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THESIS - USE OF MICROORGANISMS AS BIOINDICATORS

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					USE OF MICROORGANISMS AS BIOINDICATORS FOR DETECTION OF
                    HEAVY METALS




                FAZURIANA BINTI AHMAD




                   MASTER OF SCIENCE
               UNIVERSITI PUTRA MALAYSIA

                         2006




                           1
USE OF MICROORGANISMS AS BIOINDICATORS FOR DETECTION OF
                    HEAVY METALS




                                     By

                       FAZURIANA BINTI AHMAD




Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
     in Fulfilment of the Requirement for the Degree of Master of Science


                                  May 2006

                                      i
Dedicated to my beloved family and friends…..




                      ii
 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
                of the requirement for the degree of Master of Science



USE OF MICROORGANISMS AS BIOINDICATORS FOR DETECTION OF
                    HEAVY METALS

                                          By

                          FAZURIANA BINTI AHMAD

                                      May 2006


Chairman : Professor Mohd Arif Syed, PhD

Faculty    : Biotechnology and Biomolecular Sciences


In this study, soil bacteria were isolated and were then screened for their sensitivity

to heavy metals. This study employs the tetrazolium dye MTT (3-(4,5-demethyl-

thiazol-2-yl)-2,5-diphenyltetrazolium bromide) where bacteria reduced the dye,

causing the dye to precipitate and to become intensely coloured. In the presence of

heavy metals, the reduction will be inhibited and become colourless. A total of 250

bacterial isolates were successfully obtained from 10 different locations in Peninsular

Malaysia which were then screened with six selected heavy metals in the presence of

common divalent cations such as calcium and magnesium at the highest

concentration of 25 mg/L and 50mg/L respectively using a MTT assay. An isolate

designated as isolate SC27 at 8 hours growth and isolate S8 at 12 hours growth were

found to be most sensitive to mercury and silver respectively.        The IC50 (50%

inhibitory concentration) of mercury and silver are 0.2698 mg/L and 0.073 mg/L

respectively after data was analyzed using the Graphpad Prism™ version 4.0

software. The assay was found to be unaffected by interference from other tested

xenobiotics. Preliminary field study tests showed the ability of these two bacterial


                                          iii
isolates to detect mercury and silver after comparison with AAS analysis. Isolate

SC27 was identified as Uncultured bacterium strain Dr.Y13 (DQ 226214) which is

related to Enterobacter sp. using Microbact™ kit and was confirmed using 16S

rRNA gene analysis while isolate S8 was identified as Serratia sp. with 90.79 %

similarity using the Microbact™ kit.




                                       iv
  Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
                   memenuhi keperluan untuk ijazah Master Sains



PENGGUNAAN MIKROORGANISMA SEBAGAI BIOINDIKATOR UNTUK
            MENGESAN LOGAM-LOGAM BERAT


                                      Oleh

                         FAZURIANA BINTI AHMAD

                                    Mei 2006



Pengerusi : Profesor Mohd Arif Syed, PhD

Fakulti   : Bioteknologi dan Sains Biomolekul



Dalam kajian ini, bakteria tanah dipencilkan dan kemudian disaringkan untuk

melihat tahap kesensitifan terhadap logam berat. Kajian ini menggunakan pewarna

tetrazolium MTT (3-(4,5-demethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) di

mana bakteria ini akan menurunkan pewarna ini menyebabkan pewarna termendak

dan sebatian menjadi berwarna. Dengan kehadiran logam berat, penurunan ini akan

direncat dan sebatian menjadi tidak berwarna. Sebanyak 250 isolat bakteria berjaya

diperolehi dari 10 kawasan yang berlainan di Semenanjung Malaysia dan seterusnya

disaring dengan enam logam berat yang dipilih dengan kehadiran kation divalen

seperti kalsium dan magnesium pada kepekatan 25 mg/L dan 50 mg/L dengan

menggunakan asai MTT. Isolat yang dikenali sebagai isolat SC27 pada pertumbuhan

8 jam dan isolat S8 pada pertumbuhan 12 jam didapati masing-masing sensitif

kepada merkuri dan argentum. IC50 (50% kepekatan perencat) merkuri dan argentum

masing-masing pada 0.2698 mg/L dan 0.073 mg/L setelah data dianalisa

menggunakan perisian Graphpad Prism™ versi 4.0. Dalam kajian ini didapati asai

                                        v
ini tidak dipengaruhi oleh lain-lain xenobiotik yang dipilih. Kajian percubaan awal

terhadap sampel air menggunakan asai ini menunjukkan kedua-dua isolat bakteria ini

berupaya untuk mengesan merkuri dan argentum setelah dibandingkan dengan

analisa AAS. Isolat SC27 dikenalpasti sebagai Uncultured bacterium strain Dr. Y13

(DQ 226214) dengan menggunakan analisa molecular filogenetik 16S rRNA

walaupun kit Microbact™ mengkelaskan bakteria ini sebagai Enterobacter sp. Isolat

S8 pula dikelaskan kepada Serratia sp. dengan kepercayaan sebanyak 90.70%

dengan menggunakan kit Microbact™.




                                        vi
                            ACKNOWLEDGEMENTS



First and foremost, I am most grateful to Allah S.W.T. for enabling me to come this

far. Life has its ups and downs, but in time of needs, You have always been there for

me. And in one way or another, I have always managed to go through. I could not

have done it without Your help and blessings.



After what I thought to be the most challenging time of my master years, I managed

to summarize it all into this black book. And there are many people out there I most

thank for that. Firstly, my deepest gratitude to my lovable supervisor, Prof. Mohd.

Arif Syed for his invaluable guidance throughout the completion of this project.

Special thanks also dedicated to my co-supervisor, Dr. Mohd Yunus Abd. Shukor,

who has always, always been there to guide, teach and support me throughout my

project. He has made me a better person in many, many ways. I’m sure anybody

who has had the honor to know him will say the same. I pray that this respectful man

will make it big out there, which I’m sure is not very far ahead.



I would also like to take this opportunity to thank all the wonderful people in the lab,

who are Abg. Ariff, Kak Sue, Kak Ilah, Surini and Sim. You guys are the most

labmates anyone can ask for. Not forgetting all lab members 204 as well as the

undergraduates of Enzymology and Bioremediation Lab (115 and 204) for their kind

assistance and for sharing their experiences and knowledge, directly or indirectly.

And most of all, I owe it all to my beloved parents and family for their undying

support and faith in me. Thank you for being understanding even though you don’t

really understand what on earth I was working on. Only God has the wealth to


                                          vii
reward these wonderful people. I shall not go out there and be successful without

being grateful to each and everyone of them.




