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BIOREMEDIATION OF HEAVY METAL POLLUTED WATER USING IMMOBILIZED
FRESHWATER GREEN MICROALGA, BOTRYOCOCCUS sp.
ONALO JOAN IYE
A thesis submitted in
Fulfilment of the requirement for the award of the
Degree of Master of Master of Science
Faculty of Science, Technology and Human Development
Universiti Tun Hussein Onn Malaysia
MAY 2015
vi
ABSTRACT
Heavy metal containing wastewater are regarded as highly toxic to the aquatic
environment and to life in general due to their bio-accumulating, cytotoxic,
mutagenic and carcinogenic effects on life. Bioremediation is the use of biological
materials (e.g. microalgae) in the removal of toxic compounds from the environment
such as the heavy metals which is considered more cost effective and
environmentally friendly when compared to the physical and chemical methods. The
present study was undertaken to check for the heavy metal bioremoval efficiency of
free and immobilized Botryococcus sp. Four heavy metals were studied and the free
cells efficiently reduced Chromium which is equivalent to 94%, followed by Copper
(45%), Arsenic (9%) and Cadmium (2%). For the immobilized biomass, the highest
(P<0.05) removal efficiency was recorded in the highest biomass concentration (i.e.
15 beads/ml) for Cadmium, Arsenic and Chromium at 76%, 68% and 67%. Whereas,
the highest (P<0.05) removal of copper was observed in the blank alginate beads at
84%. The positive control (free cells) recorded the highest (P<0.05) reduction for
biological oxygen demand (BOD) whereas, the 15 beads/ml gave the highest
(P<0.05) reduction for control gave the highest (P<0.05) reduction for the Chemical
oxygen demand (COD). In the LD50 experiment, immobilized biomass harvested
from the bioremoval study experiment were used on fishes for toxicity testing. A
total of 100% mortality was recorded in the positive control after 24 hours whereas,
3% mortality was observed in negative control and in the 10 beads/ml treatment after
72 hours. No mortality was found in any other treatment after a period of 96 hours.
The results obtained from this study suggests that, immobilized cells of
Botryococcus sp. is efficient in the bioremoval of heavy metals from contaminated
waters and also have great potential in the biotransformation of toxic compounds to
less-toxic forms.
vii
ABSTRAK
Logam berat yang terkandung dalam air sisa dianggap sangat bertoksik kepada
persekitaran dan kehidupan akuatik yang disebabkan oleh kesan bio-accumulating,
cytotoxic, mutagenic dan carcinogenic dalam kehidupan. Bioremediasi adalah
merupakan penggunaan bahan biologi (eg. mikroalga) dalam penyingkiran bahan
toksik seperti logam berat secara lebih efektif dari segi kos serta mesra alam jika
dibandingkan dengan kaedah konvensional. Kajian ini telah dijalankan untuk menilai
kecekapan penyingkiran logam berat dengan menggunakan Botryococcus sp. Empat
logam berat telah dikaji menggunakan free sel yang mana Kromium dikurangkan
sebanyak 94%. Diikuti oleh Copper (45%), Arsenik (9%) dan Kadmium (2%).
Manakala untuk biojisim bergerak, yang paling tinggi (P <0.05) penyingkiran
dicatatkan pada kepekatan biojisim yang tertinggi (iaitu 15 manik/ml) dan untuk
Kadmium, Arsenik serta Kromium masing-masing pada 76%, 68% dan 67%.
Manakala, yang tertinggi (P <0.05) penyingkiran kuprum diperhatikan dalam manik
alginat kosong pada 84%. Kawalan positif (sel percuma) merekodkan jumlah
tertinggi (P<0.05) pengurangan permintaan oksigen biologi (BOD) manakala, 15
manik/ml memberi nilai tertinggi (P<0.05) pengurangan untuk memberi kawalan
yang paling tinggi (P<0.05) pengurangan permintaan oksigen kimia (COD). Dalam
eksperimen LD50, biomas bergerak dituai dari eksperimen kajian bioremoval
digunakan pada ikan untuk ujian ketoksikan. Sebanyak 100% kematian dicatatkan
pada kawalan positif selepas 24 jam sedangkan, 3% kematian diperhatikan dalam
kawalan negatif dan dalam 10 manik / rawatan ml selepas 72 jam. Tiada kematian
didapati dalam mana-mana rawatan yang lain selepas tempoh 96 jam. Keputusan
yang diperolehi daripada kajian ini menunjukkan bahawa, sel-sel Botryococcus sp.
bergerak. cekap dalam menyingkirkan logam berat daripada air yang tercemar dan
juga mempunyai potensi besar dalam bio-transformasikan sebatian toksik kepada
bentuk yang kurang bertoksik.
viii
TABLE OF CONTENTS
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT vi
ABSTRAK vii
TABLE OF CONTENTS viii
LIST OF TABLES xv
LIST OF FIGURES xvi
LIST OF SYMBOLS xix
LIST OF ABBREVIATIONS xx
CHAPTER 1 INTRODUCTION
1.1 Background of Study 1
1.2 Problem Statement 4
1.3 Objectives of Study 5
ix
1.4 Scope of Study 6
1.5 Significance of Study 7
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction 9
2.2 Water Pollution 9
2.2.1 The Textile Industry as a Source of Water Pollution 10
2.2.2 Effects of Water Pollution 12
2.3 Heavy Metals 13
2.3.1 Some Important Heavy Metals 13
2.3.1.1 Arsenic 13
2.3.1.2 Cadmium 14
2.3.1.3 Chromium 15
2.3.1.4 Copper 16
2.3.2 General Toxicity of Heavy Metals 16
2.3.3 Water Pollution by Heavy Metals 17
2.4 Bioremediation 19
2.4.1 Types of Bioremediation 20
2.4.1.1 In Situ Bioremediation 20
2.4.1.2 Ex Situ Bioremediation 21
2.4.2 The Principles of Bioremediation 22
2.5 Microalgae 23
2.5.1 Biotechnological Applications of Microalgae 24
x
2.6 Microalgae as Bioremediators 24
2.6.1 Mechanism of Heavy Metal Removal by
Microalgae 26
2.7 Botryococcus sp. 29
2.7.1 Some Biotechnological Applications of
Botryococcus sp. 31
2.8 The Use of Immobilized Microalgae for
Bioremediation 31
2.8.1 Alginate as an Immobilization Matrix 34
2.9 Bioremediation of Water Polluted by Heavy
Metals 34
2.10 Standard for Wastewater Effluent 36
2.11 Conclusion 37
CHAPTER 3 METHODOLOGY
3.1 Introduction 38
3.2 Sample Organism 40
3.2.1 Preparation of Botryococcus sp. 40
3.3 Algal Cell Count 43
3.3.1 Calculation of Cell Concentration 44
3.3.2 Specific Growth Rate (µ/day) 45
3.4 Experimental Setup for the Growth of Botryococcus
sp. In the Wastewater Containing Heavy Metals 45
xi
3.5 Immobilization of Botryococcus sp. 46
