Faculty of Resource Science and Technology - ir.unimas.my OF SOIL IMPACTED … · polisiklik (HAP)...

PHYTOREMEDIATION OF SOIL IMPACTED BY HYDROCARBONS

AND HEAVY METALS

Aisyaidil binti Hanri

Master of Science (Chemistry)

2014

Faculty of Resource Science and Technology

PHYTOREMEDIATION OF SOIL IMPACTED BY HYDROCARBONS

AND HEAVY METALS

Aisyaidil binti Hanri, BSc (Hons.) (UNIMAS)

A thesis submitted

In fulfillment of the requirements for the degree of

Master of Science (Chemistry)

Department of Chemistry

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2014

ii

DECLARATION

The work presented in this thesis entitled “Phytoremediation of Soil Impacted by

Hydrocarbons and Heavy Metals” is the results of my own research except as cited in

references. I hereby declare that this thesis is my own work and effort and that it has not

been submitted anywhere for any award. Where other sources of information have been

used, they have been acknowledged.

__________________________


07021287

Faculty of Science and Technology

Universiti Malaysia Sarawak

iii

ACKNOWLEDGEMENT

Alhamdulillah, thank to Allah for his blessing I can successfully completing this

research. I would like to thank Prof Dr Zaini bin Assim for giving me support and

guidance in doing this research project. My co-supervisors, Prof Dr Isa bin Ipor, thank you

for guiding me in selecting suitable plants and occupying space in the green house for my

research, as well as, Assoc Prof Dr Awg Sallehhin Awg Hussaini, thank you for guiding

me in bacteria isolation and culture enrichment process and allowing me to use the Genetic

Molecular Laboratory (GML) to perform bacteria isolation and culture enrichment. Not

forgotten, I am grateful to Universiti Malaysia Sarawak for your generous fellowship

support. It has helped me greatly by allowing me to concentrate more on my research

without having to worry about finances. I would not be as far along in my research without

your support. Again, I am very thankful for your contribution.

I would like to express my special thanks to my friends and family for their

continuous support and help throughout my study. You have been the best personal

cheering squad anyone could ask for.

Special recognition to Dr Ihab Lubbad, Mdm Ting Woei and Mr Tommy Bakeh for

guiding and helping me in ICP-MS analysis. Also to Mr Leo Bulin, Mr Rajuna Tahir and

Mohammed Akkbik for assisting me during HPLC analysis, as well as Mr. Azzuddin

Shebli for assisting me during GC-FID analysis. Not forgotten, Mdm Dyg Fatimawati

Awang Alli, thank you for your assist and support throughout my research. Last but not

least, I am indebted to Nur Hafizah Azizan for teaching me and sharing her knowledge on

bacteria isolation and culture enrichment techniques, as well as guiding me to perform

experimental work at her laboratory (GML).

iv

Phytoremediation of Soil Impacted With Hydrocarbons and Heavy Metals


ABSTRACT

Phytoremediation is the application of plants and their associated microorganisms to degrade, sequester or

contain contaminants in soil. Four plant species namely Kaempheria rotunda, Jatropha podagrica, Asystasia

coromandeliana and Phyllanthus amarus were evaluated for their ability to survive and adapt in soil

contaminated with heavy metals and mixture of aliphatic hydrocarbons and polycyclic aromatic

hydrocarbons (PAHs). These plants were also evaluated for the ability as metal hyperaccumulator and their

ability to degrade aliphatic hydrocarbons and PAHs in their rhizosphere. Bacteria were isolated from the soil

rhizosphere to determine whether degradation of aliphatic hydrocarbons and PAHs are related to the number

of bacteria in the rhizosphere. P. amarus could not survive in soil contaminated with heavy metals and a

mixture of aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs). The bioconcentration

factor (BCF) of the plants showed that K. rotunda is more potential as cadmium and zinc accumulator with

BCF values of 5.28 and 0.90, respectively. Addition of ethylenediaminetetraacetic acid (EDTA) as

chelating/mobilizing agent only affect the accumulation of chromium in J. podagrica, and chromium

accumulated was 2-3 times higher in EDTA-treated plants compared to non-EDTA-treated plants. The

colony forming unit present in the rhizosphere soil of K. rotunda, J. podagrica and A. coromandeliana were

9.6 x 104, 9.3 x 10

4 and 3.8 x 10

4, respectively. K. rotunda, J. podagrica, A. coromandeliana planted in soil

contaminated with aliphatic hydrocarbons and PAHs have shown the ability to degrade hydrocarbons in their

rhizosphere. K. rotunda showed better potential for the degradation of aliphatic hydrocarbons in the

rhizosphere due to the high number of bacteria present in the rhizosphere and consistently decreased

concentration of aliphatic hydrocarbons throughout the experimental period. A. coromandeliana, in the other

hand poorly degraded aliphatic hydrocarbons, although the plant highly adapted in the contaminated soil.