       “Always aim for the sky, for if u fail, at least u can reach the clouds”




                                         viii
I certify that an Examination Committee has met on 9 May 2006 to conduct the final
examination of Fazuriana Binti Ahmad on her Master of Science thesis entitled “Use
of Microorganisms as Bioindicators for Detection of Heavy Metals” in accordance
with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti
Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends
that the candidate be awarded the relevant degree. Members of the Examination
Committee are as follows:


Norhani Abdullah, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)

Abu Bakar Salleh, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)

Mohammad Ismail Yaziz, PhD
Associate Professor
Faculty of Environmental Studies
Universiti Putra Malaysia
(Internal Examiner)

Wan Azlina Ahmad, PhD
Associate Professor
Faculty of Science
Universiti Technology Malaysia
(External Examiner)




                                   _________________________________
                                   HASANAH MOHD. GHAZALI, PhD
                                   Professor/Deputy Dean
                                   School of Graduate Studies
                                   Universiti Putra Malaysia

                                   Date :




                                        ix
This thesis submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee are as follows:



Mohd Arif Syed, PhD
Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)


Mohd Yunus Abdul Shukor, PhD
Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)




                                            ___________________________
                                            AINI IDERIS, PhD
                                            Professor/Dean
                                            School of Graduate Studies
                                            Universiti Putra Malaysia

                                            Date :




                                        x
                                 DECLARATION

I hereby declare that the thesis in based on my original work except for quotation and
citations, which have been duly acknowledged. I also declare that it has been not
been previously or concurrently for any other degree at UPM or other institutions.




                                               ___________________________

                                               FAZURIANA BINTI AHMAD

                                               Date:




                                          xi
                        TABLE OF CONTENTS

                                                                      Page

DEDICATION                                                            ii
ABSTRACT                                                              iii
ABSTRAK                                                               v
ACKNOWLEDGEMENTS                                                      vii
APPROVAL                                                              ix
DECLARATION                                                           xi
LIST OF TABLES                                                        xiv
LIST OF FIGURES                                                       xv
LIST OF ABBREVIATIONS                                                 xvii


CHAPTER

1    INTRODUCTION                                                     1

2    LITERATURE REVIEW                                                4
     2.1  Heavy metal Pollutions                                      4
     2.2  Heavy metals in the Malaysian Environment                   6
     2.3  Definition of Heavy Metals                                  10
     2.4  The Chemistry of Several Heavy Metals and Their Toxicity    12
          2.4.1 Silver (Ag)                                           15
          2.4.2 Mercury (Hg)                                          16
          2.4.3 Arsenic (As)                                          17
          2.4.4 Cadmium (Cd)                                          19
          2.4.5 Lead (Pb)                                             20
          2.4.6 Copper (Cu)                                           21
     2.5  Biochemistry of Heavy Metals                                21
          2.5.1 Heavy Metals in Soils and Plants                      21
          2.5.2 Heavy Metals in Animals                               23
     2.6  Uses of Heavy Metals                                        24
     2.7  Determination/Bioassay of Heavy Metals in the Environment   27
          2.7.1 Classical Bioassay/ Bioindicator                      29
          2.7.2 Modern Bioassay/ Bioindicator of Heavy Metals         30
          2.7.3 Microtox™ Bioluminescence Assay                       32
          2.7.4 Polytox™ Bioassay                                     33
          2.7.5 DeltaTox™ Bioassay                                    34
          2.7.6 Lux-Fluoro Test Bioassay                              34
          2.7.7 Enzyme Bioassay                                       36
          2.7.8 Metabolic/ Microbial Bioassay                         37
          2.7.9 Bioassay Using Antibodies                             45
          2.7.10 Biosensors for Heavy Metals Detection                46

3    MATERIALS AND METHODS                                            48
     3.1 Chemicals and Equipments                                     48
     3.2 Preparation of Solutions                                     48
         3.2.1 MTT Dye Stock Solution                                 48

                                   xii
           3.2.2   Heavy Metals Stock Solutions                             48
           3.2.3   Pesticides and Miscellaneous Xenobiotics Stock           49
                   Solutions
    3.3    Sample Collection                                                49
    3.4    Isolation and Culture of Bacteria                                51
    3.5    MTT Assay of Bacterial Inhibition Studies                        52
           3.5.1 Preliminary Screening of Bacterial Respiration Inhibited   52
                   by Divalent Cations and Heavy Metals
    3.6    Effect of Different Stages of Microbial Growth on Inhibitory     54
           Effect of Heavy Metals
    3.7    Determination of the IC50 Value of the Mercury and Silver        55
    3.8    Effect of Different Buffers System                               56
    3.9    Interfering Effects of Other Xenobiotics on Selective Bacteria   57
    3.10   Preliminary Testing on Water Samples from Polluted Areas         58
    3.11   Identification of Bacteria                                       59
           3.11.1 Biochemical Test Using Microbact™ Kit                     59
           3.11.2 Partial Sequence of 16S rRNA for the Identification of    61
                   Bacteria

4   RESULTS AND DISCUSSION                                                  68
    4.1  Sample Collection                                                  68
    4.2  MTT Assay of Bacterial Inhibition Studies                          70
         4.2.1 Preliminary Screening of Bacterial Respiration Inhibited     70
                 by Divalent Cations and Heavy Metals
    4.3  Effect of Different Stages of Microbial Growth on Inhibitory       74
         Effect of Heavy Metals
    4.4  Determination of the IC50 Value of Mercury and Silver              83
    4.5  Effect of Different Buffers System                                 89
         4.6     Interfering Effect of Other Xenobiotics on the MTT         92
                 Assay by Isolate SC27 and S8
         4.6.1 Interfering Effects of Xenobiotics on Isolate SC27           92
         4.6.2 Interfering Effects of Xenobiotics on Isolate S8             96
    4.7  Preliminary Testing on Water Samples from Polluted Areas           100
    4.8  Identification of Bacteria                                         104
         4.8.1 Colony Examination of Isolate SC27 and S8                    104
         4.8.2 Biochemical Test Using Microbact™ Kit                        107
                 4.8.2.1 Identification of Isolate SC27 and S8              107
         4.8.3 Partial Sequence of 16S rRNA for the Identification of       111
                 Bacteria

5   CONCLUSION                                                              123

    REFERENCES                                                              125
    APPENDICES                                                              136
    BIODATA OF THE AUTHOR                                                   146




                                    xiii
                                 LIST OF TABLES



Table                                                                           Page

1       Summary of heavy metals data for the west coast of Peninsular            9
        Malaysia in 1992

2       Malaysia: Status of Marine Water Quality, 2003                           10

3       Characterization of heavy metals                                         11

4       Classification of elements according to toxicity and their uptake        12

5       Formulae of commonly occurring arsenicals                                18

6       LC50 of heavy metals which inhibits the reduction of tetrazolium dye     32
        MTT by R. meloliti

7       Location of sample collection, pH of the soil, sample type, GPS          69
        location and total number of isolates from each location

8       The inhibition of MTT dye reduction at different periods of bacterial    76
        growth by different concentrations of heavy metal

9       The percent inhibition of the MTT dye reduction of Hg and Ag on          84
        Isolate SC27 and S8 respectively

10      The sensitivity of isolate SC27 and S8 to Hg and Ag respectively     85
        in comparison to immobilized urease, free urease, Microtox™, Daphnia
        magna and fish bioassay (rainbow trout)