3.6 Wastewater Sample Collection, Handling
and Preservation 47
3.7 Determination of pH, Temperature, DO and
Conductivity 48
3.8 Chemical Oxygen Demand (COD) Test 49
3.9 Biochemical Oxygen Demand (BOD) Test 51
3.10 Total Solids (TS) 52
3.10.1 Procedure for the Determination of Total
Dissolved Solids (TDS) 52
3.10.1.1TDS Calculation 53
3.10.2 Procedure for the Determination of Total
Suspended Solids (TSS) 54
3.10.2.1TSS Calculation 54
3.11 Determination of Total Organic Carbon (TOC),
Total Carbon (TC), Inorganic Carbon (IC)
and Total Nitrogen (TN) 55
3.12 Determination of Orthophosphate (PO43-
) 56
3.13 Determination of Heavy Metal in the Water
Sample 57
3.14 Experimental Setup 58
3.15 Determination of Heavy Metal Toxicity after
xii
Removal by the Microalga 59
3.15.1 Procedure for the LD50 Test 60
3.17 Statistical Analysis 61
3.18 Conclusion 61
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Introduction 62
4.2 Characteristics of the Textile Wastewater 62
4.3 Heavy Metal Content of the Textile Wastewater 64
4.4 Growth of Botryococcus sp. in the Wastewater 66
4.5 Size Distribution and Characteristics of Beads 70
4.6 Heavy Metal Reduction/Removal 71
4.6.1 Reduction of Arsenic 72
4.6.2 Reduction of Cadmium 76
4.6.3 Reduction of Chromium 80
4.6.4 Reduction of Copper 83
4.6.5 Effect of Biomass Concentration on the Reduction
of Arsenic Cadmium, Chromium and Copper 86
4.6.6 Effects of Immobilization 88
4.6.7 Effect of Time 92
4.7 Physical and Chemical Parameters Reduction 93
4.7.1 Reduction of COD, BOD and TDS 94
4.7.2 Reduction of TC, IC and TOC 97
xiii
4.8 Toxicity Testing 98
4.8.1 Median Lethal Dose (LD50) Analysis 98
4.8.1.1 Treatment of the synthetic wastewater 106
CHAPTER 5 CONCLUSION AND RECOMMENDATIONS
5.1 Introduction 109
5.2 Conclusion 109
5.3 Recommendations 112
REFERENCES 113
APPENDIX 152
VITA 179
xiv
LIST OF TABLES
2.1 Typical Characteristics of Raw Textile Wastewater 12
2.2 Some Microalgal Strains used in the Removal of Heavy
Metals from Contaminated Solutions 26
2.3 Some Biomass Immobilized in Different Matrixes that have been
used in the Removal of Heavy Metals from Wastewater 33
2.4 Standards A & B for Effluent Parameters 37
3.1 Composition of Bold’s Basal Medium (BBM) 42
3.2 Experimental Analysis/Parameter Testing 48
4.1 Physio-Chemical Characteristics of the Sample Wastewater (Textile) 63
4.2 Heavy Metal of Importance Present in the Textile Wastewater 65
4.3 Characteristics of the Water used for the Test 101
4.4 Average Weight ± SD and Length ± SD (standard deviation) of the
Fishes after 24 Hours. 102
4.5 Average Weight ± SD and Length ± SD (standard deviation) of the
Fishes after 48 Hours. 103
4.6 Average Weight ± SD and Length ± SD (standard deviation) of the
Fishes after 72 Hours. 103
4.7 Average Weight ± SD and Length ± SD (standard deviation) of the
xv
Fishes after 96 Hours. 104
xvi
LIST OF FIGURES
2.1 Process of Heavy Metal Contamination 18
3.1 Methodology Flow Chart 40
3.2 Botryococcus sp. 43
3.3 Growth of Botryococcus sp. 43
3.4 Microscope & Haemocytometer 44
3.5 Area in the Haemocytometer 44
3.6 Immobilized Botryococcus sp. 47
3.7 pH, Temperature, Conductivity and DO Meter 49
3.8 DRD Reactor for COD Test 50
3.9 BOD Bottles (300 ml each) 51
3.10 Vacuum Filter Pump 55
3.11 TOC Analyser 56
3.12 Atomic Absorption Spectrophometer (AAS) 57
3.13 Flasks Containing the Different Concentrations of the Immobilized
Biomass 59
3.14 Treatment Flasks Inoculated with the Immobilized Biomass and the
Wastewater 59
3.15 LD50 Test Aquarium 61
xvii
4.1 Daily Growth Rate (x 103) cells/ml of Botryococcus sp. in
Different Concentration of the Wastewater 67
4.2 Specific Growth Rate (µ/day) 68
4.3 Heavy Metal Reduction 69
4.4 Botryococcus sp. Immobilized in Sodium Alginate 70
4.5 Size Distribution of Beads 71
4.6 Percentage Reduction of Arsenic by Botryococcus sp. 73
4.7 Mean ± Standard Error for Arsenic Reduction on
Day 0, 3, 5, 7 and 10 75
4.8 Percentage Reduction of Cadmium by Botryococcus sp. 77
4.9 Mean ± Standard Error for Cadmium reduction on
Day 0, 3, 5, 7 and 10 79
4.10 Percentage Reduction of Chromium by Botryococcus sp. 80
4.11 Mean ± Standard Error for Chromium Reduction on
Day 0, 3, 5, 7 and 10 82
4.12 Percentage Reduction of Copper by Botryococcus sp. 84
4.13 Mean ± Standard Error for Copper Reduction on
Day 0, 3, 5, 7 and 10 85
4.14 Effect of Biomass Concentrations 87
4.15 Effect of Immobilization 90
4.16 Effect of Time on the Bioremoval 92
4.17 COD, BOD and TDS Reduction for a Period of 7 days 95
4.18 Reductions of TC, TOC and IC for a Period of 7 days 97
xviii
4.19 Silver Bard (Barponymus gonionotus) 100
4.20 Percentage Mortality in the Treatments after a Period of 96 hours 102
4.21 Changes in the Concentration of DO 106
4.22 Heavy Metal Reduction in the Synthetic Wastewater 107
xix
LIST OF SYMBOLS
% - Percentage
Mg/L - Milligram per Litre
oC - Degree Celsius
rpm - Rounds per Minute
psi - Per Square Inch
g - Grams
ml - Millilitre
L - Litre
g/L - Grams per Litre
Cells/Square - Cells per Square
Cells/ml - Cells per Millilitre
Beads/ml - Beads per Millilitre
dH20 - Distilled Water
xx
LIST OF ABBREVIATIONS
TN - Total Nitrogen
PO43- -
Orthophosphate
TOC - Total Organic Carbon
TC - Total Carbon
TC - Inorganic Carbon
TS - Total Solids
DO - Dissolved Oxygen
TDS - Total Dissolved Solids
TSS - Total Suspended Solids
AAS - Atomic Absorption Spectrophotometer
As - Arsenic
Cd - Cadmium
Cr - Chromium
Cu - Copper
< - Less than
NaCl - Sodium Chloride
K2Cr2O7 - Potassium dichromate
xxi
AgSO4 - Silver Sulphate
HgSO4 - Mercuric Sulphate
H2SO4 - Sulphuric Acid
CO2 - Carbon di Oxide
H2O - Water
SEM - Scanning Electron Microscopy
LD50 - Median Lethal Dose
UTHM - Universiti Tun Hussein Onn Malaysia
FSTPi - Faculti Sains, Teknology dan Pembangunan Insan
xxii
LIST OF APPENDIXES
APPENDIX A Growth of Botryococcus sp. in the Wastewater 151
APPENDIX B Percentage Removal of Arsenic (Day 0-10) 152
APPENDIX C Percentage Removal of Chromium (Day 0-10) 153
APPENDIX D Percentage Removal of Cadmium (Day 0-10) 154
APPENDIX E Percentage Removal of Copper (Day 0-10) 155
