such as Acanthus ilicifolius) would accelerate the degradation process
if the sediment was nutrient deficient or the environment was reduced and anoxic. The

Chapter 6
General discussion and conclusions 151
present research demonstrated that the microorganisms especially the oil-degraders in
rhizosphere sediment played more important role in the clean-up of spent lubricating oil
in mangrove sediment than the uptake of oil by mangrove plants.

Reference 151
Reference
Agency for Toxic Substances and Disease Registry (ATSDR), Toxicological Profile for
total petroleum hydrocarbons (TPH) (1999). Atlanta, GA: U.S. Department of Health
and Human Services, Public Health Service.
Al-Arfaj, A.A.; Alam, I. A., Chemical characterization of sediments from the Gulf area
after the 1991 oil spill. Marine Pollution Bulletin, 1993, 27, 97–101.
Albaigés, J.; Algaba, J.; Clavell, E.; Grimalt, J. O., Petroleum geochemistry of the
Tarragona Basin (Spanish Mediterranean offshore). Organic Geochemistry 1985, 10,
441–450.
Alef, K.; Nannipiere, P., In: Methods in Applied Soil Microbiology and Biochemistry
(1995). San Diego: Academic Press: p228-231.
Alkorta, I.; Garbisu, C., Phytoremediation of organic contaminants in soils. Bioresource
Technology 2001, 79, (3), 273-276.
Allan, K. A.; Herbert, B. E.; Morris, P. J.; McDonald, T. J., Biodegradation of
petroleum in two soils: implications for hydrocarbon bioavailability. In Situ On-Site
Bioremediation 1997, 5, 629–634.
Alongi, D. M.; Wattayakorn, G.; Pfitzner, J.; Tirendi, F.; Zagorskis, I.; Brunskill, G. J.;
Davidson, A.; Clough, B. F., Organic carbon accumulation and metabolic pathways in
sediments of mangrove forests in southern Thailand. In: Marine Geology (2001).
p85-103.
Al-Sayed, H. A.; Ghanem, E. H.; Saleh, K. M., Bacterial community and some
physico-chemical characteristics in a subtropical mangrove environment in Bahrain.
Marine Pollution Bulletin 2005, 50, (2), 147-155.
Anderson, T. A.; Guthrie, E. A.; Walton, B. T., Bioremediation in the rhizosphere.
Environmental Science & Technology 1993, 27, (13), 2630-2636.
Atlas, R. M., Microbial degradation of petroleum hydrocarbons: an environmental
perspective. Microbiology Review 1981, 45, (1), 180-209.

Reference 152
Atlas, R. M., Petroleum biodegradation and oil spill bioremediation. Marine Pollution
Bulletin 1995, 31, (4-12), 178-182.
Augustinus, P. G. E. F., Geomorphology and sedimentology of mangroves. In:
Geomorphology and Sedimentology of Estuaries (1995), Developments in
Sedimentology 53. Amsterdam: Elsevier Science.
Banks, M. K.; Govindaraju, R. S.; Schwab, A. P.; Kulakow, P.; Finn, J.,
Phytoremediation of Hydrocarbon-Contaminated Soil (2000). Boca Rotan, FL: Lewis
Publishers.
Banks, M. K.; Kulakow, P.; Schwab, A. P.; Chen, Z.; Rathbone, K., Degradation of
crude oil in the rhizosphere of Sorghum bicolor. International Journal of
Phytoremediation 2003, 5, (3), 225-234.
Barahona, L. M.; Vázquez, R. R.; Velasco, M. H.; Jarquín, C. V.; Pérez, O. Z.; Cantú, A.
M.; Albores, A., Diesel removal from contaminated soils by biostimulation and
supplementation with crop residues. Applied Soil Ecology 2004, 27, (2), 165-175.
Bence, A. E.; Kvenvolden, K. A.; Kennicutt II, M. C., Organic geochemistry applied to
environmental assessments of Prince William Sound, Alaska, after the Exxon Valdez oil
spill—a review. Organic Geochemistry 1996, 24, (1), 7-42.
Bento, F. M.; Camargo, F. A. O.; Okeke, B. C.; Frankenberger, W. T., Comparative
bioremediation of soils contaminated with diesel oil by natural attenuation,
biostimulation and bioaugmentation. Bioresource Technology 2005, 96, (9), 1049-1055.
Boopathy, R., Factors limiting bioremediation technologies. Bioresource Technology
2000, 74, (1), 63-67.
Bosma, T. N. P.; Middeldorp, P. J. M.; Schraa, G.; Zehnder, A. J. B., Mass transfer
limitation of biotransformation: quantifying bioavailability. Environmental Science &
Technology 1997, 31, 248-252.
Bragg, J. R.; Prince, R. C.; Harner, E. J.; Atlas, R. M., Effectiveness of bioremediation
for the Exxon-Valdez oil-spill. Nature 1994, 368, (6470), 413-418.