11      Locations of water samples tested taken from Pulau Pinang river          100

12      Microscopic and macroscopic observation of isolate SC27 and S8           105

13      Biochemical test result using Microbact™ 24E (12A+12B) kit for           107
        isolate SC27

14      Biochemical test result using Microbact™ 24E (12A+12B) kit for           109
        isolate S8




                                           xiv
                                  LIST OF FIGURES



Figure                                                                              Page

1        The sources of heavy metals contamination in the environment                4

2        Disposition of metals in human                                             14

3        Molecular structure of MTT                                                 40

4        NADH assay principle with a tetrazolium salt and PMS                       42
         (phenozine methosulfate) as an electron carrier

5        Diagram the flow of NADH production in the glycolysis and                   43
         Kreb’s cycle to the electron transport chain

6        The inhibition of the reduction of the MTT dye by isolate SC27 in          77
         Hg using five different growth periods

7        The inhibition of the reduction of the MTT dye by isolate S8 in Ag         79
         using five different growth periods

8        The inhibition of the reduction of the MTT dye by isolate S7 in Ag          80
         using five different growth periods

9        The inhibition of the reduction of the MTT dye by isolate S1 in Ag          81
         using five different growth periods

10       The inhibition of the reduction of the MTT dye by isolate K104 in Ag       82
         using five different growth periods

11       Inhibition effect of isolate SC27 by Hg at eight hours bacterial growth     87
         as measured using the MTT assay

12       Inhibition effect of isolate S8 by Ag at 12 hours bacterial growth as      88
         measured using the MTT assay

13       Effect of different buffers system on inhibition of MTT reduction          90
         isolate SC7 by Hg at the final concentration of 0.3 mg/L

14       Effect of different buffers system on inhibition of MTT reduction          91
         isolate S8 by Ag at the final concentration of 0.2 mg/L

15       Effects of inhibition of isolate SC27 respiration by heavy metals at the   93
         final concentration of 5 mg/L using MTT assay

16       Effects of inhibition of isolate SC27 respiration by xenobiotics at the    94
         final concentration of 0.4 % using MTT assay

                                            xv
17   Effects of inhibition of isolate SC27 respiration by pesticides at the   95
     final concentration of 4 mg/L using MTT assay

18   Effects of inhibition of isolate S8 respiration by heavy metals at the   97
     final concentration of 5 mg/L using MTT assay

19   Effects of inhibition of isolate S8 respiration by xenobiotics at the    98
     final concentration of 0.4% using MTT assay

20   Effects of inhibition of isolate S8 respiration by pesticides at the     99
     final concentration of 4 mg/L using MTT assay

21   The inhibition studies on pre-treated water samples from polluted        102
     areas by isolate SC27 and S8 for the detection of Hg and Ag
     respectively using MTT assay

22   Concentrations of heavy metals taken from Pulau Pinang as determined 103
     using AES (Perkin Elmer Optima 3000)

23   Photomicrograph of Gram-negative rod isolate SC27 by observation     106
     under light microscope 1000 x magnification (Olympus BX40.F4, Japan)

24   Photomicrograph of Gram-neagtive rod isolate S8 by observation       106
     under light microscope 1000 x magnification (Olympus BX40.F4, Japan)

25   Extraction of genomic DNA from bacterial isolate SC27 and S8             113

26   16S rRNA gene (~ 1500 bp) of bacterial isolate SC27 and S8 gene          115
     amplified via PCR

27   The region of homology between the forward and reverse complement         117
     of isolate SC27

28   The 16S rRNA sequences of isolate SC27                                    118

29   The 16S rRNA sequence of isolate SC27 and its accession number as         119
     deposited in GenBank

30   Phylogenetic tree of newly isolated bacteria for bioindicator of Hg      122




                                        xvi
                LIST OF ABBREVIATIONS




%       percent

°C      degree Celsius

    g   microgram

    l   microliter

bp      basepair

DNA     deoxyribonucleic acid

dNTP    deoxyribonucleic triphosphate

EDTA    ethylenediaminetetraacetic acid

EGTA    etilena glikol-bis-(β-aminoetilether)N,N,N’N’-acid tetraacetic)

EtBr    ethidium bromide

g       gram

GPS     Global Positioning System

HCl     hydrochloric acid

kb      kilobase

L       liter

M       molar

mg      milligram

min     minute

mL      milliliter

mM      milimolar

MTT     3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide

NCBI    National Center for Biotechnology Information

PBS     phosphate buffered saline

                            xvii
PCR   polymerase chain reaction

pH    - log concentration of H+ ion (Puissance hydrogen)

RNA   ribonucleic acid

rpm   revolutions per minute

Taq   Thermus aquaticus

TBE   tris-borate-EDTA

U     units

v/v   volume/volume

w/v   weight/volume

x     times




                         xviii
                                    CHAPTER 1



                                 INTRODUCTION



Heavy metals are considered to be one of the main pollutants in the environment

since they have a significant effect on ecological quality. Unlike organic pollutants,

heavy metals cannot be detoxified via degradation and thus they persist in the

ecosystem. The aquatic ecosystem is the last recipient for these pollutants where

they would accumulate and become concentrated as they go up into the food chain,

causing a serious potential risk to human health as humans complete the chain as the

terminal consumer (Tüzen, 2003).



Past studies have shown that heavy metals are produced from industrial activities,

urbanization, transportation, agricultural activities and other sources. Heavy metals

are roughly defined as metals having an atomic density of over 6 gcm-3. However,

the term is indistinguishable and includes metals and some metalloids, such as

arsenite (Bruins et al., 2000). As an example of famous cases of heavy metal

pollutions are mercury poisoning occurred in Minamata, Japan which is caused fatal

to the infants and childrens (Kjellstrom et al., 1986; Mendola et al., 2002) and Itai-

itai disease represents a tragic case of cadmium poisoning (Jarup et al., 2000).



In Malaysia, with the rapid growth of industries, contamination by heavy metals

could not be avoided. Many cases of heavy metals toxicity have been reported. In

response, the discharge limit of these metals under the Environmental Quality

(Sewage and Industrial Effluents) Regulations, 1979 was legislated. Specifically, the


                                           1
standard B for Cd is 0.02 mg/L, 0.50 mg/L for Pb, 1.0 mg/L each for Zn, Cu and Cr,

Hg is 0.005 mg/L and As is 0.10 mg/L. Because Hg, Ag, Cd, As, Pb and Cu are

hazardous heavy metals that have no beneficial function in the biological system and

are highly toxic, these heavy metals were selected for the study. This study was

carried out to determine the potential of using bacteria as an indicator for the

presence of heavy metals.



Biosensors and bioindicators provide rapid detection of heavy metal compounds

without the disadvantages of the classical methods such as atomic absorption

spectroscopy and potentiometric method. A bioindicator can be defined as a plant,

animal or microorganisms species whose function, population or status that can be

reliably used to indicate changes in the environment such as chemical, physiological

or behavioural and thus determine the ecosystem level (Vedagiri, 1998). Several

configurations have been described in the past including enzyme biosensors, whole

cell biosensors and genetically modified biosensors for the determination of heavy

metals   (Krawczyn’ski      et   al.,   2000).   Other   than   that,    the   microbial

bioindicator/bioassay provides a simpler and less expensive method, since no

specialized equipment is required.