APPENDIX F Arsenic Reduction on Days 3, 5, 7 and 10 in the
Different Treatments 157
APPENDIX G Homogeneity of Variance and ANOVA for Arsenic
Reduction 159
APPENDIX H Chromium Reduction on Days 3, 5, 7 and 10
In the Different Treatments 161
APPENDIX I Homogeneity of Variance and ANOVA for
Chromium Reduction 163
APPENDIX J Cadmium Reduction on Days 3, 5, 7 and 10
In the Different Treatments 165
APPENDIX K Homogeneity of Variance and ANOVA
For Cadmium Reduction 167
APPENDIX L Copper Reduction on Days 3, 5, 7 and 10
xxiii
In the Different Treatments 169
APPENDIX M Homogeneity of Variance and ANOVA
For Copper Reduction 171
APPENDIX N Reductions of the Parameters (BOD, COD,
TDS, TOC, TC, IC) 173
APPENDIX O Homogeneity of Variance and ANOVA
For BOD Reduction 174
APPENDIX P Homogeneity of Variance and ANOVA
For COD Reduction 175
APPENDIX Q Homogeneity of Variance and ANOVA
For TDS Reduction 176
APPENDIX R Homogeneity of Variance and ANOVA
For TOC Reduction 177
1
CHAPTER 1
INTRODUCTION
1.1 Background of Study
Increase in human activities such as industrialization and civilization has resulted in
the issue of enviornmental pollution and in most cases the pollution of water bodies.
This issue has for long been a great problem that requires adequate attention.
Pollution according to Abdel-Raouf et al. (2012) is referred to as a non-natural
occurrence usually associated with elevated concentrations of naturally existing
materials or synthetically made substances that are released into a particular
environment. Heavy metals are environmental pollutants of inorganic origin with
atomic weights of about 63.5 to 200.6 g/mol (Srivastava & Majumder, 2008). Heavy
metals are known to be emptied into freshwaters and their resources via industrial
effluents (Tripathi, 2014).
They tend mainly to pollute surface water and groundwater due to natural
events or human activities. They are carcinogenic, mutagenic and cytotoxic and
therefore, pose a great and deadly effect on the environment and on living things
(Moo-Young, 2011). The release of both organic and inorganic contaminants into the
environment by either industrial activities or other form of anthropogenic practices
has led to the case of environmental pollution (Mouchet, 1986; Lim et al., 2010).
2
Heavy metal discharge into the environment has rather become a rapid practice
owing to the increasing trend in technology and also their use in different industrial
processes (Tripathi, 2014). According to Vidal (2001), environmental quality has a
lot to do with the quality of life on earth as this go hand in hand. Recently, the
problem of environmental pollution has been accepted as a general or global issue as
the evaluated contaminated areas is significant (Cairney, 1993).
Among the major new technologies that have appeared since the 1960s,
bioremediation applications have attracted a great deal of attention and interest. The
application of microbial metabolic potential (bioremediation) is accepted as an
environmentally benign and economical measure for the decontamination of polluted
environments. Bioremediation methods are generally categorized into ex situ and in
situ bioremediation. The bioremediation of heavy/toxic metals is focused on the
reduction in the level or amount of hazardous wastes as the immoderate
accumulation of these metallic ions can be highly toxic to plants, aquatic life and the
environment as a whole (Singh et al., 2002). Mohee & Mudhoo, (2012), stated that
the limited achievements in using these biological materials in the process of
bioremediation have recently been ascribed to the reduction in biological system
productivity and diversity under environmental conditions. Considering the many
adverse effects that accompany environmental pollution by heavy metals comes a
better and an effective remedial approach, a process known as bioremediation.
Bioremediation is a branch of biotechnology that utilizes biological processes
to render harmless the environmental contaminants (Boopathy, 2000). There are a
number of agents used for bioremediation out of which are the bacteria, fungi and the
microalgae but the microalgae presenting unique characteristics out of which is their
ability to distinguish between the essential and the non-essential substances that are
present in the environment. In addition, these set of organisms are single celled and
as a result they do not mutate. Microalgae are unicellular and colonial organisms that
are highly diversified and are made up of the eukaryotic protists and the prokaryotic
blue-green algae (cyanobacteria). These set of organisms are known to be
outstanding in assessing environmental quality and are thus, considered unique (Day
et al, 1999).
Microorganisms, especially the microalgae present a relatively great size for
heavy metal ion binding at an eco-friendly rate of production and have been
therefore, applied in the bioremoval of metallic ions from contaminated
3
environments (Bitton, 2011). In addition, they are also greatly recognized for their
capacity and efficiency in the process of bioaccumulation and biosorption of toxic
metals (Moo-Young, 2011).
Microalgae are known for their capability in the production of several
important biological materials either at a viable or non-viable state or when
immobilized and the ease at which algae can be cultured or grown have made them
influential useful biomass when treating environmental problems which has captured
a lot of public concern (Kaur & Bhatnager, 2002). The ability of microalgae in the
uptake of heavy metals from wastewater has now been acknowledged (Travieso et
al., 2002) and this has been the major focus on the use of microalgae in
bioremediation (Abdel Hameed & Ebrahim, 2007). Some microalgal strains which
have demonstrated heavy metal removal ability and efficiency from wastewater
include; Chlorella, Scenedesmus, Botryococcus, Phormidium, Desmodesmus
pleiomorphus, Chlamydomonas and Spirulina (Sandau et al., 1996; Rangsayatorn et
al., 2004; Monteiro et al., 2009; Ruiz-Marin et al., 2010; Rawat et al., 2011;
Kshirasagar, 2013).