Reference 153
Burns, K. A.; Codi, S.; Duke, N. C., Gladston, Australia field studies: weathering and
degradation of hydrocarbons in oiled mangrove and salt marsh sediments with and
without the application of an experimental bioremediation protocol. Marine Pollution
Bulletin 2000, 41, 392-402.
Burns, K. A.; Codi, S.; Swannell, R. J. P.; Duke, N. C., Assessing the oil degradation
potential of endogenous micro-organisms in tropical marine wetlands. Mangroves &
Salt Marshes 1999, 3 (2), 67-84.
Burns, K. A.; Garrity, S. D.; Jorissen, D.; Macpherson, J.; Stoelting, M.; Tierney, J.;
Yellesimmons, L., The Galeta oil-spill .2. Unexpected persistence of oil trapped in
mangrove sediments. Estuarine Coastal & Shelf Science 1994, 38, (4), 349-364.
Burns, W. A.; Mankiewicz, P. J.; Bence, A. E.; Page, D. S.; Parker, K., A
principal-component and least-square method for allocating sediment hydrocarbons to
multiple source. Environmental Toxicology & Chemistry 1997, 16, 1119-1131.
Carls, M. G.; Harris, P. M.; Rice, S. D., Restoration of oiled mussel beds in Prince
William Sound, Alaska. Marine Environmental Research 2004, 57, (5), 359-376.
Carlton, J. M., Land-building and stabilization by mangroves. Environmental
Conservation 1974, 1, (4), 285-294.
Chaîneau, C. H.; Rougeux, G.; Yepremian, C.; Oudot, J., Effects of nutrient
concentration on the biodegradation of crude oil and associated microbial populations in
the soil. Soil Biology & Biochemistry 2005, 37, (8), 1490-1497.
Charrie-Duhaut, A.; Lemoine, S.; Adam, P.; Connan, J.; Albrecht, P., Abiotic oxidation
of petroleum bitumens under natural conditions. Organic Geochemistry 2000, 31, (10),
977-1003.
Chekol, T.; Vough, L. R.; Chaney, R. L., Phytoremediation of polychlorinated
biphenyl-contaminated soils: the rhizosphere effect. Environment International 2004, 30,
(6), 799-804.

Reference 154
Choi, S. C.; Kwon, K. K.; Sohn, J. H.; Kim, S, J, Evaluation of fertilizer additions to
stimulate oil biodegradation in sand seashore mescocosms. Journal of Microbiology &
Biotechnology 2002, 12, 431-436.
Clark, R. B., Marine pollution (2001), 5 th ed. Oxford: Oxford University Press: p64-95.
Coulon, F.; Pelletier, E.; Gourhant, L.; Delille, D., Effects of nutrient and temperature
on degradation of petroleum hydrocarbons in contaminated sub-Antarctic soil.
Chemosphere 2005, 58, (10), 1439-1448.
Corredor, J. E.; Morell, J. M.; Delcastillo, C. E., Persistence of spilled crude-oil in a
tropical intertidal environment. Marine Pollution Bulletin 1990, 21, (8), 385-388.
Cozzarelli, I. M.; Baedecker, M. J.; Eganhouse, R. P.; Goerlitz, D.F., The geochemical
evolution of low-molecular-weight organic acids derived from the degradation of
petroleum contaminants in groundwater. Geochim Cosmochim Acta 1994, 58, 863–877.
Cunningham, S. D.; Anderson, T. A.; Schwab, A. P.; Hsu, F. C., Phytoremediation of
soils contaminated with organic pollutants. Advances in Agronomy 1996, 56, 56-114.
Cunningham, S. D.; Berti, W. R., Remediation of contaminated soils with green plants -
an overview. In Vitro Cellular & Developmental Biology-Plant 1993, 29, (4), 207-212.
Cunningham, S. D.; Ow, D. W., Promises and prospects of phytoremediation. Plant
Physiology 1996, 110, (3), 715-719.
Daane, L. L.; Harjono, I.; Zylstra, G. J.; Haggblom, M. M., Isolation and
characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with
the rhizosphere of salt marsh plants. Applied & Environmental Microbiology 2001, 67,
(6), 2683–2691.
Dave, H.; Ramakrishna, C.; Bhatt, B. D.; Desai, J. D, Biodegradation of slope oil from
petroleum Industry and bioreclamation of slop oil contaminated soil. World Journal of
Microbiology & Biotechnology 1994, 10, 653–656.

Reference 155
de Hemptinne, J. C.; Peumery, R.; Ruffier-Meray, V.; Moracchini, G.; Naiglin, J.;
Carpentier, B., Compositional changes resulting from the water-washing of a petroleum
fluid. Journal of Petroleum Science & Engineering 2001, 29, 39–51.
Del'Arco, J. P.; de Franca, F. P., Biodegradation of crude oil in sandy sediment.
International Biodeterioration & Biodegradation 1999, 44, (2-3), 87-92.
DeLaune, R. D.; Devai, I.; Mulbah, C.; Crozier, C.; Lindau, C. W., The influence of soil
redox conditions on atrazine degradation in wetlands. Agriculture Ecosystems &
Environment 1997, 66, (1), 41-46.
Dowty, R. A.; Shaffer, G. P.; Hester, M. W.; Childers, G. W.; Campo, F. M.; Greene, M.
C., Phytoremediation of small-scale oil spills in fresh marsh environments: a mesocosm
simulation. Marine Environmental Research 2001, 52, (3), 195-211.
Duke, N. C., Mangrove floristics and biogeography. In: Robertson, A. I.; Alongi, D. M.
(Eds.), Tropical mangrove ecosystems (1992). Washington D. C.: The American
Geophysical Union: p63-100.
Duke, N. C.; Ellison, J. C.; Burns, K. A., Surveys of oil spill incidents affecting
mangrove habitat in Austrialia: a preliminary assessment of incidents, impacts on
mangroves, and recovery of deforested area. In: C. Beck (Ed.), Canberra: APPEA
Journal (1998). Australian Petroleum Production and Exploration Association:
p646-654.
Dutta, T. K.; Harayama, S., Fate of crude oil by the combination of photooxidation and
biodegradation. Environmental Science & Technology 2000, 34, (8), 1500-1505.
Environmental Oil Ltd., (2000) The environmental challenge. Homepage:
http://www.ozramp.net.au/~enviro/challenge.htm
Escalante-Espinosa, E.; Gallegos-Martínez, M. E.; Favela-Torres, E.; Gutiérrez-Rojas,
M., Improvement of the hydrocarbon phytoremediation rate by Cyperus laxus Lam.
inoculated with a microbial consortium in a model system. Chemosphere 2005, 59,
(3), 405-413.