In this study, the focus is on the toxicity of heavy metals to microbes where the

microbial inhibition was detected using MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assay. Thus, the usage of this simple colorimetric

microbial bioindicator of heavy metals could be simplified down to the use of a

colour chart to qualitatively detect heavy metals. This potentially could be used on

site to monitor the health of an environment or ecosystem.              Furthermore, the


                                            2
information obtained from this study also can be used for the development of a heavy

metals bioindicator/bioassay using microbes. Otherwise, this will enable us to

monitor heavy metals contamination in the environment especially in Malaysia.



The objectives of this study are:



   1. To isolate soil bacteria from different areas in Peninsular Malaysia.

   2. To screen for bacteria by measuring the inhibition of their respiratory activity

       in the presence of heavy metals measured using the MTT assay.

   3. To determine the critical concentrations of heavy metals that inhibits

       reduction of MTT dye by 50% (IC50).

   4. To carry out preliminary field studies on the efficacy of the microbial

       bioassay.




                                         3
                                   CHAPTER 2



                            LITERATURE REVIEW



2.1    Heavy Metal Pollutions.



The presence of heavy metals in potable water, domestic wastewater, industrial

effluents and receiving water is a matter of serious concern because of the toxic

properties of these metals. The level of contamination has reached a point that it is

no longer a question of whether one has been exposed to heavy metals but rather the

level of exposure. Figure 1 shows the sources of heavy metals contamination in the

environment.




Figure 1: The sources of heavy metals contamination in the environment
(Kakkar and Jaffery, 2005).




                                         4
Soil contamination by heavy metals is particularly problematic because they are not

degraded in soil and cannot be permanently eliminated. For example, a very serious

heavy metal pollution incident was occurred in Minamata Bay and Niigata, Japan

during the 1950s and 1960s where of mercury (Hg) was released in effluents from a

plastics manufacturing plant and this become a problem when were ingested by fish.

People eating the fish developed serious neurological maladies and in some cases

caused fatal. At Minamata, several years elapsed between the appearance of

neurological symptoms and identification of the causative agent (Marsh, 1994).

Infants and childrens were particularly vulnerable to this “Minamata disease”. All

the children had severe disabilities including mental retardation, cerebral palsy and

seizures (Kjellstrom et al., 1986; Mendola et al., 2002).



On the other hand, Itai-itai disease is caused by cadmium (Cd) poisoning. It was

initially diagnosed in farm workers in northern Japan who regularly drank water

from the Junzu river and consumed rice grown in paddy fields irrigated by the same

river. The disease was named Itai-itai (“ouch-ouch”) due to the pain caused by the

decalcification and final fracturing of bones symptomatic of Cd poisoning. Up until

1965, it was estimated that 200 people, mostly farm women over middle age had

suffered from the disease and half of these cases had proved fatal (Jarup et al., 2000).



Lead (Pb) was a problem in urban areas with dense traffic because leaded gasoline

was used for many years. In Canada, it has been estimated that drinking water in

hard-water areas can contribute 15% and in soft-water areas 46% of the Pb in the diet

(Links et al., 2001). Due to concerns about human health and the environment, Pb

was phased out of gasoline from 1973 to 1990. While it was being phased out, Pb


                                           5
levels in the atmosphere dropped significantly and they continue to decrease today

(Bellinger and Needleman, 1992).



Meanwhile, exposure of millions of people to arsenic-contaminated water from hand

tube wells is a major concern in many Asiatic countries (Chatterjee et al., 1995).

The largest population at risk by groundwater arsenic (As) contamination is in

Bangladesh (Chowdhury et al., 2000; Ahsan et al., 2000), West Bengal in India

(Bagla and Kaiser, 1996; Rahman et al., 2001, 2002) and other countries including

Cambodia, Myanmar, Nepal and Vietnam (Berg et al., 2001). Nearly one quarter of

the 11,000 villagers surveyed in Bangladesh suffered skin lesions. Researchers

observed that As concentrations in excess of 300 g/L were associated with arsenical

lesions, which depended a lot on nutritional conditions, water consumption and other

susceptibility factors (Breslin, 2000). Thousands more people in Taiwan, Mongolia,

Chile and Argentina suffer from the same health problem. Concerns about the

cancer risk from As contaminated drinking water caused the WHO to revise the

guideline from 50 to 10 parts per billion (ppb) in 1993 (Kakkar and Jaffery, 2005).



2.2    Heavy Metals in the Malaysian Environment.



Malaysia has emerged as one of the fast-track developing countries in the last 20

years. The industrial sector has contributed much to the country’s development.

But, as in other developing countries, Malaysia is not exempt from facing problems

related to environmental pollution. Thus, the Environmental Quality (Sewage and

Industrial Effluents) Regulations, 1979 is the most referred law related to heavy

metals pollution of the environment. The Third Schedule of the regulations is shown


                                          6
in Appendix I. There are two different standards, namely A and B. Standard A

applies to all areas upstream of water intakes whereas Standard B applies to all areas

downstream of all water intakes.       A survey by the Malaysian Department of

Environment showed that 10.4% of the 420, 000 tonnes of scheduled wastes

generated in 2001 contained heavy metals mainly in the form of sludges (DOE,

2001).



In Malaysia, contaminated land can be found at many places such as motor

workshops, petrol stations, fuel oil depots, railway yards, bus depots, landfills,

industrial sites and sites with underground storage tanks. They become a problem

because the used lubricating oil contains other contaminants especially heavy metals

such as As, Ba (barium), Cd, Cr (chromium), Pb, Fe (iron) and Zn (zinc). Spent

diesel is usually contaminated with carbon particles and heavy metals (Malaysia

Environmental Quality Report, 2002).



In 1997, the Department of Environment, Malaysia revealed that 25 rivers in

Malaysia were considered to be highly polluted.         The number has increased

compared to 13 rivers in 1996. The polluted rivers include Sungai Merbok, Kedah;

Sungai Juru, Sungai Perai and Sungai Jawi, Pulau Pinang; Sungai Raja Hitam/

Manjung, Perak; Sungai Buloh, Sungai Sepang and Sungai Lukut Besar, Selangor;

Sungai Duyong, Melaka; Sungai Air Baloi, Sungai Kempas, Sungai Danga Sungai

Rambah, Sungai Tukang Batu and Sungai Sedili Kecil, Johor; Sungai Balok, Sungai

Cherating, Sungai Ibai and Sungai Landas, Pahang and Sungai Batang Kemena Niah,

Sarawak (Utusan Malaysia, 1998). Meanwhile, the four most polluted rivers that

have been reported in the 2004 Malaysian Environment Quality Report are


                                          7
Menggaris in Sabah, Jelutong in Pahang and Buluh and Tukang Batu in Johor which

are identified as their waters described as unsafe for drinking and irrigation (News

Strait Times, 2005).



In Terengganu, there is a small amount of Hg in the gas produced from the offshore

field of the oil and gas industry. These problems also occur in Vietnam, Indonesia

and some of them are discharging other toxic substances such as Ba, Cu (copper) and

Zn into the sea (DOE, 2001).