However, A. coromandeliana showed potential to degrade PAHs in the rhizosphere, where dissipation of

PAHs in soil planted with A. coromandeliana was in the range of 81.82 – 100%. Moreover, A.

coromandeliana showed higher tolerance to PAHs compared to other plants tested, as well as fibrous root

that could contribute good maintenance of the rhizosphere.

v

Fitoremediasi Tanah yang Dicemari oleh Hidrokarbon dan Logam Berat


ABSTRAK

Fitoremediasi adalah penggunaan tumbuhan dan mikroorganisma berkaitan dengannya untuk mengurai,

mengasing atau mengambil pencemar dalam tanah. Keupayaan empat spesies tumbuhan iaitu Kaempheria

rotunda, Jatropha podagrica, Asystasia coromandeliana dan Phyllanthus amarus untuk menyesuaikan diri

pada tanah yang tercemar dengan logam berat dan campuran hidrokarbon alifatik serta hidrokarbon aromatik

polisiklik (HAP) telah dinilai. Keupayaan tumbuhan ini sebagai penumpuk hiper logam di dalam tisu serta

keupayaannya untuk mengurai hidrokarbon alifatik dan HAP dalam rhizosfera juga telah dinilai. Bakteria

telah dipencilkan daripada rhizosfera tanah untuk menentukan sama ada penguraian hidrokarbon alifatik dan

HAP adalah disebabkan oleh bilangan bakteria dalam rhizosfera. P. amarus tidak dapat hidup pada tanah

yang tercemar dengan logam berat dan juga campuran hidrokarbon alifatik dan HAP. Faktor pemekatan

biologi (BCF) tumbuhan menunjukkan K. rotunda berpotensi untuk menumpukkan kadmium dan zink

dengan nilai BCF 5.28 dan 0.90, masing-masingnya. Penambahan asid etilenadiaminatetraasetik (EDTA)

sebagai agen pengkelat/penggerak hanya memberi kesan terhadap penumpukan kromium pada tisu J.

podagrica, di mana penumpukkan kromium adalah 2-3 kali ganda lebih tinggi pada tanah yang dirawat

EDTA berbanding pada tanah yang tidak dirawat. Unit pembentukan koloni pada rhizosfera K. rotunda, J.

podagrica dan A. coromandeliana adalah 9.6 x 104, 9.3 x 10

4 and 3.8 x 10

4, masing-masingnya. K. rotunda,

J. podagrica dan A. coromandeliana yang ditanam pada tanah yang tercemar dengan hidrokarbon alifatik

dan HAP menunjukkan keupayaan untuk mengurai hidrokarbon pada rhizosfera masing-masing. K. rotunda

lebih berpotensi untuk mengurai hidrokarbon alifatik pada rhizosfera disebabkan oleh bilangan bakteria yang

lebih tinggi pada rhizosferanya dan penurunan kepekatan hidrokarbon alifatik yang konsisten sepanjang

eksperimen dijalankan. Sebaliknya, penguraian hidrokarbon alifatik oleh A. coromandeliana adalah rendah

walaupun ia dapat menyesuaikan diri pada tanah yang tercemar dengan hidrokarbon. Walaubagaimanapun,

A. coromandeliana menunjukkan potensi yang tinggi dalam penguraian HAP pada rhizosferanya dimana

kehilangan HAP pada tanah yang ditanam dengan A. coromandeliana adalah dalam julat 81.82 – 100%.