Reference 156
Euliss, K.; Ho, C.; Schwab, A. P.; Rock, S.; Banks, M. K., Greenhouse and field
assessment of phytoremediation for petroleum contaminants in a riparian zone.
Bioresource Technology 2008, 99, (6), 1961-1971.
Fabre, A.; Fromard, Fr.; Trichon, V., Fractionation of phosphate in sediments of four
representative mangrove stages (French Guiana). Hydrobiologia 1999, 392, (1), 13-19.
Feller, I. C., Effects of nutrient enrichment on growth and herbivory of dwarf red
mangrove (Rhizophora mangle). Ecological Monographs 1995, 65, 477-505.
Fernández-Álvarez, P.; Vila, J.; Garrido-Fernández, J. M.; Grifoll, M.; Lema, J. M.,
Trials of bioremediation on a beach affected by the heavy oil spill of the Prestige.
Journal of Hazardous Materials 2006, 137, (3), 1523-1531.
Forsyth, J. V.; Tsao, Y. M.; Bleam, R. D., Bioremediation: When is augmentation
needed? In: Hinchee, R. E.; Fredrickson, J.; Alleman, B. C. (Eds.), Bioaugmentation for
site remediation (1995). Columbus: Battelle Press: p1-14.
Frick, C. M.; Germida, J. J.; Farrell, R. E., Assessment of Phytoremediation as an
in-situ technique for cleaning oil-contaminated sites. In: Proceedings of the
Phytoremediation Technical Seminar (1999). Ottawa: Environment Canada: p105-124.
Garcia-Blanco, S.; Suidan, M. T.; Venosa, A. D.; Huang, T.; Cacho-Rivero, J.,
Microcosm study of effect of different nutrient addition on bioremediation of fuel oil #2
in soil from Nova Scotia coastal marshes. In: Proceedings of International oil spill
conference (2001). Washington D. C.: America Petroleum Institute.
Garret, R. M.; Pickering, I. J.; Haith, C. E.; Prince, R.C., Photooxidation of crude oils.
Environment Science & Technology 1998, 32, 3719-3723.
Garrity, S. D.; Levings, S. C.; Burns, K. A., The Galeta oil-spill .1. Long-term effects
on the physical structure of the mangrove fringe. Estuarine Coastal & Shelf Science
1994, 38, (4), 327-348.
Getter, C. D.; Cintron, G.; Dicks, B.; Lewis III, R. R.; Seneca, E. D., The recovery and
restoration of salt marshes and mangroves following an oil spill. In: Cairns Jr., J. and

Reference 157
Buikema Jr., A., (Eds.), Restoration of Habitats Impacted by Oil Spills (1984), Boston:
Butterworth Publishers: p65–113.
Getter, C. D., Scott, G. I., Michel, J., The effects of oil spills on mangrove forests: a
comparison of five oil spill sites in the Gulf of Mexico and the Caribbean Sea. In:
Proceeding of the Oil Spill Conference (1981). Washington, D. C.: American Petroleum
Institute: p535-540.
Gleason, S. M.; Ewel, K. C.; Hue, N., Soil redox conditions and plant–soil relationships
in a micronesian mangrove forest. Estuarine, Coastal & Shelf Science 2003, 56, (5-6),
1065-1074.
Glick, B. R., Phytoremediation: synergistic use of plants and bacteria to clean up the
environment. Biotechnology Advances 2003, 21, (5), 383-393.
Gonneea, M. E.; Paytan, A.; Herrera-Silveira, J. A., Tracing organic matter sources and
carbon burial in mangrove sediments over the past 160 years. Estuarine Coastal & Shelf
Science 2004, 61, (2), 211-227.
Grant, D. L.; Clarke, P. J.; Allaway, W. G., The response of gray mangrove
(Avicennia-Marina (Forsk) Vierh) seedlings to spills of crude-oil. Journal of
Experimental Marine Biology & Ecology 1993, 171, (2), 273-295.
Gundlach, E. R.; Boehm, P. D.; Marchand, M.; Atlas, R. M.; Ward D. M.; Wolfe, D. A,
Fate of Amoco Cadiz oil. Science 1983, 221, 122–129.
Gundlach, E. R.; Hayes, M., Classification of coastal environments in terms of potential
vulnerability to oil spill damage. Marine Technology Society Journal 1978, 12, (4),
18-27.
Guo, C. L.; Zhou, H. W.; Wong, Y. S.; Tam, N. F. Y., Isolation of PAH-degrading
bacteria from mangrove sediments and their biodegradation potential. Marine Pollution
Bulletin 2005, 51, (8-12), 1054-1061.
Hart, S., In situ bioremediation: Defining the limits. Environmental Science &
Technology 1996, 30, (9), A398-A401.

Reference 158
Head, I. M.; Swannell, R. P. J., Bioremediation of petroleum hydrocarbon contaminants
in marine habitats. Current Opinion in Biotechnology 1999, 10, (3), 234-239.
Hughes, J. B.; Shanks, J.; Vanderford, M.; Lauritzen, J.; Bhadra, R., Transformation of
TNT by aquatic plants and plant tissue cultures. Environmental Science & Technology
1997, 31, (1), 266-271.
Junoy, J.; Castellanos, C.; Vieitez, J. M.; de la Huz, M. R.; Lastra, M., The
macroinfauna of the Galician sandy beaches (NW Spain) affected by the Prestige
oil-spill. Marine Pollution Bulletin 2005, 50, (5), 526-536.
Kaimi, E.; Mukaidani, T.; Miyoshi, S.; Tamaki, M., Ryegrass enhancement of
biodegradation in diesel-contaminated soil. Environmental & Experimental Botany
2006, 55, (1-2), 110-119.
Kathiresan, K.; Bingham, B. L., Biology of mangroves and mangrove ecosystems.
Advances in Marine Biology, 2001, 40, 81-251.
Kato, T.; Haruki, M.; Imanaka, T.; Morikawa, M.; Kanaya, S., Isolation and
characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep
subterranean petroleum reservoirs. Journal of Bioscience & Bioengineering 2001, 91,
(1), 64-70.
Ke, L.; Wang, W. Q.; Wong, T. W. Y.; Wong, Y. S.; Tam, N. F. Y., Removal of pyrene
from contaminated sediments by mangrove microcosms. Chemosphere 2003, 51, (1),
25-34.
Ke, L.; Wong, T. W. Y.; Wong, Y. S.; Tam, N. F. Y., Fate of polycyclic aromatic
hydrocarbon (PAH) contamination in a mangrove swamp in Hong Kong following an
oil spill. Marine Pollution Bulletin 2002, 45, (1-12), 339-347.
Ke, L.; Wong, T. W. Y.; Wong, A. H. Y.; Wong, Y. S.; Tam, N. F. Y., Negative effects
of humic acid addition on phytoremediation of pyrene-contaminated sediments by
mangrove seedlings. Chemosphere 2003, 52, (9), 1581-1591.