Generally, pollution in the Straits of Malacca is linked to high population density,

heavy industrialization and changing land use patterns on the west coast of

Peninsular Malaysia. Industrial, domestic and agricultural sources are the major

contributors of the pollution in the Straits. Industrial activities are the main

contributor of commonly generated heavy metals such as Hg, As, Cd, Cr and Pb in

west coast of Penisular Malaysia (Table 1). States with high industrial development

(Pulau Pinang, Selangor, Johor and Perak) have high levels of heavy metals (Pb and

Hg) contamination (DOE, 1994). Table 2 shows the status of marine water quality in

Malaysia in 2003 showing parameters exceeding interim standards (%).



As a consequence all of these contributors of pollution in Malaysia, the government

will spend more than RM20 million in year 2006 to clear rivers of these wastes

(News Strait Times, 2006).




                                         8
Table 1: Summary of heavy metals data for the west coast of Peninsular
Malaysia in 1992 (Dow, 1995).


Metals         State with samples     Highest       Lowest     No. of samples
(limit allowed above standard     reported level   reported   above standard
by DOE
standard A)

Arsenic(As)     Johor (entire state)   1.83          0.037          2
0.05 mg/L       Perak                  0.38           ND           31

Lead (Pb)       Perak                  0.33          0.14          43
0.09 mg/L

Chromium (Cr) Perak                    0.62          0.02           8
0.1 mg/L

Mercury (Hg)     Johor                  2.01         0.2           23
0.001 mg/L       Pulau Pinang           0.05           ND          66
                 Perak                  0.08           ND          20
                 Kedah                  0.04            0          14
                 Melaka                 0.018        0.003          6
                 Negeri Sembilan        0.004           0          16

Cadmium (Cd)     Pulau Pinang           0.2           0             2
0.005 mg/L       Johor                  0.07          ND            4
                 Selangor               0.27          0             1
                 Kedah                  0.01          ND            6
                 Perak                  0.02          ND           33

Note:
ND = Not Detected




                                       9
Table 2: Malaysia: Status of Marine Water Quality, 2003 (Malaysia
Environmental Quality Report, 2003).

State            No. of       No. of Parameter exceeding interim standard (%)
                Station       sample      Cd       Cr Hg Pb          As    Cu
Perlis             2            24         0       0    25     13     0     42
P. Langkawi        7            42         0       0    12      0     0     81
Kedah              3            12         0       0      0     0     0     92
P. Pinang         23           136         0       0    13      1     1     82
Perak             13            52         0       0      5    33     0       0
Selangor          14            69         0       0      4     0     0       0
N. Sembilan       13            77         0       0      7     0     0       0
Melaka             9            54         0       0      0     4     6       0
Johor             45           151         0       0      0     0     1       0
Pahang            11            80         0       0      0     1     0       1
Terengganu        19            76        58       1     18    70     0     58
Kelantan          10            30        43        -     0    73     0     63
W.P Labuan         5            15          -      -      -    -     -      -
Sabah             26            65          -       2     94 13       0       0
Sarawak           19            89          -       -       0 16      0       3
Malaysia (total) 219           972
Average (%)                               6.7      0.2 12.7 16.1 0.5 30



2.3     Definition of Heavy Metals.



Various classifications of heavy metals have been made based on either their

chemical or biological properties. The term “heavy metals” has sometimes been

restricted to metallic elements, which normally have atomic weights between of 63

and 201 and specific gravity above 5.0 and comprise approximately 65 metallic

elements. A more general classification is that they are metals or metalloids found in

the periodic table of elements from Group IIA through VIA, including the semi-

metallic elements: boron (B), As, selenium (Se) and tellurium (Te)(OSHA, 2000).



According to the environmental encyclopedia (Cunningham et al., 1998) heavy

metals are generally defined as environmentally stable elements of high specific

gravity and atomic weight.     They have such characteristics as shine, ductility,
                                         10
malleability and high electric and thermal conductivity. The category “heavy metal”

is somewhat subjective and highly non-specific because it can refer to approximately

80 of the 130 elements in the periodic table. The characterization of heavy metals

due to its atomic number and weight is shown in Table 3.



Table 3: Characterization of heavy metals (Periodic Table).

Heavy metals           Groups in Periodic          Atom              Atomic
                       Table                       number            weight
Cr                      VIb                          24                52
Mn                       VIIb                        25                 54.9
Co                      VIII                         27                 58.9
Ni                       VIII                        28                 59.71
Cu                        Ib                         29                 63.54
Zn                       IIb                         30                 65.37
As                       Va                          33                 74.92
Se                       VIa                         34                 79
Ag                        Ib                         47                108
Cd                        IIb                        48                112.40
Hg                        IIb                        80                201
Pb                       IVa                         82                207.19




Some have classified metalloid and transition metals as heavy metals based on their

importance in biological functions. Transition metals are essential in low

concentrations for biological functions but are toxic if present in high concentrations.

Example of transition metals include Fe, Cu, cobalt (Co) and manganese (Mn). On

the other hand, metals in the metalloid group such as Hg, Pb and As, have no known

biological functions and are toxic to cells even in low concentrations (Badri, 1987).

The classification of elements in the periodic table according to the toxicity and their

uptake where easily exposed to organisms or not is presented in Table 4.
                                          11
Table 4: Classification of elements according to toxicity and their uptake (Badri,
1987).

Not critical                    Toxic, partially dissolved    Very toxic and easily
                                or easily exposed             exposed

Na C F K S Sr H Cl               Ti Ga Hf Rh Nb Ir           Be As Au Cu Pd Pb

P     Li Mg Al O Br Si           La Zr Os Ta Ru Re           Co Se   Hg Zn Ag Sb

Fe Rb Ca N                       W                           Ni Te Tl Pt Sn Cd Bi




2.4       The Chemistry of Several Heavy Metals and Their Toxicity.



Identification of heavy metals is based on their chemistry. Each heavy metal has

their own characteristics and their properties make them toxic to organisms if in high

concentrations. In this study, six heavy metals were chosen due to their prevalence

in the environment. These heavy metals are silver (Ag), mercury (Hg), arsenic (As),

cadmium (Cd), lead (Pb) and copper (Cu) and their characteristics will be further

elaborated below.



The ultimate cause for concern about heavy metals in the environment is their

extreme toxicity towards humans. Toxicity of metals is a function of:

      •   Specific metal.

      •   Form of metal.

      •   Level of exposure.

      •   Period of exposure.

These factors play a major role in the ultimate sign of toxicity. Most of the heavy

metals bind to the sulfhydryl groups thus inhibiting enzyme activity, disrupting

                                             12
cellular transport and causing changes in protein functions (Kakkar and Jaffery,

2005).



In classical toxicology, a compound is considered toxic if it damages living

organisms and this defined in terms of the median lethal dose; LD50, i.e. the amount

of a compound, which causes the death of 50% of the test animals. It is commonly

expressed as the amount of the toxic compound per 100 grams (eg, milligrams or

kilogram) of the body weight of the test animal. LC50 is the concentration of the

toxic compound which kills 50% of the test animal in a given time usually in hours

(Vedagiri, 1998).