Tambahan pula, A. coromandeliana menunjukkan toleransi yang tinggi terhadap HAP berbanding tumbuhan

lain yang digunakan dalam kajian ini serta mempunyai akar serabut yang menyumbang kepada

penyelenggaraan rhizosfera yang lebih baik.

vi

TABLE OF CONTENTS

Contents Page

DECLARATION ii

ACKNOWLEDGEMENTS iii

ABSTRACT iv

ABSTRAK v

TABLE OF CONTENTS vi

LIST OF TABLES ix

LIST OF FIGURES x

CHAPTER 1 GENERAL INTRODUCTION

1.1 Phytoremediation 1

1.2 Problem Statement 6

1.3 Objectives 12

CHAPTER 2 LITERATURE REVIEW

2.1 Heavy Metals in the Environment 13

2.2 Chelating Agents of Heavy Metals 14

2.3 Phytoextraction of Heavy Metals 17

2.4 Aliphatic Hydrocarbons in the Environment 22

2.5 Polycyclic Aromatic Hydrocarbons in the Environment 23

2.6 Rhizodegradation of Aliphatic Hydrocarbons and Polycyclic Aromatic

Hydrocarbons 24

vii

CHAPTER 3 PHYTOEXTRACTION OF HEAVY METALS

3.1 Introduction 30

3.2 Materials and Methods

3.2.1 Experimental Set Up 32

3.2.2 Plant Analysis 33

3.2.3 Inductively-Coupled Plasma Mass-Spectrometer (ICP-MS)

Analysis 34

3.3 Results and Discussion

3.3.1 ICP-MS Calibration Curves 35

3.3.2 Plant Growth and Survival in Heavy Metals 37

3.3.3 Heavy Metals Accumulation in Plants 37

3.3.4 Bioconcentration Factor (BCF) 49

3.4 Conclusion 54

CHAPTER 4 PHYTOREMEDIATION OF ALIPHATIC AND

POLYCYCLIC AROMATIC HYDROCARBONS

4.1 Introduction 56

4.2 Materials and Methods

4.2.1 Chemicals 58

4.2.2 Experimental Set Up 58

4.2.3 Soil Extraction and Crude Extract Fractionation 61

4.2.4 Gas Chromatography-Flame Ionization Detector (GC-FID)

Analysis

63

4.2.5 High Performance Liquid Chromatography (HPLC) Analysis 63

viii

4.2.6 Response Factor and Sample Concentration Calculation 64

4.2.7 Isolation of Bacteria from Rhizosphere Soil 65

4.3 Results and Discussion

4.3.1 Plant Growth and Survival in Hydrocarbons Contaminated

Soil

66

4.3.2 Isolation of Bacteria from Rhizosphere Soil 66

4.3.3 Degradation of Aliphatic Hydrocarbons 69

4.3.4 Degradation of PAHs 78

4.4 Conclusion 90

CHAPTER 5 GENERAL CONCLUSION

5.1 Conclusion 93

5.2 Recommendation 96

References 97

ix

LIST OF TABLES

Tables Page

Table 1.1 : Information of the selected plant species 10

Table 3.1 : Initial metal concentrations in soil after one week equilibration

before plant transplanting

33

Table 3.2 : Concentration of standard solution for calibration analysis 35

Table 3.3 : Metals accumulation in plants at different sampling times 38

Table 4.1 : Initial concentration of aliphatic hydrocarbons in spiked soils on

a dry weight basis

60

Table 4.2 : Initial concentration of PAHs in spiked soils on a dry weight

basis

61

Table 4.3 : HPLC parameters for the PAHs analysis of soil 64

Table 4.4 : Composition of MSM in a liter solution 66

Table 4.5 : Colony forming unit of bacteria isolated from plant rhizosphere

throughout the incubation period

68

x

LIST OF FIGURES

Figures Page

Figure 3.1 : Calibration curves of Ni, Pb, Cd, As, Cr and Zn 36

Figure 3.2 : Accumulation of Cr in K. rotunda and J. podagrica 40

Figure 3.3 : Accumulation of Cd in K. rotunda and J. podagrica 42

Figure 3.4 : Accumulation of Zn in K. rotunda and J. podagrica 44

Figure 3.5 : Accumulation of Pb in K. rotunda and J. podagrica 46

Figure 3.6 : Accumulation of Ni in K. rotunda and J. podagrica 47

Figure 3.7 : Accumulation of As in K. rotunda and J. podagrica 49

Figure 3.8 : Comparative BCF of Cd and Zn in K. rotunda and J.

podagrica at different sampling time

51

Figure 3.9 : Comparative BCF of Pb and Ni in K. rotunda and J.