Reference 159
Ke, L.; Yu, K. S. H.; Wong, Y. S.; Tam, N. F. Y., Spatial and vertical distribution of
polycyclic aromatic hydrocarbons in mangrove sediments. Science of the Total
Environment 2005, 340, (1-3), 177-187.
Kennish, M. J., Ecology of estuaries: Anthropogenic effects (2002). Boca Raton, FL:
CRC Press: p63-175.
Kim, S. J.; Choi, D. H.; Sim, D. S.; Oh, Y. S., Evaluation of bioremediation
effectiveness on crude oil-contaminated sand. Chemosphere 2005, 59, (6), 845-852.
Knight, R. L.; Kadlec, R. H.; Harry, M., The use of treatment wetlands for petroleum
industry effluents. Environmental Science & Technology 1999, 33, 973–980.
Ko, J. Y.; Day, J. W., A review of ecological impacts of oil and gas development on
coastal ecosystems in the Mississippi Delta. Ocean & Coastal Management 2004, 47,
(11-12), 597-623.
Kraigher, B.; Stres, B.; Hacin, J.; Ausec, L.; Mahne, I.; van Elsas, J. D.; Mandic-Mulec,
I., Microbial activity and community structure in two drained fen soils in the Ljubljana
Marsh. Soil Biology & Biochemistry 2006, 38, (9), 2762-2771.
Kuo, L.C., An experimental study of crude oil alteration in reservoir rocks by water
washing. Organic Geochemistry 1994, 21, 465–479.
LaRiviere, D. J.; Autenrieth, R. L.; Bonner, J. S., Redox dynamics during recovery of
an oil-impacted estuarine wetland. Water Research 2003, 37, (14), 3307-3318.
Lau-Wong, M. M. M.; Rootham, R. C.; Bradley, G. C., A strategy for the management
of contaminated dredged sediment in Hong Kong. Journal of Environmental
Management 1993, 38, 9–114.
Leahy, J. G.; Colwell, R. R., Microbial degradation of hydrocarbons in the environment.
Microbiology & Molecular Biology Review 1990, 54, (3), 305-315.

Reference 160
Lee, K.; Doe, K. G.; Lee, L. E. J.; Suidan, M. T.; Venosa, A. D., Remediation of an
oil-contaminated experimental freshwater wetland: Habitat recovery and toxicity
reduction. In: Proceedings of the International Oil Spill Conference (2001). Washington
D. C.: American Petroleum Institute: p323-328.
Lee, K.; Tremblay, G. H.; Gauthier, J.; Cobanli, S. E.; Griffin, M., Bioaugmentation
biostimulation: a paradox between laboratory and field results. In: Proceedings of
International Oil Spill Conference (1997). Washington D. C.: American Petroleum
Institute: p697-705.
Lee, S. H.; Oh, B. I.; Kim, J. G., Effects of various amendments on heavy mineral oil
bioremediation and soil microbial activity. Bioresource Technology 2008, 99, (7),
2578-2587.
Lessard, R. R.; Demarco, G., The significance of oil spill dispersants. Spill Science &
Technology Bulletin 2000, 6, (1), 59-68.
Levings, S.C.; Garrity, S. D., Oiling of mangrove keys in the 1993 Tampa Bay oil spill,
Proceedings of the International Oil Spill Conference (1995). Washington, D. C.:
American Petroleum Institute.
Lewis, R. R., Impacts of oil spills on mangrove forests. In: H. J. Teas (Ed.), Tasks for
vegetation science vol 8, Biology and Ecology of mangroves (1983). The Hague: Dr W.
Junk Publishers: p171-183.
Leys, N. M.; Ryngaert, A.; Bastiaens, L.; Verstraete, W.; Top, E. M.; Springael, D.,
Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated
with polycyclic aromatic hydrocarbons. Applied & Environmental Microbiology 2004,
70, (4), 1944–1955.
Lin, Q. X.; Mendelssohn, I. A., A comparative investigation of the effects of Louisiana
crude oil on the vegetation of fresh, brackish, and salt marsh. Marine Pollution Bulletin
1996, 32, (2), 202–209.