In addition, there are also some other toxicity dose terms such as LD01 which stands

for lethal dose for 1% of the animal test, LDL0 which is the lowest dose causing

lethality to the test animal and also TDL0 which is the lowest dose causing toxic

effect to the animal test population (Vedagiri, 1998).



The toxicity of any chemical depends on a number of factors including its

absorption, distribution, metabolism and excretion. Figure 2 depicts the deposition

of metals in humans.




                                          13
       Figure 2: Deposition of metals in human (Kakkar and Jaffery, 2005).



Different levels of exposure to heavy metals can lead to three different effects. The

first is called acute effects in which symptoms appear immediately after exposure.

These effects are generally caused by fairly high concentrations of heavy metals

during a short exposure period. The second is called chronic effects in which the

effects are delayed but long-lasting and may occur months to years after exposure.

Generally, this is a result of low-level exposure over a long period. Lastly, lethal

effects can be defined as responses that occur when physical or chemical agents

interfere with cellular and subcellular processes in the organism at the high level thus

causing death (Kakkar and Jaffery, 2005).




                                          14
2.4.1   Silver (Ag).



Silver (Ag) belongs to the transition group 1B with an atomic number of 47. Ag is a

white, ductile metal occurring naturally in the pure form and in ores. Ag has the

highest electrical and thermal conductivity of all metals. Some Ag compounds are

extremely photosensitive but are stable in air and water except for being readily

tarnished when exposed to sulfur compounds. It is also a normal trace constituent of

many organisms (Malik, 2004).



There are several compounds of Ag which are potential explosion hazards; silver

acetylide (Ag2C2) is sensitive to explode on contact and silver azide (AgN3) explodes

spontaneously under certain conditions (Irwin, 1997). Soluble Ag salts are in general

more toxic than insoluble salts. In natural waters, the soluble monovalent (Ag+)

species is the form which is of environmental concern. Signs of chronic Ag

intoxication in tested birds and mammals included cardiac enlargement, vascular

hypertension, hepatic necrosis, anemia, lowered immunological activity, altered

membrane permeability, kidney pathology, enzyme inhibition, growth retardation

and a shortened life span (Vangronsveld, 1994)



In humans, skin contact with Ag compounds may cause mild allergic reactions such

as rash, swelling, and inflammation while industrial and medicinal exposures to Ag

may cause lesions of the kidneys and lungs, and arteriosclerosis. Otherwise, the

colloidal Ag compounds may interfere with nasal ciliary activity and exposure to

dust containing high levels of silver compounds, such as silver nitrate or silver oxide,

may cause breathing problems, lung and throat irritation, and stomach pain. The


                                          15
major problem in humans arising from overexposure to silver is called argyria,

which is characterized by blue-gray colouration of the skin, mucous membranes and

internal organs (Irwin, 1997). According to World Health Organization in 1987, a

continuous daily dose 0.4 mg of silver intake may produce argyria. This compound

is also a threat not only to human but to fish because it is widely distributed in the

water systems.



2.4.2   Mercury (Hg).



Mercury (Hg) is naturally present in the earth’s crust and exists as a silvery-grey

liquid (quicksilver) at normal temperature and pressure. Its symbol corresponds to

the Latin word hydragyrum. Weathering and degassing through volcanoes and by

evaporation from oceans releases Hg into the geochemical cycle (Steinnes 1990;

Boening 2000). Soil with concentrations below 0.05 mg Hg kg-1 is considered as

clean soil in Finland (Puolanne et al., 1994).



This element can be divided into two major categories, organic and inorganic.

Inorganic Hg includes the elemental silvery liquid metal (Hgo) (melting point, 38oC;

boiling point, 357oC) as well as mercurous ion (Hg+), mercuric ion (Hg2+) and their

compounds. Organic mercury includes chemical compounds which contain carbon

atoms that are covalently bound to a mercury atom, such as methylmercury (CH3-

Hg+). The mercurous salts are less soluble than the mercuric salts and are

consequently less toxic (Cunningham et al., 1998).




                                          16
Over the centuries, the symptoms of inorganic Hg poisoning were well documented

by the exposure of miners and industrial workers to Hg accumulation in their brains,

kidneys and livers. Once inorganic Hg is absorbed, it accumulates in the kidney and

liver, with the renal cortex containing the highest concentrations. However, the

effects of organic alkyl mercurials such as methylmercury were more severe (Kakkar

and Jaffery, 2005).



2.4.3   Arsenic (As).



Arsenic (As) is an element having an atomic number of 33 and an atomic weight of

74.92. It is listed by the U.S. Environmental Protection Agency as a hazardous

substance (Hazardous waste numbers P010, P012) and as a carcinogen. This

metalloid occurs all over the place in nature and belongs to the VA elements group

(N, P, As, Sb and Bi) and is mostly insoluble in water (Cunningham et al., 1998).



In nature, it is found in areas where silver or antimony is deposited. It occurs

naturally in the environment where it is released from As-bearing rocks through

weathering, by volcanic action or during mining. Human activities such as coal

burning and refining of sulphide-rich minerals add arsenic to the atmosphere

(Wildfang et al., 2000).



Arsenic can exist in three possible oxidation states: elemental (0), trivalent (+3, e.g.

arsenite; or -3, e.g. arsine) and pentavalent (+5, e.g. arsenate). In general, inorganic

forms of arsenic (e.g. arsenite or arsenate) are more toxic than organic forms, for




                                          17
example, methylarsonate, dimethylarsinate or arsenobetaine (Wildfang et al., 2000).

The chemical formulae of some common arsenicals are shown in Table 5.



Table 5: Formulae of commonly occurring arsenicals (Wildfang et al., 2000).

         Arsenicals                                 Formulae

Arsenite                                            AsO2-, AsO33-
Arsenate                                            H2AsO4-, HAsO42-, AsO43-
Arsenous acid                                       H3AsO3
Arsenic acid                                        H3AsO4
Arsenic trioxide                                    As2O3
Arsenic pentoxide                                   As2O5
Gallium arsenide                                    GaAs
Indium arsenide                                     InAs
Arsine                                              AsH3
Monomethylarsonic acid (MMA)                        CH3AsO(OH)2
Dimethylarsinic acid                                (CH3)2AsO(OH)




The toxicity of As depends on the state of oxidation. Absorbed As affects tissues

rich in oxidative systems, such as the alimentary tract, liver, kidney, lung and

epidermis. Individuals consuming drinking water containing 0.41 mg/L of As were

found to have higher concentrations in the blood, hair and nails than an unexposed

population (Raven et al., 1998). Other clinical signs in acute cases include fever,

emaciation, anorexia, depression and hair loss (Kakkar and Jaffery, 2005).




                                         18
2.4.4   Cadmium (Cd).