Both living and non-living microalgae are capable in the accumulation,
elimination and biosorption of heavy metals with no effects of toxicity (Sandau et al.,
1996). Either suspended cells of microalgae or immobilized cells encapsulated in a
particular matrix can be used for waste water treatment. The use of immobilized
microalgae however, in the removal of pollutants has proffered some advantages
over the use of its counterparts (i.e. the free cells). Some of which include; easy
separation of biomass from the wastewater, potential for reusability in further
absorption cycles and increased bioaccumulation capacity (Abdel Hameed &
Ebrahim, 2007; Ruiz-Marin et al., 2010). Furthermore, immobilized algal system had
more sorption efficiency, stronger and healthier cellular structural composition,
greater electro-kinetic efficiency and increased chlorophyll content as compared to
the free and suspended algal system (Maznah et al., 2012; Zeng et al., 2013). Zeng et
al (2013), put forward for consideration the need for the application and exploration
of immobilized microalgal growth bioreactors in the different areas of
biotechnological applications. This is due to the great differences observed between
the immobilized and free microalgal bioreactor. Several immobilizing polymer have
been used in cell encapsulation. Alginate polymer in particular, has been extensively
used for the removal of heavy metal from contaminated solutions (Hadiyanto et al.,
4
2014). Immobilized algal biomass of Spirulina platensis and Chlorella vulgaris has
been used in the removal of Chromium and Copper from textile wastewater by
Hadiyanto et al. (2014). Botryococcus majorly has been extensively studied
particularly in the production of biofuel (Amaro et al., 2011). Likewise, there is a
distinguishing feature that gives rise to the uniqueness of this organism that is the
production of lipids which is closely related to hydrocarbon (Metzger & Largeau
2005; Cheng et al., 2013). Dayananda et al. (2007) clearly expressed that
Botryococcus braunii gives rise to 85% hydrocarbon content of its dry total biomass
composition. However, there are no reports found on the use of immobilized
Botryococcus sp. in the removal of heavy metals (arsenic, cadmium, copper and
chromium) from multi heavy metal containing textile wastewater. In addition, this is
the first research on the heavy metal biodetoxification by immobilized Botryococcus
sp. after bioremediation.
In view of the generalization of the effectiveness and potentialities of
bioremediation giving consideration to the different industries in the communities of
Malaysia, it becomes overwhelming to experimentally investigate its impact on the
metal ion related water pollution. This study, therefore, was designed to determine
the potentiality of Botryococcus sp. This is freshwater green microalga indigenous
specie isolated from the heart of a tropical rainforest in Johor, Malaysia in the
process of bioremediation of heavy metal polluted water. The inoculum was
provided by STG1008 of the Microbiology laboratory, Faculty of Science,
Technology and Human Development. Universiti Tun Hussein Onn Malaysia.
1.2 Problem Statement
Malaysia is one of the developing countries with so many industries and as a result is
faced with the disposal of industrial waste directly or indirectly into the rivers, a
situation which is of a great concern of the government, community dwellers,
operating companies, stakeholders, supervisory and regulatory agencies. The major
source of heavy metal pollution out of the several sources is the industry as a result
of their extensive use in industrial processes. One of the recognized potential
industries known for heavy metal production in their waste is the textile industry
5
(Deepali & Gangwar, 2010). This industrial sector is known to be highly diversified
in terms of production, raw materials, products and industrial chain (Savin &
Butnaru, 2008).
The rise in the establishment of more textile industries and the enlargement of
existing ones could be attributed to the growing population trend as well as the
continuous change in fashion sense and style. It is in no doubt a fact that the textile
industries contribute very highly to the issue of water pollution most especially by
heavy metals (Imtiazuddin et al., 2012) considering their extensive use of water in
the various industrial chains of processes (Solanki et al., 2013). According to
Hadiyanto et al. (2014), the increase in the growth of textile industries has led to
elevated chances of the environment being polluted by heavy metals. These toxic
elements are usually present in their wastes disposed into the environment
particularly, when the wastes are not appropriately taken care of. In addition, due to
the large amount of water produced as waste in this industry, the problem of
negligence in pollution control may arise (Tüfekci et al., 2007). Furthermore, the
most challenging problem of the textile industry on the environment is concomitantly
attributed to the large water usage as well as wastewater discharge (Savin & Butnaru,
2008).
Considering, the toxic effects of heavy metals on the environment which is a
great on problem on lives generally, there is therefore, a need for proper treatment of
industrial wastewater before discharging them into the water bodies. Methods
conventionally used are either too expensive or inefficient. In addition, application of
non-biological wastewater treatment techniques have sometimes led to the generation
of additional pollutants or the formation of toxic sludge as a result of the many
chemicals involved in this process (Domnez & Aksu, 1990; Topuz & Macit, 2010)..
1.3 Objectives of Study
The main objective of this study is to explore the use of bioremediation processes in
the treatment of water containing heavy metals by Botryococcus sp.
6
To determine the physical and chemical characteristics of the raw textile
wastewater.
To determine the heavy metal bioaccumulation capacity of Botryococcus sp.
and also the efficiency of bioremoval by immobilized Botryococcus sp.
To determine the biodetoxification of the heavy metals by immobilized
Botryococcus sp. after bioremediation.
1.4 Scope of Study
This research was carried out using the microalgae, Botryococcus sp. that was
collected and isolated from the tropical rain forest, Johor, Malaysia. The inoculum
was provided by STG1008 of Microbiology laboratory, FSTPi, UTHM. The sample
organism was immobilized in sodium alginate. Meanwhile, the wastewater is an
industrial wastewater obtained from a textile industry in Batu Pahat, Johor, Malaysia.
The Dissolved Oxygen (DO), Temperature, conductivity, pH, chemical oxygen
demand (COD), biochemical oxygen demand (BOD), Total Solids (TSs), Total
Organic Carbon (TOC), Total Nitrogen (TN), orthophosphate (PO43-
) and heavy
metal test (Flame Atomic Absorption Spectroscopy, AAS) were all carried out
according to the Standard Methods and Examination of Wastewater (APHA, 2012).
The flasks containing the treatment media were maintained at standard temperature
in the course of the experiment. The algal (Botryococcus sp.) cells after harvesting
were counted using the haemocytometer before the immobilization of the biomass in
alginate. The Median Lethal Dose (LD50) was used to test for the toxicity of the
heavy metals present in the wastewater that has been bioaccumulated by the
immobilized biomass on fishes after bioremediation.
7
1.5 Significance of Study
Increase in modernization has led to greater degree of industrialization and increased
load of toxic pollutants (such as the heavy metals) in the environment. The process of
bioremediation is environmentally friendly as it makes use of naturally existing
biological materials in the treatment of wastewater. Likewise, biological wastewater
treatment of wastewater with special preference to the microalgae is regarded as a
better option in bioremediation considering their photosynthetic abilities as they
absorb atmospheric CO2 and converts solar energy into important biomass (Kumar et
al., 2014). Also, they can achieve greater performance in wastewater treatment by
incorporating the nutrients and other pollutants present in the wastewater and thereby
reducing the contaminant load present in the wastewater most especially, when
immobilized.
The immobilized biomass proffers some advantages such as the easy
separation of the biomass from the wastewater after treatment. This is in no doubt, a
promising technology in dealing with environmental pollution. This study is thus,
designed to explore the process of bioremediation with experimental investigation of
the effectiveness of the green freshwater microalga, Botryococcus sp. (chlorophyta)
immobilized in sodium alginate in the bioremediation of water contaminated with
heavy metals. The applications of biological materials for dealing with heavy metal
contamination and removing these pollutants from contaminated waters has the
efficiency in obtaining higher degree of accomplishment at an eco-friendly rate. In
addition, this method is considered more environmentally friendly when measured
with the use of the existing methods (physical and chemical wastewater treatment
technologies) for the removal of toxic metal from polluted waters (Wilde &
Benemann, 1993). Furthermore, it has the ability to achieve greater performance
considering the fact that the microalgae can utilized contaminants present in
wastewater as nutrients thereby reducing the pollutant load.