Reference 161
Lin, Q. X.; Mendelssohn, I. A., The combined effects of phytoremediation and
biostimulation in enhancing habitat restoration and oil degradation of petroleum
contaminated wetlands. Ecological Engineering 1998, 10, (3), 263-274.
Lin, Q. X.; Mendelssohn, I. A.; Suidan, M. T.; Lee, K.; Venosa, A. D., The
dose-response relationship between No. 2 fuel oil and the growth of the salt marsh grass,
Spartina alterniflora. Marine Pollution Bulletin 2002, 44, (9), 897-902.
Liste, H. H.; Alexander M., Plant-promoted pyrene degradation in soil. Chemosphere
2000, 40, (1), 7-10.
Liste, H. H.; Felgentreu, D., Crop growth, culturable bacteria, and degradation of petrol
hydrocarbons (PHCs) in a long-term contaminated field soil. Applied Soil Ecology
2006, 31, (1-2), 43-52.
Liste, H. H.; Prutz, I., Plant performance, dioxygenase-expressing rhizosphere bacteria,
and biodegradation of weathered hydrocarbons in contaminated soil. Chemosphere
2006, 62, (9), 1411-1420.
Loehr, R. C.; McMillen, S. J.; Webster, M. T., Predictions of biotreatability and actual
results: soils with petroleum hydrocarbons. Practice Periodical of Hazardous, Toxic, &
Radioactive Waste Management 2001, 5, 79-87.
Macek, T.; Mackova, M.; Kas, J., Exploitation of plants for the removal of organics in
environmental remediation. Biotechnology Advances 2000, 18, 23–34.
Madsen, T.; Kristensen, P., Effects of bacterial inoculation and non-ionic surfactants on
degradation of polycyclic aromatic hydrocarbons in soil. Environmental Toxicology &
Chemistry 1997, 16, 631-637.
Maki, H.; Sasaki, T.; Harayama, S., Photo-oxidation of biodegraded crude oil and
toxicity of the photo-oxidized products. Chemosphere 2001, 44, 1145–1151.
Marin, J. A.; Hernandez, T.; Garcia, C., Bioremediation of oil refinery sludge by
landfarming in semiarid conditions: Influence on soil microbial activity. Environmental
Research 2005, 98, 185–195.

Reference 162
Marja, R. T. P.; John, P.; Jaakko, A. P., Phytoremediation of subarctic soil contaminated
with diesel fuel. Bioresource Technology 2005, 84, 221–228.
Massachusetts Department of Environmental Protection, Method for the determination
of extractable petroleum hydrocarbons (EPH) (1998). Massachusetts: Commonwealth
of Massachusetts: p41.
Meharg, A. A.; Cairney, J. W. G., Ectomycorrhizas - extending the capabilities of
rhizosphere remediation? Soil Biology & Biochemistry 2000, 32 (11-12), 1475-1484.
Mendelssohn, I. A.; Hester, M. W.; Hill, J. M., Effects of oil spills on coastal wetlands
and their recovery (1993). New Orleans: U.S. Department of the Interior, Minerals
Management Service, Gulf of Mexico OCS Regional Office. OCS Study MMS 93-0045:
p46.
Merkl, N.; Schultze-Kraft, R.; Infante, C., Phytoremediation in the tropics – influence of
heavy crude oil on root morphological characteristics of graminoids. Environmental
Pollution 2005, 138, (1), 86-91.
Miller, C. D.; Hall, K.; Liang, Y. N.; Nieman, K.; Sorensen, D.; Issa, B.; Anderson, A.
J.; Sims, R. C., Isolation and characterization of polycyclic aromatic
hydrocarbon-degrading Mycobacterium isolates from soil. Microbial Ecology 2004, 48,
(2), 230–238.
Mills, M. A.; Bonner, J. S.; McDonald, T. J.; Page, C. A.; Autenrieth, R. L., Intrinsic
bioremediation of a petroleum-impacted wetland. Marine Pollution Bulletin 2003, 46,
(7), 887-899.
Mitsch, W. J.; Gosselink, J. G., Wetlands (2000). New York: John Wiley & Sons, Inc.
Morin, T. C., Enhanced intrinsic bioremediation speeds site cleanups. Pollution
Engineering 1997, 29, (2), 44-47.
Muratova, A.; Hubner, T.; Narula, N.; Wand, H.; Turkovskaya, O.; Kuschk, P.; Jahn, R.;
Merbach, W., Rhizosphere microflora of plants used for the phytoremediation of
bitumen-contaminated soil. Microbiological Research 2003, 158, (2), 151-161.

Reference 163
Muratova, A.; Hubner, T.; Tischer, S.; Turkovskaya, O.; Moder, M.; Kuschk, P., Plant -
Rhizosphere-microflora association during phytoremediation of PAH-contaminated soil.
International Journal of Phytoremediation 2003, 5, (2), 137-151.
National Research Council, Oil in the Sea III: Inputs, Fates, and Effects (2003).
Washington D. C.: National Academy Press: p69.
Ngabe, B.; Bidleman, T. F.; Scott, G. I., Polycyclic aromatic hydrocarbons in storm
runoff from urban and coastal South Carolina. Science of the Total Environment 2000,
255, (1-3), 1-9.
NOAA, Oil spills in Mangroves: Planning & response considerations, National Oceanic
and Atmospheric Administration, Office of Response and Restoration 2002 In: Hoff, R.
(Ed.).
Office of Technology Assessment, Coping with an oiled area: An analysis of oil apill
response technologies (1990). Washington, D. C. OTA-BP-O-63.
Oh, Y. S.; Sim, D. S.; Kim, S. J., Effects of nutrients on crude oil biodegradation in the
upper intertidal zone. Marine Pollution Bulletin 2001, 42, (12), 1367-1372.
Page, A. L.; Miller, R. H.; Keeney, D. R., Method of Soil Analysis, Part 2 - Chemical
and Microbiological Properties (1982), 2 nd Edition. Agronomy, No 9, Madson,
Wisconsin: American Society of Agronomy and Soil Science Society of America.
Paquin, D.; Ogoshi, R.; Campbell, S.; Li, Q. X., Bench-scale phytoremediation of
polycyclic aromatic hydrocarbon-contaminated marine sediment with tropical plants.
International Journal of Phytoremediation 2002, 4, (4), 297-313.
Pastor, D.; Sanchez, J.; Porte, C.; Albaiges, J., The Aegean Sea oil spill in the Galicia
coast (NW Spain). I. Distribution and fate of the crude oil and combustion products in
subtidal sediments. Marine Pollution Bulletin 2001, 42, (10), 895-904.