52

Figure 3.10 : Comparative BCF of Cr and As in K. rotunda and J.


53

Figure 4.1 : Chromatogram of n-alkanes and internal standard (eicosene)

in spiked soil at day 0

59

Figure 4.2 : Chromatogram of PAH and internal standard (d10-pyrene) in

spiked soil at day 0

59

Figure 4.3 : Schematic diagram of soil extraction and fractionation 62

Figure 4.4 : Chromatogram of n-alkanes and internal standard (eicosene) 69

Figure 4.5 : Gas chromatograms showing changes of aliphatic

hydrocarbons profile in soil planted with K. rotunda at 60,

120 and 180 days

71


hydrocarbons profile in soil planted with J. podagrica at 60,

120 and 180 days.

72

xi

Figures Page


hydrocarbons profile in soil planted with A. coromandeliana

at 60, 120 and 180 days.

75


hydrocarbons profile in unplanted spiked soil at 60, 120 and

180 days.

76

Figure 4.9 : Concentration of TAH at different sampling time in soil

planted with K. rotunda, J. podagrica and A. coromandeliana.

77

Figure 4.10 : HPLC chromatogram of PAHs standard and d10-pyrene

(internal standard)

79

Figure 4.11 : Concentration variation of acenaphthene in planted and

unplanted spiked soils at different time of sampling

79

Figure 4.12 : Concentration variation of phenanthrene in planted and


81

Figure 4.13 : Concentration variation of fluorene in planted and unplanted

spiked soils at different time of sampling

82

Figure 4.14 : Concentration variation of anthracene in planted and


83

Figure 4.15 : Concentration variation of fluoranthene in planted and


84

Figure 4.16 : Concentration variation of pyrene in planted and unplanted

spiked soils at different time of sampling

85

Figure 4.17 : HPLC chromatograms showing changes of PAHs profile in

soil planted with A. coromandeliana at 60, 120 and 180 days

86


unplanted spiked soil at 60, 120 and 180 days.

87


soil planted with K. rotunda at 60, 120 and 180 days.

88

Figure 4.20 : HPLCs chromatograms showing changes of PAHs profile in

soil planted with J. podagrica at 60, 120 and 180days

89

1

CHAPTER 1

GENERAL INTRODUCTION

1.1 Phytoremediation

‘Phytoremediation’ consist of the Greek prefix “phyto” which means plants and

“remedium” which means to correct or remove an evil (Cunningham et al., 1996). It is a

technique that uses plant to remediate contaminated soil and water (US EPA, 2000). The

idea of using metal accumulating plants to remove heavy metals and other compounds was

first introduced in 1983. However, the concept has actually been implemented for the past

300 years (Chaney et al., 1997). Phytoremediation includes all biological, chemical and

physical processes using plants, including rhizosphere for in situ or ex situ removal,

transfer, stabilization or destruction of contaminants in soils, sludges, sediments, other

solids or groundwater. Plants mineralize some toxic organic compounds and accumulate

heavy metals and other inorganic compounds from soil into aboveground shoots (US EPA,

2000). These techniques include phytodegradation, phytovolatilization, rhizodegradation,

phytostabilization, rhizofiltration and phytoextraction (Kevin, 2006; Ghosh and Singh,

2005a; US EPA, 2000).

Phytodegradation, also known as phytotransformation (US EPA, 2000) is the

uptake, metabolism and breakdown of contaminants within the plant to simpler molecules

that are incorporated into the plant tissues (Chaudhry et al., 1998), or degradation of

contaminants in the soil, sediments, sludges, groundwater by enzymes produced and

released by the plant (US EPA, 2001). Enzymes involved are usually dehalogenases,

oxygenases and reductases (Black, 1995). Several compounds subjected to

2

phytodegradation are organic compounds (trinitrotoluene and trichloroethylene) and

herbicides (atrazine and benzaton) (US EPA, 2001).

Phytovolatilization is the uptake of organic compounds such as trichloroethylene,

toluene and ethylbenzene, and inorganic contaminants such as Hg and Se by the plant from

soil, water or a mixed soil and water matrix, converts it to a volatile form and release it to

the atmosphere, usually through the leaf stomata (US EPA, 2001; Aitchison et al., 2000;

Burken and Schnoor, 1998). This technique can be applied in the removal of contaminants

in groundwater, soils, sediments and sludges (US EPA, 2001). However, this technique is

only suitable for contaminants that do not pose a significant air pollution hazard.