The technology involve in this process is easily applicable as it does not
require the use of heavy equipment or high labour. Likewise, the immobilized
biomass can be used for further biotechnological applications as they are renewable.
These studies however have been focused on developed nations with limited studies
addressing the impact of this process in an emerging economies and their
8
environment. The significance of this study therefore, is to investigate the
effectiveness of bioremediation processes in eliminating heavy metal ions from
waters by the use of microalgae immobilized in sodium alginate.
9
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
This is the second chapter of this thesis. The main objective of this research is to
explore the use of immobilized Botryococcus sp. in the bioremediation of wastewater
loaded with heavy metals. As the presence of these toxic pollutants in water can be
detrimental to human health and the environment in general. This chapter therefore,
aims to review the keywords in the title as this will help in better understanding and
application of this technique in accordance to the objective of the study.
2.2 Water Pollution
Pollution refers to the introduction of unwanted materials into a system. Water
pollution (either by natural means or as a result of human activities) has become a
general issue that requires fast action in handling such a situation. This is a result of
the several hazardous effects that accompany pollutions as well as the many
economical harms that can be caused by it (Nollet, 2007). According to Boopathy
(2000), groundwater pollution with deadly substances is one of the serious
10
challenges that the civilized and industrialized world is faced with today.
Furthermore, Sasikumar & Papinazath (2003) and Sharma (2012) stated the fact that
increase in population goes hand in hand with increase in the load of environmental
pollutant.
The need to meet the daily needs of individuals has led to the development of
more industries and has therefore, led to increase in environmental pollutants
resulting from the activity of humans (Verma et al., 2014). In addition, in the quest to
meet the world’s icreasing population needs, there is thus, an expeditious act in the
enlargement of various industries which has resulted in the impediment of life
quality and which is also detrimental to the environment (Kumar et al., 2011). And
this has emanated in the increasing load of environmental toxicants from the
resulting industrial effluent discharge (Vidali, 2001).
Water pollution majorly occurs as a result of untreated industrial waste
disposal into the environment and can lead to so many dangerous effects on the
receiving biota. The pollution of groundwater can be generally categorised into three
namely; contamination by organic compounds, by microorganisms and lastly by
inorganic contaminants such as the toxic heavy metals (Zouboulis & Katsoyiannis,
2005). The emptying of several inorganic and organic contaminants from different
sources which include; industrial, agricultural and municipal points has resulted into
the long lasting problem of water pollution and also in the gradual gathering of
chemicals in the food chain (Verma et al., 2014). Additionally, these situations have
also led to the insufficiency in obtaining water of high quality (Kamaludeen et al.,
2003).
2.2.1 The Textile Industry as Source of Water Pollution
Increase in clothing and apparel demands is believed to be attributed to the
increasing fashion sense and style thus, leading to higher productivity of textile
materials to meet this growing trend (Desta, 2013). The textile industry is one
industry that makes extensive use of water in the cause of their production (Savin &
Butnaru, 2008) which can cause laxity in the control of pollution (Tüfekci et al.,
2007). Based on these, the textile industry has been identified as a great source of
11
environmental pollution due to water consumption and pollutants discharge (Lacasse
& Baumann, 2006).
Textile industry is considered one of the industrial divisions that have great
deal of variety in the areas of the raw materials as well as the interconnected
processes involved in the industrial processes (Savin & Butnaru, 2008). According to
Mahmood et al. (2005), there is slim feasibility in the use of reported literature in
forecasting the characteristics of textile wastewater as the production, technology and
chemicals used varies industry by industry. This type of industry is very verse and
one of the most fast growing industries in the world. There is no doubt that the
wastewater produced from the textile industrial processes are loaded with heavy
metal pollutants. These pollutants needs to be reduced to permissible limits before
they are been released into the environment as they are known to be non-
biodegradable toxicants (Mahmood et al., 2005).
Water containing these toxic heavy metals could be toxic to the receiving
biota and the mankind in general. Furthermore, these toxicants have the ability to
move from one level to another in the food chain, a process known as
bioaccumulation which is regarded as a serious threat to life. According to Savin &
Butnaru (2008), the major point of pollution in the textile wastewater comes from the
dyeing and the finishing processes as these processes necessitates the extensive use
of different forms of chemicals and dyeing agents and it has also been discovered
that the heavy metal pollution occur at this points in the industrial processes (NIIR
Board, 2003).
The inability in the proper handling of such wastes is what has resulted into
the problem of heavy metal pollution. Conventional methods used by the textile
industries in treating their waste include; chemical precipitation, coagulation,
adsorption and membrane separation (Hadiyanto et al., 2014). These approaches,
however, are both expensive and can cause final sludge treatment problems (Lau et
al., 1998) and this could be as a result of high suspended solids present in the
wastewater. Some of the heavy metals shown to be present in textile wastewater
include; Cu, Cd, Cr, Zn (Mahmood et al., 2005; Imtiazuddin et al., 2012; Hadiyanto
et al., 2014).
12
Table 2.1: Typical Characteristics of Raw Textile Wastewater
Parameter Value References
BOD
150-350 mg/L
Mahmood et al., 2005
COD 712 mg/L Solanki et al., 2013
pH 5.5-10.5 Mahmood et al., 2005
TDS 1500-2200 mg/L Mahmood et al., 2005
TSS 200-1100 mg/L Mahmood et al., 2005
Temperature 40oC Imtiazuddin et al., 2012
Chlorides 121.6 mg/L Savin & Butnaru, 2008
Cu 80 mg/L Hadiyanto et al., 2014
Cd 0.1 mg/L Imtiazuddin et al., 2012
Cr 1.0mg/L Imtiazuddin et al., 2012
As 100 ppb Chethana, 2014
2.2.2 Effects of Water Pollution
When living things take in contaminated water, they might be exposed to different
kind of illnesses which can be either long-term or short-term (Gopalan, 2012).The
edible aquatic creatures such as fish, crabs, and prawns may sometime take in these
water contaminants and when these are consumed by human beings, various illness
that causes lethality follow (Gopalan, 2012). Polluted water has adverse effects on all
forms of life present in the environment. The pollutants in the environment tend to
reduce the level of Dissolved Oxygen (DO) in the ecosystem and this can as a
consequence lead to decreased level of aquatic lives or reduced biodiversity
(Gopalan, 2012). Marcos & Carlos (2007) thus, stated the need for proper treatment
of wastewater before releasing into the environment or been used for other purposes.
13
2.3 Heavy Metals
Heavy metals are contaminants from inorganic source that tend mainly to
contaminate water as a result of natural events and human activities (Moo-Young,
2011). Metals are known to be found everywhere in the environment via air, water
and soil when compared to other form of pollutants. And they can neither be broken
down nor metabolized (Sodhi, 2009). According to Ahalya et al. (2003), heavy
metals of concern include; Lead Chromium, Zinc, Selenium, Cadmium, Mercury,
Gold, Nickel, Uranium, Silver and Arsenic. Most of these toxic substances that are
inclined to pollute the environment are known to be in soluble forms (Rani et al.,
2010). These elements (heavy metals) are considered highly distributed in the
environment and therefore lead to water and soil pollution and in some cases, air
pollution. Their increasing load in the ecosystem has led to great concern in the view
of the general public (Mane & Bhosle, 2012) as a result of their toxicity.