Reference 164
Peacock, E. E.; Hampson, G. R.; Nelson, R. K.; Xu, L.; Frysinger, G. S.; Gaines, R. B.;
Farrington, J. W.; Tripp, B. W.; Reddy, C. M., The 1974 spill of the Bouchard 65 oil
barge: Petroleum hydrocarbons persist in Winsor Cove salt marsh sediments. Marine
Pollution Bulletin 2007, 54, (2), 214-225.
Pearce, E., Living sea walls keep floods at bay. New Scientist 1996, 150, 7.
Pelletier, E.; Delille, D.; Delille, B., Crude oil bioremediation in sub-Antarctic intertidal
sediments: chemistry and toxicity of oiled residues. Marine Environmental Research
2004, 57, (4), 311-327.
Peralta-Videa, J. R.; de la Rosa, G.; Gonzalez, J. H.; Gardea-Torresdey, J. L., Effects of
the growth stage on the heavy metal tolerance of alfalfa plants. Advances in
Environmental Research 2004, 8, (3-4), 679-685.
Peters, K. E. A. M.; Moldowan, J. M., The biomarker guide. In: Interpreting molecular
fossils in petroleum and ancient sediments (1993). Englewood Cliffs: Prentice Hall.
Potter, T. L.; Simmons, K. E., Total petroleum hydrocarbon criteria working group
series vol.2, Composition of petroleum mixtures (1998). Amherst, Massachusetts:
Amherst Scientific Publishers: p1-17.
Prince, R. C., Petroleum spill bioremediation in marine environments. Critical Reviews
in Microbiology 1993, 19, (4), 217-242.
Prince, R. C.; Clark, J. R.; Lindstrom, J. E.; Butler, E. L.; Brown, E. J.; Winter, G.;
Grossman, M. J.; Parrish, P. R.; Bare, R. E.; Braddock, J. F.; Steinhauer, W. G.;
Douglas, G. S.; Kennedy, J. M.; Barter, P. J.; Bragg, J. R.; Harner, E. J.; Atlas, R. M.,
Bioremediation of the Exxon Valdez oil spill: monitoring safety and efficacy. In: R.E.
Hinchee et al. (Eds.), Hydrocarbon Bioremediation (1994). Boca Raton, Florida: Lewis
Publishers: p107-124.
Prince, R. C.; Owens, E. H.; Sergy, G. A., Weathering of an Arctic oil spill over 20
years: the BIOS experiment revisited. Marine Pollution Bulletin 2002, 44, 1236–1242.

Reference 165
Pritchard, P. H.; Costa, C. F., EPA's Alaska Oil Spill Bioremediation Project.
Environmental Science & Technology 1991, 25, (3), 372-379.
Rahman, K. S. M.; Rahman, T. J.; Kourkoutas, Y.; Petsas, I.; Marchant, R.; Banat, I. M.,
Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium
amended with rhamnolipid and micronutrients. Bioresource Technology 2003, 90, (2),
159-168.
Rahman, K. S. M.; Rahman, T. J.; Lakshmanaperumalsamy, P.; Banat, I. M., Towards
efficient crude oil degradation by a mixed bacterial consortium. Bioresource
Technology 2002, 85, (3), 257-261.
Ramsay, M. A.; Swannell, R. P. J.; Shipton, W. A.; Duke, N. C.; Hill, R. T., Effect of
bioremediation on the microbial community in oiled mangrove sediments. Marine
Pollution Bulletin 2000, 41, 413-419.
Reddy, C. M.; Eglinton, T. I.; Hounshell, A.; White, H. K.; Xu, L.; Gaines, R. B.;
Frysinger, G. S., The west falmouth oil spill after thirty years: The persistence of
petroleum hydrocarbons in marsh sediments. Environmental Science & Technology
2002, 36, (22), 4754-4760.
Rentz, J. A.; Alvarez, P. J. J.; Schnoor, J. L., Benzo[a]pyrene co-metabolism in the
presence of plant root extracts and exudates: Implications for phytoremediation.
Environmental Pollution 2005, 136, (3), 477-484.
Richard, J. Y.; Vogel, T. M., Characterization of a soil bacterial consortium capable of
degrading diesel fuel. International Biodeterioration & Biodegradation 1999, 44, (2-3),
93-100.
Riser-Roberts, E., Remediation of petroleum contaminated soils. In: Biological,
Physical and Chemical Processes (1998). Boca Raton, FL: CRC Press: p78-113.
Salt, D. E.; Blaylock, M.; Kumar, N. P. B. A.; Dushenkov, V.; Ensley, B. D.; Chet, I.;
Raskin, I., Phytoremediation - a novel strategy for the removal of toxic metals from the
environment using plants. Bio-Technology 1995, 13, (5), 468-474.

Reference 166
Salt, D. E.; Smith, R. D.; Raskin, I., Phytoremediation. Annual Review of Plant
Physiology & Plant Molecular Biology 1998, 49, 643-668.
Sarkar, D.; Ferguson, M.; Datta, R.; Birnbaum, S., Bioremediation of petroleum
hydrocarbons in contaminated soils: Comparison of biosolids addition, carbon
supplementation, and monitored natural attenuation. Environmental Pollution 2005, 136,
(1), 187-195.
Santas, R.; Santas, P., Effects of wave action on the bioremediation of crude oil
saturated hydrocarbons. Marine Pollution Bulletin 2000, 40, 434–439.
Sauer, T. C., Hydrocarbon source identification and weathering characterization of
intertidal and subtidal sediments along the Saudi Arabian coast after the Gulf War oil
spill. Marine Pollution Bulletin 1993, 27, 117–134.
Sauer, T. S.; Michel, J.; Hayes, M. O.; Aurand, D. V., Hydrocarbon characterization and
weathering of oiled intertidal sediments along the Saudi Arabian coast two years after
the Gulf war oil spill. Environmental International 1998, 24, 43–60.
Schaefer, M.; Juliane, F., The influence of earthworms and organic additives on the
biodegradation of oil contaminated soil. Applied Soil Ecology 2007, 36, 53–62.
Schnoor, J. L.; Licht, L. A.; Mccutcheon, S. C.; Wolfe, N. L.; Carreira, L. H.,
Phytoremediation of organic and nutrient contaminants. Environmental Science &
Technology 1995, 29, (7), A318-A323.
Sherman, R. E.; Fahey, T. J.; Howarth, R. W., Soil-plant interactions in a neotropical
mangrove forest: iron, phosphorus and sulfur dynamics. Oecologia 1998, 115, (4),
553-563.
Short, J. W.; Lindeberg, M. R.; Harris, P. M.; Maselko, J. M.; Pella, J. J.; Rice, S. D.,
Estimate of oil persisting on the beaches of Prince William Sound 12 years after the
Exxon Valdez oil spill. Environmental Science & Technology 2004, 38, (1), 19-25.