Phytovolatilization has been primarily used for the removal of Hg. Unfortunately, the less

toxic elemental transformed mercury when released to the atmosphere is likely to be

recycled by precipitation and then deposit back into ecosystem (US EPA, 2000). Banuelos

et al. (2000) reported that some plants which grow in high Se media produce volatile Se in

the form of dimethylselenide and dimethyldiselenide. Dushenkov (2003) reported that

tritium (3H), a radioactive isotope of H has been successfully decayed to a stable He with a

half-life of about 12 years through phytovolatilization.

Rhizodegradation process takes place at the intersection of bioremediation and

phytoremediation. In rhizodegradation, the organic contaminants in soil are breakdown

through microbial activity of the root zone (rhizosphere). Yeast, fungi, bacteria and other

microorganisms consume and digest organic substances like fuels and solvent (Ghosh and

Singh, 2005a). The rhizosphere zone is more amenable to the microbes that degrade the

contaminants. Root exudates such as organic acids and ketones may promote microbial

growth, as may the increase in soil organic matter caused by the roots (Waters, 2003). This

technique is effective for compounds such as petroleum hydrocarbons, chlorinated

3

solvents, pesticides, polychlorinated biphenyls (PCBs) and surfactants (US EPA, 2001),

though it is a much slower process than phytodegradation (Ghosh and Singh, 2005a). This

rhizodegradation enhancement is also known as plant-assisted degradation, plant-assisted

bioremediation, plant-aided in situ biodegradation and enhanced rhizosphere degradation

(US EPA, 2000).

Phytostabilization is mostly used for the remediation of soil, sediment and sludges

(US EPA, 2000) and depends on the roots ability to limit contaminant mobility and

bioavailability in the soil. This technique use plants to stabilize contaminants through

chemical, biological and physical modification directly (in situ) in the soil.

Phytostabilization can occur through the sorption, precipitation, complexation or metal

valence reduction. The plants primary purpose is to decrease the amount of water

percolating through the soil matrix, which may result in the formation of hazardous

leachate and prevent soil erosion and distribution of the toxic metal to other areas. A dense

root system stabilizes the soil and prevents erosion (Berti and Cunningham, 2000) besides

reducing the mobility of contaminants such as Pb, Cr, Hg, Cu or Zn by the accumulation of

contaminants by plant roots, absorption onto root or precipitation within the root zone (US

EPA, 2001). This technique is very effective when rapid immobilization is needed to

preserve ground and surface water and disposal of biomass is not required (Ghosh and

Singh, 2005a). In phytostabilization, the contaminant is not destroyed but simply prevented

from migrating offsite or causing further damage to the ecosystem. However, a

disadvantage of this technique is that it might involve monitoring a site for the foreseeable

future to ensure that contaminant migration does not occur (Waters, 2003).

Rhizofiltration is defined as the use of plants, both terrestrial and aquatic to absorb,

concentrate and precipitate contaminants from polluted aqueous sources with low

4

contaminant concentration into their roots (US EPA, 2000; Chaudhry et al., 1998), or

adsorption or precipitation of contaminants onto plant roots. These processes can be biotic

or abiotic. Plant uptake, concentration and translocation might occur depending on the

contaminant. Exudates from the plant roots might cause precipitation of some metals (US

EPA, 2000). Rhizofiltration can partially treat industrial discharge, agricultural runoff or

acid mine drainage. It can be used for Pb, Cd, Cu, Fe, Ni, Mn, Zn, Cr and radionuclides,

which are primarily retained within the roots (US EPA, 2001; Chaudhry et al., 1998). This

technology has been tested in the field with uranium (U) contaminated water at

concentrations of 21-874 μg/L. The treated U concentration as reported by Dushenkov et

al. (1997) was < 20 μg/L before discharge into the environment.

Phytoextraction is the best approach to remove the contamination primarily from

soil and isolate it, without destroying the soil structure and fertility. It is also known as

phytoaccumulation, phytoabsorbtion or phytosequestration (US EPA, 2000; Schnoor,

1995). This technique was developed specifically for inorganic pollutants such as metals

(Ag, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Zn), metalloids (As, Se), radionuclides (90

Sr,

137Cs,

243U,

238U) and nonmetals (Schnoor, 1995). In this process, the removal of

contaminants is achieved through the root network and the accumulation potential into the

plant biomass. The biomass is then harvested to complete the extraction of contaminants

from the environment (McIntyre, 2003). According to Salt et al. (1995) and Rulkens et al.