2.3.1 Some Important Heavy Metals
2.3.1.1 Arsenic
Arsenic is recognised as the number two most frequent pollutant with great deal of
interest by USEPA (2002) as it tends to pollute the water bodies around the globe
(Bhumbla and Keefer, 1994; USEPA, 2001; Mead, 2005). In addition, increased
level of these toxic compounds in drinking water is regarded as the most
predominant cause of arsenic poisoning worldwide (Mazumder, 2008). According to
Mazumder et al. (1988), the arsenical dermatitis that occurred in India was associated
with elevated levels of this element present in the well waters having the
concentration range between 0.2-2.0 mg/l. Arsenic in its most dangerous state can
cause carcinogenic damage to the skin and other parts of the body (Mandal &
Suzuki, 2002; Igwe & Abia, 2006).
14
Arsenic is considered one of the most predominating constituents of
industrial effluents that are released into the environment and subsequently causing
the problem of pollution (Verma et al., 2014).The major route of exposure of humans
to arsenic is via inhalation of contaminated air, ingestion of contaminated food and
water (Mazumder, 2008) and as well as consumption of crops irrigated by arsenic
contaminated waters. Little or no risk is caused by arsenic to human health through
skin exposure to this element such as the washing of hands and the bathing because
there is minimal dermal adsorption of arsenic (Meharg, 2005). However, it becomes
highly toxic when waters or food contaminated with arsenic is ingested or
contaminated air is inhaled. It has been estimated that arsenic is the 20th
abundant
element in the earth crust, 22nd
in the sea water and 12th
in the body of humans
(Woolson, 1975; Brown et al., 1991; Mandal &Suzuki, 2002). This non-essential
element is considered to be primarily released into the environment either by natural
means or by anthropogenic activities.
A total of one hundred million people by estimation have been shown
globally to have chronically been exposed to high levels of arsenic from drinking
water (Bang et al., 2005; Parvez et al., 2006). According to Meharg (2005), arsenic
is considered particularly dangerous and deadly considering the fact that there is high
environmental influence contributing to its presence in the environment and its great
toxicity therefore; its presence in drinking water has become a major public health
issue.
2.3.1.2 Cadmium
Cadmium is one form of heavy metal that is not regarded as an essential element for
the biological function of life and is therefore not present in uncontaminated water
but could be present in industrial wastewater (Shyong & Chen, 2000; Lamai et al.,
2005). It is known to be extensively used in paints, dyes, cement and phosphate
fertilizers (Tripathi, 2014). The presence of this element in the water bodies
according to Sangalang & O’Halloran (1972) and Bengtsson et al. (1975), can lead to
dangerous situations in fishes among which is the steroid hormones modification,
contractions of the skeletal and longitudinal body as well as the collapse of the
15
vertebrae. In addition, cadmium has been recognized to constantly causing decrease
in the global fish stock as it is highly toxic and can lead to harmful effect on the
aquatic environment most especially, on fishes (Wright & Welbourn, 1994; Tripathi,
2014).
This heavy metal also, has the potency of altering dynamicity in the aquatic
ecosystem being a usual water pollutant (Shukla et al., 2013). Cadmium is among the
so called “big three” toxic heavy metals that has attracted a great deal of concern
considering its dangerous effect arising from industrial activities (Forstner &
Wittmann, 1979). They are highly toxic to life even at very low and minute
concentrations (Srianga et al., 2010). Cadmium has the potency in the displacement
of essential elements and chronic exposure to elevated level of cadmium can lead to
unfavourable conditions in humans such as bone damage, liver damage, kidney
failure and in some cases cancer (Doshi et al., 2007; Solisio et al., 2008).
2.3.1.3 Chromium
This element is considered as a micronutrient and on the other hand regarded as a
deadly metal. The hexavalent chromium is considered highly toxic than the trivalent
chromium (Smith & Lec, 1972; Michalak, 2007). Additionally, Cr (VI) which is the
toxic form of the element is usually released into the environment by industrial
operations (Faisal & Hasnain, 2004). It is highly mobile through the soil and the
water bodies and has a great capability of been taken in via the skin (Park & Jung,
2001).
The following are some of the unpleasant conditions caused by this toxic
heavy metal; carcinogenicity (Shumilla et al., 1999), teratogenicity (Asmatulla et al.,
1998) and Mutagenicity (Cheng et al., 1998). According to Kowalski (1994),
considering its carcinogenicity and teratogenicity, chromium is one of the top 16
toxic environmental contaminants. Based on this, the EPA (US Environmental
Protection Agency) has therefore, proposed the need for its removal from industrial
effluents before finally releasing the industrial wastewaters into the environment.
16
2.3.1.4 Copper
The toxicity of copper is considered relatively high when compared to other heavy
metals although its concentration is usually low (Gibson & Mitchel, 2005) and it
plays a major role in the contamination of the environment (Subhashini et al., 2011).
It is regarded as one of the most extensively used form of heavy metal in the
industries which can become greatly and extremely deadly to life at high
concentrations (Igwe & Abia, 2006). Davis et al. (2000) stated the fact that copper
has been found to set down in the liver, pancreas, skin, brain and the myocardium
(the muscular tissue of the heart) exerting unfavourable conditions to life.
2.3.2 General Toxicity of Heavy Metals
Increased level of heavy metals in the environment causes some toxic effects in the
biota such as the damaging of cells as well as the inhibition of enzyme activity (Jadia
& Fulekar, 2009). The deadly effect of metallic toxins on health intensely depends on
their absorption, distribution, excretion and metabolic rate in living organisms. These
toxins can get into the human body through skin, lungs and the gastrointestinal tracts.
Once they get to the bloodstream, they may be carried to the target organs (such as
the liver or kidney) or some may be deposited in bones or muscles (Gopalan, 2012).
Mathis & Cummings (1973) ascertained that heavy metals have the ability to
generate different health difficulties to humans by taking in the contaminated water
or the edible aquatic resources.
Some heavy metals are known to be immunosuppressive, cause neurological
disorder, anaemia, gout, kidney damage, gastro-enteritis, and high blood pressure.
The cations of mercury, lead, cadmium and arsenic possess a very great affinity for
sulphur. As a result of this, they are able to bind with the sulfhydryl group (~SH) in
enzymes which influences the rate of metabolic activities in the human body
(Gopalan, 2012), such a reaction can be represented as follows;
CH3Hg+
+ Enz-SH CH3Hg-S-enz + H+
17
Once this displacement happens, there are some changes made in the active site of
the enzyme and this make the enzyme to lose its proficiency as a reaction booster
(Gopalan, 2012). In addition, due to the non-biodegradable nature and the long
biological half-lives required for their elimination from biological tissues, the
presence of heavy metals in the biota has therefore, become a great concern (Olatunji
et al., 2009). Generally, considering all the effects of these toxic substances it can be
inferred that their presence in the environment presents more harm than good on the
receiving ecosystem.