Cadmium (Cd) is a soft, ductible, silver-white metal that belongs together with zinc

and mercury in group IIB in the Periodic Table. It has relatively low melting

(320.9oC) and boiling points (765oC) and a relatively high vapour pressure (WHO,

2000). In the air, Cd is rapidly oxidized into cadmium oxide. However, when

reactive gases or vapor such as carbon dioxide, water vapour, sulfur dioxide, sulfur

trioxide or hydrogen chloride are present, it will produce cadmium carbonate,

hydroxide, sulfite, sulfate or chloride respectively. These compounds may be formed

in chimney stacks and emitted to the environment (Kakkar and Jaffery, 2005).



Cadmium is a relatively rare element (0.2 mg/kg in the earth’s crust) and is not found

in the pure state in nature. It occurs mainly in association with the sulfide ores of Zn,

Pb and Cu. It was thought to be less hazardous after Hg, Pb, As and cynide. It is an

especially toxic heavy metal that is very mobile in soil. Once adsorbed, Cd is

persistently retained in human and accumulates mainly in the liver and kidney with

the concentration being directly related to the oral dose administered (Kakkar and

Jaffery, 2005). The kidney is the critical organ after long-term occupational or

environmental exposure to Cd. Otherwise, smoking is the largest source of Cd for

people. Cd is absorbed by inhaling cigarette smoke. Once it enters the body, it stays

there for many years (Malik, 2004).




                                           19
2.4.5   Lead (Pb).



Lead (Pb) is a member of group IVB in the Periodic Table of the elements and one of

the oldest metals known to humans. Its compounds were used by Egyptians to

varnish pottery as far as back 7000 B.C (Cunningham et al., 1998). It is a soft, grey,

heavy metal produced by roasting the ore galena [lead sulfide (PbS)] and has four

natural isotopes, three of which are the end products of the radioactive decay of

uranium (U) and thorium (Th) (David, 1998).



Lead can exist in several valence states such as Pb(0), Pb(I), Pb(II) and Pb(IV). It

naturally occurs as the sulfide, carbonate, sulfate and chlorophosphate. In its

elemental state, Pb is a dense (11.3 pg/cm3) blue-grey coloured metal which melts at

37oC and boils at 1744oC (WHO, 1995).



The human toxicology and epidemiology of Pb exposure has been reviewed by the

World Health Organization, the Department of Health and Society Security, United

State and the Royal Commission on Environmental Pollution, United Kingdom. Pb

is a strong toxicant that harmfully affects many systems in the body. Acute toxicity

of Pb is rare but may occur with contaminated food and drink. Other effects include

reduced life span of erythrocytes, impairment of proximal tubular function, reduced

birth weights, reduced sperm count and motility. It is also implicated in miscarriages

and stillbirths (Borja-Aburto et al., 1999). It was reported about 50% of Pb is

absorbed with inhalation of dusts, 10-15% absorbed orally, out of which 90% is

distributed to bones (Links et al., 2001).




                                             20
2.4.6    Copper (Cu).



Copper (Cu) is a reddish-brown, soft and ductile metal with an atomic number of 29.

It has an atomic weight of 63.546 and widely distributed in nature in ores containing

sulfides, arsenides, chlorides and carbonates. It occurs naturally in rock, soil, water,

sediment and air. Cu also occurs naturally in animals and plants (Cunningham et al.,

1998).



Copper is necessary for good health. However, excess ingestion can harm human

life and has toxic effects. Acute over exposure causes an immediate metallic taste,

followed by epigastric burning, nausea, vomiting and diarrhea. Other symptoms

include ulcers and other damage to the gastrointestinal tract, jaundice and

suppression of urine production. Fatal cases often include secondary effects, such as

hypertension, shock and coma. If the Cu present in drinking water is more than

normal levels, people who drink it may develop liver dysfunction and suffer diarrhea,

vomiting, stomach cramps and nausea (Brierley, 1990).



2.5      Biochemistry of Heavy Metals.

2.5.1    Heavy Metals in Soils and Plants.



Heavy metals are natural components of soil and introduced into the ecosystem by

the manufacture and use of materials containing heavy metals as well as the disposal

of these wastes.    Meanwhile, most elements are only present in minimal and

insignificant eco-toxicological concentrations in undisturbed locations in certain

areas.


                                          21
The accumulation of contaminants is aided by the capability of soil to bind with clay

minerals or organic substances. Heavy metals may occur in soils in the following

chemical forms:

   i)      in ionic or complexed form in the soil solution.

   ii)     as readily exchangeable ions in organic and inorganic exchange active

           material.

   iii)    as more firmly bound ions in the exchange complexes.

   iv)     as chelated ions in an organic or organomineral complex.

   v)      incorporated in precipitated sequioxides or insoluble salts.

   vi)     incorporated into microorganisms and their biological residues.

   vii)    held in the crystal lattice structure of primary and secondary minerals.

                                                              (Matthews, 1983)



According to Kabata, Pendias and Pendias (1984), the uptake of heavy metals is

lower in alkaline soils than acid soils. The majority of heavy metals is present as

cations in soils and partially depends on the density or charge at the soil colloidal

surface. Organic matter have many active sites where heavy metals can be trapped.

Increasing the organic matter content will increase absorption of heavy metals in the

soils (Alloway, 1990).



In contrast, plants take up metal trace elements from the soil through the root. Some

of the trace elements are essential as plant nutrients but plants growing in a polluted

environment can accumulate metals at high concentrations, causing a serious risk to

human health when the plant enters the food chain (Qian et al., 1996; Wenzel and

Jockwer, 1999). It is known that high concentrations of metals in plant can interfere


                                          22
with physiologically important functions of the plants which can cause imbalance of

nutrients and have harmful effects on the synthesis and functioning of biologically

important compounds such as enzymes, vitamins, hormones, etc. (Vangronsveld and

Clijsters, 1994; Luo and Rimmer, 1995). Therefore, soils containing heavy metals

could serve as the first indicator for heavy metals transitioning to higher level of

trophic levels in the soil-plant/human system (Kubota et al., 1992).



Factors that influence the uptake of heavy metals by plants are organic matter

contained in the soils, pH and the presence of other ions in solution. Heavy metals

found in plants are dependent on the uptake rate and the plant species. Therefore, if

there are two plant species living under the same condition, the heavy metals content

maybe different (Haque and Subramaniam, 1982).



2.5.2   Heavy Metals in Animals.



Several heavy metal pollutants are believed to have the ability to suppress the

immune system of marine mammals. Heavy metals, like Cd, Pb, and Hg are highly

toxic at relatively low concentrations and can accumulate in mammalian tissues over

long periods of time (Hu, 2002). Cd accumulates in the kidneys and liver of marine

mammals and with a half-life of decades, leaves the body extremely slowly. Kidney

damage and a decalcification of the skeleton are the serious chronic effects of high

cadmium exposure (Kakkar and Jaffery, 2005). Damage to the nervous system and

gastrointestinal symptoms are the main signs of Pb poisoning. Pb accumulates in the

liver, kidney, spleen, and skeleton. Once it has been integrated into the skeleton, it

takes several years to leave the body (Links et al., 2001). Hg is a nerve toxin and


                                          23
affects the brain, particularly in the growing fetus and the young, and it can also

damage reproduction in mammals (Bontidean et al., 2004).