(1998), two basic strategies of phytoextraction have been developed, that are chelates

assisted phytoextraction or induced phytoextraction, where artificial chelates are added to

increase the mobility and uptake of metal contaminants, and continuous phytoextraction in

the removal of metal which depends on the natural ability of the plant to remediate; only

the numbers of plant growth repetitions are controlled. The main advantage of this

5

technique is the ability to concentrate the pollutant into small volume and preventing the

pollutant from extended interaction with the ecosystem. The disadvantages of this

technique are the contaminant is not destroyed and suitable disposal method and location

must be established for the contaminated plant tissue (Waters, 2003).

Phytodegradation can occur simultaneously with phytovolatilization (US EPA,

2000). Orchard et al. (2000) reported that trichloroethylene could be taken up by the plant

roots, transported through the roots and stem into the leaves, and then transpired through

the leaves into the atmosphere. This process was demonstrated in a dual chamber system

with plants and trichloroethylene.

The efficiency of phytoremediation is dependent on the morphology and depth of

the root, growth rate, evapotranspiration rate, assimilation and metabolism of contaminant,

production of exudates and detoxifying enzymes (Karthikeyan and Kulakow, 2003;

Schnoor, 1995). In addition, plant biomass production and plant elemental uptake are two

key factors for successful application of phytoremediation (Reeves and Baker, 2000).

6

1.2 Problem Statement

Over centuries, human industrial, mining and military activities as well as farming

and waste practices have contaminated large areas of developed countries with high

concentrations of heavy metals and organic pollutants. These sites not only cause negative

effects to ecosystem and other natural resources, but also pose a great danger to public

health as the pollutants can enter food through agricultural products or leach into drinking

water (EEA, 2003; EC, 2002). Land contamination in Malaysia was generally caused by

improper waste and waste treatment facilities management such as illegal dumping waste

and abandoned waste treatment facilities. Chemical industries also contribute on land

contamination from the leaking of underground petroleum storage tanks, improper and

illegal storage of fuels and chemicals within industrial premises, as well as gradual

accumulation of chemicals within industrial premises. Land contamination then

subsequently cause water contamination, besides other activities such as sewage discharge

and industrial effluents discharge that also contribute to water contamination on Malaysia

(Yin et al., 2006).

Compared to other developed countries, the implementation of established

technologies for land remediation in Malaysia is rather limited. This may be due to the

majority of engineers and environmental experts in Malaysia that are unaware of such

technologies that could restrict the efforts in redevelopment of contaminated sites (Yin et

al., 2006). Despite the limited implementation of technologies for land remediation, some

remediation works has been done in Malaysia during the past decades such as the aerobic

and anaerobic treatment of sludge and waste waters that was successfully applied for the

agro based industries, as well as in secondary and tertiary treatment of industrial effluents

7

with some organic content. Other remediation works such as the use of water plants for

removal of heavy metals in wastewater by Standards and Industrial Research Institute of

Malaysia (SIRIM) and initial research on specialized microorganisms for treatment of oily

wastewater and other wastewater had faced numerous problems (Tan, 2007). However,

with the oncoming creation of a National Register for contaminated sites and formulation

of comprehensive policies on redevelopment of such sites attribute under the 9th

Malaysia

Plan, these technologies will be essential in assisting sustainable development initiatives in

the country (Yin et al., 2006).

Recent remediation techniques in Malaysia have been focused on conventional

remediation methods that include soil vapour extraction, remedial natural attenuation,

containment, solidification and stabilization and contaminated soil excavation.

Bioremediation and phytoremediation methods are still new and mainly limited.

Bioremediation, for example is generally conducted after a primary clean up activity where

monitoring of soil conditions is initiated, while phytoremediation is limited to bench-scale

research in academic institutions. However, phytoremediation has shown to be an

attractive remediation option as it increases the aesthetic values of the contaminated site

and requires less equipment and labour than any other remediation methods (Yin et al.,

2006).