Reference 167
Sien, C. L., Coastal management in Singapore: institutional arrangements and
implementation. In: Ocean & Coastal Management 38 (2 special issue: Integrated
Coastal Management Practices in East Asia) (1998): p111-118.
Singh, N.; Megharaj, M.; Kookana, R. S.; Naidu, R.; Sethunathan, N., Atrazine and
simazine degradation in Pennisetum rhizosphere. Chemosphere 2004, 56, (3), 257-263.
Skipper, H. D., Bioremediation of contaminated soils. In: Sylvia, D.M. (Ed.), Principles
and application of soil microbiology (1999). Upper Saddle River, NJ: Prentice Hall:
p469-481.
Smith, V. H.; Graham, D. W.; Cleland, D. D., Application of resource ratio theory to
hydrocarbon biodegradation. Environmental Science & Technology 1998, 32,
3386-3395.
Suominen, L.; Jussila, M. M.; Makelainen, K.; Romantschuk, M.; Lindstrom, K.,
Evaluation of the Galega-Rhizobium galegae system for the bioremediation of
oil-contaminated soil. Environmental Pollution 2000, 107, (2), 239-244.
Swannell, R. P. J.; Lee, K.; McDonagh, M., Field evaluations of marine oil spill
bioremediation. Microbiological Reviews 1996, 60, (2), 342-365.
Tam, N. F. Y.; Guo, C. L.; Yau, W. Y.; Wong, Y. S., Preliminary study on
biodegradation of phenanthrene by bacteria isolated from mangrove sediments in Hong
Kong. Marine Pollution Bulletin 2002, 45, (1-12), 316-324.
Tam, N. F. Y.; Wong, T. W. Y.; Wong, Y. S., A case study on fuel oil contamination in
a mangrove swamp in Hong Kong. Marine Pollution Bulletin 2005, 51, (8-12),
1092-1100.
Tam, N. F. Y.; Wong, Y. S., Mangrove soils as sinks for waste-water-borne pollutants.
Hydrobiologia 1995, 295, (1-3), 231-241.
Tam, N. F. Y.; Wong, Y. S., Retention of wastewater-borne nitrogen and phosphorus in
mangrove soils. Environmental Technology 1996, 17, (8), 851-859.

Reference 168
Tam, N. F. Y.; Wong, Y. S., Variations of soil nutrient and organic matter content in a
subtropical mangrove ecosystem. Water Air & Soil Pollution 1998, 103, (1-4), 245-261.
Tam, N. F. Y.; Wong, Y. S., Effectiveness of bacterial inoculum and mangrove plants
on remediation of sediment contaminated with polycyclic aromatic hydrocarbons.
Marine Pollution Bulletin 2008, In press.
Taylor, P.; Bennet, B.; Jones, M.; Larter, S. R., The effect of biodegradation and water
washing on the occurrence of alkylphenols in crude oils. Organic Geochemistry 2001,
32, 341–358.
Teruhisa, K.; Masahiro, N.; Hiroshi, K.; Tomoko, Y., Marine Life Research Group of
Takeno; Kouichi, O., Impacts of the Nakhodka heavy-oil spill on an intertidal
ecosystem: an approach to impact evaluation using geographical information system.
Marine Pollution Bulletin 2003, 47, (1-6), 99-104.
Total Petroleum Hydrocarbon Criteria Working Group Series (1998), Volume 1
Analysis of Petroleum Hydrocarbons in Environmental Media. Wade Weisman (Ed.)
Trindade, P. V. O.; Sobral, L. G.; Rizzo, A. C. L.; Leite, S. G. F.; Soriano, A. U.,
Bioremediation of a weathered and a recently oil-contaminated soils from Brazil: a
comparison study. Chemosphere 2005, 58, (4), 515-522.
USEPA (1992), The Superfund Innovative Technology Evaluation Program:
Technology Profiles, 5 th ed. Washington, D. C. EPA/540/R-92/077.
USEPA (1999), Monitored Natural Attenuation of Petroleum Hydrocarbons, Office of
Research and Development, U.S. Environmental Protection Agency. EPA
600-F-98-021.
USEPA (2000), NCP Product Schedule, http://www.epa.gov/oilspill
US Fish and Wildlife Service, Classification of wetlands and deepwater habitats of the
United States (1979), Cowardin, L. M.; Carter, V.; Golet, F. C.; LaRoe, E. T. (Eds.).
FWS/OBS 79/31: p103.