2.3.3 Water Pollution by Heavy Metals
For a long time now, heavy metals have been acknowledged as sincere, serious,
deadly and deteriorated pollutants of the aquatic ecosystem with an unfavourable
effect on the biota (Calabrese et al., 1973). The contamination of water bodies by
heavy metals is as a result of natural factors or human activities such as; agricultural
run-off, mining activities and wastes discharged by various industries (Gharedaashi
et al., 2013). Among the various materials that pollute the aquatic ecosystem, heavy
metal pollution is given more concern due to their high toxicity and the hazardous
effects that accompany their existence in an environment. The chain of heavy metal
contamination arising from human/natural activities (Figure 1) goes a long way in
causing harm to all life forms that come in contact with these toxic elements.
Heavy metals are recognized as usual contaminants of water as a result of
their substantial use in various industries and their high concentration in the
environment becomes damaging to lives in that ecosystem (Kiyani et al., 2013).
Increase in industrialization and economic development goes concurrently with
increase in pollution. This in turn, poses deadly risk on the ability to obtain quality
water resources in many environments affected by pollution (Raouf et al., 2012).
Additionally, heavy metals receive specific concern when compared to other surplus
variety of pollutants affecting aquatic ecosystems owing to their strong toxicity to the
environment and life in general (Nollet, 2007). The liberation of heavy metals into
the environment especially the water bodies, presents a serious threat due to their
18
cytotoxic, mutagenic and carcinogenic effects on human beings and wild life (Moo-
Young, 2011).
Figure 2.1: Process of Heavy Metal Contamination Chain.
Human/Natural
Activities
Human Beings
Microorganisms
Fishes Animals
Crops
Soil
Food
Heavy Metals Water
19
2.4 Bioremediation
Bioremediation is a branch of biotechnology that deals with the different methods of
solving environmental problems such as cleaning the environment from
contaminants or pollutants with the use of microorganisms. It uses biological
materials to destroy or reduce the level and the concentration of hazardous wastes
such as heavy metals in a contaminated or polluted environment (Boopathy, 2000).
Bioremediation also can be defined as the technological process that makes use of
biological organisms to breakdown organic chemicals (Fiorenza et al., 1991). In
other words, it is referred to as a terminology used in giving detailed account of the
restoration of damaged environments using biological approaches (Kumar et al.,
2011).
Bioremediation has successfully cleaned up many polluted sites or
environment and has a great advantage of making use of natural and biological
processes in the cleaning of contaminated sites. Also, it is considered cost effective
when compared to the physical and chemical techniques used in cleaning up
contamination since it does not require too many equipment and labour like its
counterparts. The method of bioremediation is a technique that makes use of low
cost relativity and also low technology which has great acceptance from the public
view (Verma et al., 2014). In addition, it can also reduce the level of toxicants to the
levels lower than the limits proposed by regulation agencies (Flathman et al., 1993).
The application of bioremediation is indeed a promising technology in the area of
environmental pollution as the process can be applied in all the compartments of the
environment that have tendency of being polluted (e.g. air, water and soil) thereby,
increasing the quality of life in that particular biota.
In as much as the principles, techniques, advantages and the disadvantages of
this process are not widely and clearly understood or known, bioremediation has
successfully been applied to number of sites worldwide (Kumar et al., 2011).
Likewise, the method of bioremediation has become a growing trend with no rules
subjected to if a contaminant can be degraded or not (Kumar et al., 2011). In
addition, biological processes are often time considered highly selective and suitable
level of nutrients and contaminants are required for effective bioremediation (Vidali,
2001). Agents used for bioremediation include; Fungi (Subhashini et al., 2011),
20
Bacteria (Rani et al., 2010), Macroalgae (Davis et al., 2000; Michalak et al., 2007)
and the microalgae (Al-Rub et al., 2004; Bishnoi & Garima, 2004; Rangsayatorn et
al., 2004; Abdel Hameed, 2006; Michalak et al., 2007). According to Prescott et al.
(2002), the first bioremediating agent that was registered for a patent is the strain
Pseudomonas putida.
The microalgae however, is regarded more potential bioremediators most
especially in the biological treatment of heavy metal contamination because of their
ability to distinguish between the essential and non-essential elements needed for
their growth (Perales-Vela et al., 2006). Additionally, they have greater yield of
biomass and do not require a large area of land for cultivation. Furthermore, they
also have the ability of growing in open cultures and still remain free from
contaminants which make it cost effective and easily applicable (Barsanti &
Gualtieri, 2006).
2.4.1 Types of Bioremediation
Bioremediation, the environmental part of biotechnology that utilizes biological
potentials in dealing with environmental contaminants is broadly classified into two
major classes which are the In situ and the Ex situ methods (Boopathy, 2000).
2.4.1.1 In Situ Bioremediation
The Latin phrase “In situ” simply refers to “On site”. The In situ bioremediation is
that which is done on the contaminated site. It does not involve moving the
contaminated sample to another place for the biological treatment. This form of
bioremediation often times is used in the reduction of the level of contaminants
present in saturated soil and groundwater (Sasikumar & Papinazeth, 2003). The
process is based on the principle that involves the targeting of pollutants removal or
reduction under normal and natural environmental conditions. This is done through
the application of microbial metabolic potentials on the contaminated environment
21
without having to remove the contaminated samples from the place of contamination
(Fruchter, 2002; Farhadian et al., 2007; Jorgenson, 2007).
In situ bioremediation is regarded as a safer and cheaper method for treating
contaminations as there is no need for the excavation and the transfer of
contaminants (Vidali, 2001). Likewise, it can also bring about the contemporaneous
remediation of both soil and ground water (Sasikumar & Papinazeth, 2003).
According to Shukla et al. (2010) and Bouwer & Zehnder (1993), the In situ
bioremediation is considered the most desirable due to its cost effectiveness and less
disturbances owing to the fact that the treatment is provided on the polluted site.
However, this type of bioremediation might not yield the desired and efficient results
due to environmental factors that affect bioremediation such as the temperature, pH,
oxygen and nutrients (Sharma, 2012). In addition, the depth of the contaminated site
could also limit the effectiveness of treatment (Verma, 2014). And it has also been
reported that the method consumes time more than the other biological remedial
methods (Sasikumar & Papinazath, 2003). The various types of in situ
bioremediation include; biosparging, bioventing, in situ biodegradation,
bioaugmenation and biopilling (Vidali, 2001; Sharma, 2012).
2.4.1.2 Ex Situ Bioremediation
Ex situ bioremediation is the second form of bioremediation that is not done at the
contaminated site. It involves taking the sample from the contaminated environment
(Vidali, 2001) in order to aid microbial degradation (Kumar et al., 2011). The
principle underlying the application of the ex situ bioremediation is marked by the
intervention in the degradation of pollutants present in excavated samples (Guerin,
1999; Carberry & Wik, 2001; Prpich et al., 2006).