Conventional engineering methods to remediate contaminated soil are often very

costly, with cost ranged from $50 to $500 per ton. While certain specialized techniques can

exceed costs of $1000 per ton. With an acre of soil about 3-foot depth weight

approximately 4500 tons, the minimum cost per acre could reach a quarter million dollars

(Cunningham et al., 1995). Washing metal contaminated soil for example can cost about

$250 per cubic yard. An incineration project to clean up explosives contaminated soil at

8

the Department of Energy’s Idaho National Engineering Laboratory cost up to $4,000 per

ton to clean hot spots at the naval proving ground (Black, 1995). In European Union, the

overall cost to remediate affected sites was estimated to be between €59 and €109 billion

(EC, 2002).

In contrast, phytoremediation is a cost effective new method to remediate

contaminated soil. Phytoremediation is on average tenfold cheaper than conventional

engineering methods such as soil excavation, washing or burning, or pump and treat

systems (Glass, 1999). The cost of removing toxic metals by phytoextraction, for example

could be as low as $5 to $40 per ton of soil (Glass, 1999), or as low as $60,000 to $100,000

for phytoextraction of 50 cm depth metal polluted soil compared up to $400,000 cost of

soil excavation, transport and storage as hazardous waste (Salt et al., 1995). Furthermore,

phytoremediation could also reduce the exposure of the polluted substrate to humans,

wildlife and the environment (Pilon-Smits, 2005), besides leaving topsoil in useable

condition, as well as reducing the amount of contaminated material to be land filled or

incinerated (Black, 1995).

However, phytoremediation is a time consuming process that it may take several

growing season to clean a site (Black, 1995), or even decades to halve metal contamination

on soil (McGrath and Zhao, 2003). This could cause problems for economic development

as the contaminated sites are not available for sale or rent during phytoremediation process

(SRU, 2004). Other concern is plants that absorb toxic heavy metals could pose risk to

wildlife that eats plants, as metal can enter the food chain (Black, 1995). Furthermore,

suitable plants for phytoremediation that have characteristics such as fast growing, have

deep and wide-spreading root system, easily propagated as well as sod forming grasses that

can produce horizontal stems that might grow above or below the surface of the soil

9

(Ghosh and Singh, 2005b; Kramer and Chardonay, 2001; Aprill and Sims, 1990) are hardly

found. In addition, there are limited plants that could tolerate polluted soil and produce

high biomass at the same time as contaminants from the contaminated soil may affect plant

growth and subsequently reduced plant biomass.

Therefore, this study was done to evaluate the potential of phytoremediation to

remediate the contaminated soil. Plants were screened for the ability to survive and

remediate heavy metals and hydrocarbons contaminated soil. Four plants, Kaempferia

rotunda, Jatropha podagrica, Asystasia coromandeliana and Phyllanthus amarus have

been selected based on the characteristics of suitable plants for phytoremediation such as

fast growing, have deep and wide-spreading root system, easily propagated as well as sod

forming grasses that can produce horizontal stems that might grow above or below the

surface of the soil (Ghosh and Singh, 2005b; Kramer and Chardonay, 2001; Aprill and

Sims, 1990). The information of the selected plant species are as shown in Table 1.1.

Selected plants were evaluated for their ability to survive in soil contaminated with

heavy metals, as well as accumulate heavy metals in their tissue and consequently act as

hyperaccumulator based on the value of Bioconcentration Factor (BCF) when planted in

mixed metal contaminated soil. This study focused on mixed metal contamination as heavy

metals are usually found in soil and water. The uptake of heavy metals by plants is either

by synergistic or antagonistic interactions among metals. Synergism refers to the

phenomenon where two or more elements acting together to create a greater effect than the

effect when acting as a single element. While antagonism phenomenon is where the

combination of two or more elements gives less important effect compared when they act

as a single element. Thus, these interactions might refer to the ability of an element to

avoid or increase the absorption of other elements by the roots (Kabata-Pendias, 2000).

10

Table 1.1: Information of the selected plant species.

Plant Name Family Origin/Distribution Characteristics

Kaempferia

rotunda

Zingiberaceae1 Native to China, Taiwan, India, Indonesia,

Malaysia, Myanmar, Sri Lanka and Thailand

where it is found in open grasslands. 1

Flowered, have medicinal properties,

tuberous root. 1

Jatropha

podagrica

Euphorbiaceae2 Native to Central America

2

Flowered, cultivated for medicine and

ornamental purposes.2

Erect shrub with woody stem swollen the

base or lower part.2

Have tape root with lateral roots.3

Adapted to all kinds of soils and does not

demand any special nutritive regime4.