Reference 169
Vazquezduhalt, R., Environmental-impact of used motor oil. Science of the Total
Environment 1989, 79, (1), 1-23.
Venosa, A. D.; Lee, K.; Suidan, M. T.; Garcia-Blanco, S.; Cobanli, S.; Moteleb, M.;
Haines, J. R.; Tremblay, G. and Hazelwood, M., Bioremediation and Biorestoration of a
crude oil-contaminated freshwater wetland on the St. Lawrence River. Bioremediation
Journal 2002, 6, (3), 261-281.
Venosa, A. D.; Suidan, M. T.; Wrenn, B. A.; Strohmeier, K. L.; Haines, J. R.; Eberhart,
B. L.; King, D.; Holder, E., Bioremediation of an experimental oil spill on the shoreline
of delaware bay. Environmental Science & Technology 1996, 30, (5), 1764-1775.
Vervaeke, P.; Luyssaert, S.; Mertens, J.; Meers, E.; Tack, F. M. G.; Lust, N.,
Phytoremediation prospects of willow stands on contaminated sediment: a field trial.
Environmental Pollution 2003, 126, (2), 275-282.
Volkman, J. K.; Alexander, R.; Kagi, R. I.; Rowland, S. I.; Sheppard, P. N.,
Biodegradation of aromatic hydrocarbons in crude oils from the Barrow Sub-basin of
Western Australia. Organic Geochemistry 1984, 6, 619–632.
Wang, Z.; Fingas, M.; Blenkinsopp, S.; Sergy, G.; Landriault, M.; Sigouin, L.; Foght, J.;
Semple, K.; Westlake, D. W. S., Comparison of oil composition changes due to
biodegradation and physical weathering in different oils. Journal of Chromatographic
Analysis 1998, 809, 89-107.
Weis, J. S.; Weis, P., Metal uptake, transport and release by wetland plants:
implications for phytoremediation and restoration. Environment International 2004, 30,
(5), 685-700.
Wenzel, W. W.; Adriano, D. C.; Salt, D.; Smith, R., Phytoremediation: a
plant-microbe-based remediation system. In: Adriano, D. C., Bollag, J. M.;
Frankenberger Jr. W. T.; Sims, R. C., (Eds.). Bioremediation of Contaminated Soils,
American Society of Agronomy, Crop Science Society of America, Soil Science
Society of America (1999). (Agronomy Monograph 37): p457–508.

Reference 170
Wise, W. R.; Guven, O.; Molz, F. J.; Mccutcheon, S. C., Nutrient retention the in
high-permeability, oil-fouled beach. Journal of Environmental Engineering 1994, 120,
(6), 1361-1379.
Wolfe, D. A., The fate of the oil spilled from the Exxon Valdez. Environmental Science
& Technology 1994, 28, 561A–568A.
Wong, P. K.; Wang, J., The accumulation of polycyclic aromatic hydrocarbons in
lubricating oil over time - a comparison of supercritical fluid and liquid-liquid
extraction methods. Environmental Pollution 2001, 112, (3), 407-415.
Yamamoto, T.; Nakaoka, M.; Komatsu, T.; Kawai, H.; Ohwada, K.; Takeno, M. L. R.
G., Impacts by heavy-oil spill from the Russian tanker Nakhodka on intertidal
ecosystems: recovery of animal community. Marine Pollution Bulletin 2003, 47, (1-6),
91-98.
Ye, Y.; Tam, N. F. Y., Effects of used lubricating oil on two mangroves Aegiceras
corniculatum and Avicennia marina. Journal of Environmental Sciences 2007, 19, (11),
1355-1360.
Yerushalmi, L.; Rocheleau, S.; Cimpoia, R.; Sarrazin, M.; Sunahara, G.; Peisajovich, A.;
Leclair, G.; Guiot, S. R., Enhanced biodegradation of petroleum hydrocarbons in
contaminated soil. Bioremediation Journal 2003, 7, (1), 37-51.
Yoshitomi, K. J.; Shann, J. R., Corn (Zea mays L.) root exudates and their impact on
14 C-pyrene mineralization. Soil Biology & Biochemistry 2001, 33, (12-13), 1769-1776.
Zheng, C. G., The effect of lubricating oil pollution on growth and physiological
response of mangrove. PhD Thesis City University of Hong Kong 2006.
Zhang, C. G.; Leung, K. K.; Wong, Y. S.; Tam, N. F. Y., Germination, growth and
physiological responses of mangrove plant (Bruguiera gymnorrhiza) to lubricating oil
pollution. Environmental & Experimental Botany 2007, 60, (1), 127-136.

Reference 171
Zheng, G. J.; Man, B. K. W.; Lam, J. C. W.; Lam, M. H. W.; Lam, P. K. S., Distribution
and sources of polycyclic aromatic hydrocarbons in the sediment of a sub-tropical
coastal wetland. Water Research 2002, 36, (6), 1457-1468.
Zheng, G. J.; Richardson, B. J., Petroleum hydrocarbons and polycyclic aromatic
hydrocarbons (PAHs) in Hong Kong marine sediments. Chemosphere 1999, 38, (11),
2625-2632.
Zhou, H. W.; Guo, C. L.; Wong, Y. S.; Tam, N. F. Y., Genetic diversity of dioxygenase
genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove
sediments. FEMS Microbiology Letters 2006, 262, (2), 148–157.
Zhu, X.; Venosa, A. D.; Suidan, M. T.; Lee, K., Guideline for the bioremediation for the
oil-contaminated salt marshes. USEPA 2004, Office of Research and Development.
EPA/600/R-04/074.
Zhu, X. Q.; Venosa, A. D.; Suidan, M. T.; Lee, K., Guidelines for the bioremediation of
marine shorelines and freshwater wetland (2001). Cincinnati, OH: EPA’s Treatment and
Destruction Branch, Land Remediation and Pollution Control Division, National Risk
Management Research Laboratory.
Zinjarde, S. S.; Pant, A. A., Hydrocarbon degraders from tropical marine environments.
Marine Pollution Bulletin 2002, 44, (2), 118-121.

Appendix 172
Appendix: Conference and Publication
Conference
Leung, K. K.; Tam, N. F. Y. (2005), Phytoremediation of spent lubricating oil by
mangroves, Acanthus ilicifolius and Bruguiera gymnorrhiza. Presented at The First
Postgraduate Symposium on Marine Biology, 22 October, 2005, Hong Kong.
Publication
Zhang, C. G.; Leung, K. K.; Wong, Y. S.; Tam, N. F. Y., Germination, growth and
physiological responses of mangrove plant (Bruguiera gymnorrhiza) to lubricating oil
pollution. Environmental & Experimental Botany 2007, 60, (1), 127-136.