One of the outstanding advantages in the application of ex situ
bioremediation processes is that it requires minimum time when compared to its
counterpart, In situ bioremediation and also the firm conviction of being able to exert
control on the treatment (Pavel & Gavrilescu, 2008). Some authors are of the opinion
that ex situ bioremediation is more expensive to manage as a result of excavating the
contaminated sample from the original site (Vidali, 2001). However, some other
22
authors still are of the opinion that the ex situ bioremediation is more cost effective
because of the excavation of the contaminated site (Schacht & Ajibo, 2002; Pare,
2006; Donlon & Bauder, 2008). The efficacy of ex situ bioremediation is assured and
guaranteed as the method is independent of the environmental factors that could
affect the process adversely (Pandey et al., 2009). Besides, Physio-chemical
treatment can be added in the process to aid the degradation of pollutants because it
is carried out in the non-natural environment (Kim et al., 2005).
In a comparative study determined to evaluate the performance rate of in situ
and ex situ bioremediation for the decontamination of contaminated sample showed
that the results obtained from the in situ method showed higher variability when
compared to that obtained from the ex situ method (Carberry & Wik, 2001). This
suggests that the ex situ process can yield better bioremedial effects. The ex situ
bioremediation is divided into two main types which are the slurry-phase
bioremediation and the solid-phase bioremediation (Pavel & Gavrilescu, 2008;
Kumar et al., 2011).
2.4.2 The Principles of Bioremediation
Biotechnology is a field that has great and diverse applications. The environmental
biotechnology is a fast growing field that can be used in the actual applications of
ecosystems. Additionally, it can be applied in the transformation of pollutants into
environmentally friendly substances and as well as developing safe manufacturing
and disposal processes (Kumar et al., 2011). The effectiveness of bioremediation
greatly depends on the ability of the biological material to convert the pollutants into
harmless materials through enzymatic processes (Vidali, 2001; Sharma, 2012). This
transformation reaction takes place as part of the normal metabolic process of the
living organism (Vidali, 2001).
The techniques of bioremediation generally, are more cost effective than the
traditional methods (Kumar et al., 2011) and as such have a lot of public acceptance
(Sharma, 2012). There are no rules to the predictions on whether the contaminants
can be degraded or not (Kumar et al., 2011). Just like other technologies,
bioremediation could have its own limitations (Vidali, 2001). Referring to Colberg &
23
Young (1995), the most bioremedial systems are carried out under aerobic
conditions; however, anaerobic systems could also grant the microbial potential the
ability to deteriorate obstinate substances. The control and the optimization of the
process of bioremediation is an interrelated system of several factors. This factors
may include; the availability of microbial population capable of degrading the
pollutants, availability of contaminants to the microbial population and the
environmental factors such as the type of contaminated site, temperature, pH,
oxygen, and nutrients (Vidali, 2001; Sharma, 2012; Verma et al., 2014).
2.5 Microalgae
Algae are simple, aquatic plant-like organisms that lack true leaves, stems and roots.
They are known to be photosynthetic eukaryotes that possess great variety of shapes.
The cell walls of many algae are made up of a carbohydrate known as cellulose
(Tortora et al., 2010). Many of them are single-celled and can only be seen with the
aid of a microscope (the microalgae). Algae are generally called phytoplankton with
quite a majority of them known to be floating unicellular organisms (Bitton, 2005).
The number of algae by estimation is known to be one to ten million, with a high
percentage of the microalgae (Wan-Loy, 2012). According to Packer (2009), Algae
are known to have very high carbon capturing and photosynthetic efficiencies when
compared to terrestrial plants and are classified as either marine or freshwater plants.
Microalgae are highly diverse group of unicellular organisms (Day et al.,
1999) that do not require a large area of land for cultivation, have a short growth
period, possesses high growth rate and contains more high-lipid materials than food
crops. Recently, microorganisms (especially microalgae) have been recognised for
their capacity in the bioaccumulation and bioabsorption of heavy metals (Moo-
Young, 2011). Microalgae are uniquely characterized by being able to distinguish
between the essential heavy metals needed by the organisms from their other
counterparts. And this has been attributed to as a result of the molecular mechanism
they possess which makes them unique in the monitoring of the environment
(Perales-Vela et al., 2006).
24
2.5.1 Biotechnological Applications of Microalgae
Microalgae have been used in different biotechnological applications so far. Thay are
regarded as useful resources and biological tool used in the evaluation and
observation of environmental toxicants or pollutants including the heavy metals
(Wan-Loy, 2012). Also, microalgae also are used for the biological treatment of
contaminated water, and this process enables the efficient recycling of nutrients
within a short period of time and is known to generate valuable biological materials
that are useful in the production of bioactive compounds and biofuel (Souza et al.,
2012).
The use of algae in environmental clean-up is known to be more
environmental friendly and sustainable in that it does not produce or generate
additional pollutants (Pittman et al., 2011). In addition, microalgae are a potential
and efficient source of a great range of high value products for biotechnological use,
examples include; polyunsaturated fatty acids (PUFA), Carotenoids,
Phycobiliproteins and Polysaccharides (Wan-Loy, 2012). Microalgae are also useful
resources in the production of biodiesel with high lipids production efficiency (Halim
et al., 2012). Some microalgae (e.g. spirulina) have been injested as food or health
food due to their high nutritional value (Wan-Loy, 2012).
2.6 Microalgae as Bioremediators
Microalgae are microscopic, photosynthetic aquatic plants that can be found in
freshwater and marine ecosystems; they are highly diversified and are unicellular in
nature (Tomasellii, 2004; Priyadarshani et al., 2011). Similarly, Gill et al. (2013),
referred to them as very small and minute photosynthetic biochemical and
metabolites producers. These set of phytoplankton have shown great heavy metal
affinity, efficiency, sequestration and consistent good performance when compared
to other form of bioremediators (Volesky & Holan, 1995; Schiewer et al., 2000; Jeon
et al., 2001). And also, they have been recognized to play an important task by
controlling the level of heavy metal concentration in both lakes and oceans (Sigg,
112
5.3 Recommendations
The data obtained from this study were satisfactory. However, some observations
have been made to enhance applicability for future research. These include;
Use of higher biomass concentration particularly in the bioremoval arsenic,
cadmium and chromium.
To check for the effect of longer biosorption time in the efficiency of
bioremoval.
Intensify in more detail the composition of alginate in the bioabsorption of
heavy metals and nutrients.
To check for the incorporation of different wastewater treatment materials in
the reduction of physical and chemical parameters from high strength
wastewater.
Monitor the binding nature of copper and the right approach in the right
biomass concentration.
Demonstrate the effectiveness and the efficiency of using this immobilized
biomass in wastewater from different industries that are potential producers
of heavy metals in their waste.
Provide clear and accurate research in the bio-conversion of toxic heavy
metals by Botryococcus sp.
Intensify the biotransformation mechanism exhibited by Botryococcus sp.
Come out with different methods that can be used to check for the
biotransformation of the heavy metals
Lastly, to study deeper into the forms in which the toxic heavy metals are
biotransformed into.
113
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