Asystasia

Coromandeliana Acanthaceae

5 Native in tropical Africa, Arabia and tropical

Asia6

Widely distributed in Nigeria and throughout the

world7

Flowered, fast-growing, spreading, perennial

herb herbaceous groundcover, often rooting

at the lower nodes5,8

Phyllanthus

amarus

Euphorbiaceae9 Originates from tropical America and has spread

as weed around all tropical regions from Africa to

Asia, South America and the West indies9,10

Flowered, topical annual herbal shrub

whose stem has green capsule (fruit)11

Hard tap root12

References: 1Wu and Larsen, 2000;

2Bingtao and Gilbert, 2008;

3Nwokocha et al., 2012;

4Patil and Singh 1991;

5Saunders, 1958;

6Daziel, 1937;

7Elliot, 2004;

8Ensermu, 1994;

9Oudhia, 2008;

10Unander, 1995;

11Obianime and Uche, 2009;

12Sen et al., 2011

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In contrast, the uptake of plant will not be evaluated in plants planted in

hydrocarbons contaminated soil, but the ability of selected plants to decrease the amount of

hydrocarbons in planted soil will be evaluated instead. This is because many petroleum

hydrocarbons are large and have high molecular weight, which give the lipophilic

properties that exclude them from the plant root (Qui et al., 1997). Thus, plant uptake and

accumulation of hydrocarbons from contaminated soil is quite small and limited to low

molecular compounds (Chaineu et al., 1997). The hydrophobicity and potential for

movement of pollutant in soil is measured using the octano-water partitioning coefficient

(Kow). Hydrocarbons such as aliphatic hydrocarbons and PAHs have high Kow, are

generally bound to soil organic matter or adsorbed to roots and not substantially

translocated to the shoot (Pilon-Smits, 2005); therefore, uptake of aliphatic hydrocarbons

and PAHs into plant tissue is considered a minor pathway for their removal from soil.

Gaskin (2008) has assessed plant roots and shoots of Australian native grass species

planted in hydrocarbons contaminated soil for hydrocarbons accumulation and shown that

no hydrocarbon were accumulated by the plants. Previous study done by Binet et al. (2000)

also showed the same result where the accumulation of PAHs was found to be limited to

the root tissue and no PAHs found in shoot tissue of ryegrass (Lolium perenne). It was

noted that PAHs dissipation was likely due to biodegradation or biotransformation in the

rhizosphere (Binet et al., 2000).

Petroleum hydrocarbons are the most common organic pollutants, which include n-

alkanes and other aliphatics, aromatic compounds and other minor constituents as well

(Atlas and Philip, 2005; Sarkar et al., 2005). Microbial degradation of petroleum

hydrocarbons could be promoted by the mixture of these organic compounds, especially

when one or more components of the mixture are co-metabolite of others (Nocentini et al.,

2000). In this instance, microbes that primarily degrade one type of organic compound

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may also degrade a second compound present at concentrations too low to independently

support bacterial growth or not recognised as a substrate by chance (Gaskin, 2008). Hence,

plants were planted in soil contaminated with mixture of aliphatic hydrocarbons and PAHs

to determine the most suitable plant that can survive and decrease the amount of aliphatic

hydrocarbons and PAHs in their rhizosphere.

1.3 Objectives

Four plant species has been selected for investigation was Asystasia

coromandeliana, Kaempheria rotunda, Phyllanthus amarus and Jatropha podagrica.

These plants were choose based on the characteristics of suitable plants for

phytoremediation such as fast growing, have deep and wide-spreading root system, easily

propagated as well as sod-forming grasses which can produce horizontal stems that might

grow above or below the surface of the soil (Ghosh and Singh, 2005b; Kramer and

Chardonay, 2001; Aprill and Sims, 1990). The objectives of this project were:

a. to screen plant species for their ability to survive in soil contaminated by heavy

metal, aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs).

b. to assess the capability of selected plant species as accumulator for heavy metals.

c. to evaluate rhizosphere degradation of aliphatic hydrocarbons and PAHs

contaminated soil using the selected plant species.

d. to determine the ability of the rhizosphere microorganisms to enhance the

rhizodegradation of aliphatic hydrocarbons and PAHs.

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