Marine mammals are now well recognised to have the ability to de-methylate some

organic Hg. The liver is the most important accumulation site of Hg in both

pinnipeds and crustaceans (Hughes and Poole, 1989). Andre et al. (1990) showed

that in spotted dolphins, Stenella attenuata, three tissues (liver, skeletal muscle and

blubber) contained 95% of the Hg in the body. Accumulation of inorganic Hg in

crustaceans is usually related to age, and Law (1995) noted that the amount of

organic Hg in the livers of pilot whales, Globicephala sp., had been shown to be

negatively correlated with age and ranged from 3 to 62%. Muscle tissues in marine

mammals usually contain a high proportion of methylmercury such as in fin whales,

Balaenoptera physalus where has been found to be about 80% (Law, 1995).



An important aspect for consideration in sludge-soil-plant-animal relationships of

heavy metals is the concept of the so-called “soil-plant barrier”. Thus, Pb and Hg,

the metals most likely to cause acute toxicity to animals, do not appear to be

translocated by plants into leaves or shoots to any great amount. Unfortunately, the

“soil-plant barrier” does not protect animals from the toxic effects of metals such as

Cd, which is readily translocated by plants (Qian et al., 1996)



2.6    Uses of Heavy Metals.



Heavy metals have a huge variety of industrial applications which will to some

extent influence their appearance in wastewaters. Their widespread uses have


                                          24
resulted in environmental pollution. The uses of six selected heavy metals in this

study are described separately in paragraph below.



It has been estimated that approximately 70% of the total consumption of As is for

the manufacture of agrochemicals such as insecticides and herbicides. It is also

incorporated into paint pigments but this was discontinued when it was found that

poisonous arsine and trimethyl arsine gases formed in moist conditions (Wildfang et

al., 2000). In addition, wood preservatives, nonferrous alloys and battery grids also

contain arsenic as do fireworks, glass, ceramics and electronic products such as

semiconductors and photoconductors. Other than that, heavy industry employs

arsenic in the extraction of ores, manufacture of ammonia and sulfuric acid (Hakala

and Pyy, 1995).



Major sources of Cd contamination are the industrial production, consumption of Cd,

other nonferrous metals and the disposal of waste containing Cd (WHO, 1992).

Large quantities of Cd are mainly used in the production of nickel-cadmium batteries

or in welding. Pigments for paint are the next most common end use followed by

plastics manufacture especially as a polyvinyl chloride (PVC) stabilizer. Other uses

include alloys, solders, fungicides and chemicals in photograph, process engraving

and rubber curing (Jarup et al., 2000).



For centuries, Pb has been mined and used in industry and in household products.

Modern industrialization introduced the use of lead in mass-produced plumbing,

solder used in food cans, paint, ceramic ware and countless other products resulted in

a marked rise in population exposures in the 20th century (Hu, 2002). The principal


                                          25
use of lead is in the manufacture of batteries. Pb is also utilized for anti-knock

additions for gasoline, pigments, ammunition, solder and cable covering. Minor uses

include caulking compounds, piping and type metal. The current annual worldwide

production of Pb is approximately 5.4 million tons and continues to rise (Borja-

Aburto et al., 1999).



Mercury is a liquid with high thermal conductivity that has many unique properties

making it useful in many industrial applications. Mercuric salts have also been

extensively used by humans while mercuric sulphide has been used since the first

century as a remedy for skin and eye complaints. Metallic mercury and mercuric

chloride have been well known and used in medicine since the Middle Ages or even

earlier. More recent medical uses of mercurial compounds have included diuretics,

dental amalgam fillings and antiseptics (Hobman and Brown, 1997; Hobman et al.,

2000).



Silver has the highest thermal and electrical conductivity of any metal but it is too

expensive to be used as an electrical conductor. It is one of the most expensive

metals and valued as a penny metal. It also has been used to make jewellery and for

the reflective coating on mirrors. Beside that, it is also widely used in electroplating

photographic and ink manufacturing industries (Irwin, 1997). Ag was also used in

the Apollo space program and on Soviet spacecraft to purify drinking water and

wastewater.    In addition, numerous silver containing medications and dental

amalgam fillings have contributed to high levels of oral exposure. However, at

present these uses are banned. A few drops of silver nitrate are applied to the

conjunctiva of newborn infants to prevent ophthalmia neonatorum, a result of


                                          26
gonorrhea transmitted from the mother. Silver salt solutions and ointments are also

used to treat burns (Hu, 2002).



Copper has long been used in the development of human civilization. In the United

States, it has been used to make pennies but since 1981, new pennies have been made

from Zn with a thin Cu covering. The outer surface of the Statue of Liberty also is

made of Cu and this compound gives the statue its green colour (Haque and

Subramaniam, 1982). Otherwise, Cu is an essential trace element especially to

human nutrition because it plays a major role in enzyme functions. It is also required

along with Fe for synthesis of hemoglobin. Cu compounds are most commonly used

in agriculture to treat plant diseases like mildew for water treatment and as

preservatives for wood, leather and fabrics. Some paints contain Cu and this is really

useful on ship hulls to prevent fouling by marine organisms. It is also valued for its

high electrical conductivity and resistance to corrosion where it is used widely in

plumbing and electrical applications (Kakkar and Jaffery, 2005).



2.7    Determination/Bioassay of Heavy Metals in the Environment.



In Malaysia, heavy metal pollution has grown to a dangerous level due to their wide

usage. Thus, there is a need for simple and fast procedures to screen industrial

effluents or foodstuffs for the presence of toxic heavy metals. Bioindicators or

bioassays using growth inhibition of microorganisms and inhibition of enzyme

activity and enzyme biosynthesis are being developed and becoming increasingly

popular for the screening of the environmental toxicants. Some commercial test kits




                                         27
have even been developed for this purpose such as Microtox™ assay based on the

inhibition of light produced by bioluminescent bacteria (Guven et al., 2003).



Bioassay is defined as a biological system that could detect and quantify toxicants

(Jung et al., 1995), whereas, bioindicator is defined as one or more organisms,

population, community or ecosystem level of biological organization that indicate

toxicants. The responses of this system are linked to biological effects at one or

more of the organism, population, community or ecosystem level of biological

organization (McCarty et al., 2002).



The similarities between bioindicators and bioassays are that both of them are

involved in vivo experiments and are conducted to measure the effects of a substance

on a living organism. They may be qualitative or quantitative where the latter often

involve an estimation of the concentration or potency of a substance by measurement

of the biological response that it produces. For environmental testing, bioassays

provide an integrated picture of overall toxicity of an effluent or a sample of water,

sediment, or soil from a contaminated site.



There are several methods which have been used in the determination/bioassay of

heavy metals such as instrumental analysis, classical bioassays and modern

bioassays. Examples of the instrumental analysis are atomic absorption spectroscopy

(AAS), atomic flame spectroscopy (AFS), atomic emission spectroscopy (AES) and

inductively coupled plasma-mass spectrometry (ICP-MS). Other alternatives for the

classical methods are electrochemical methods such as ion selective electrodes (IES),

anodic stripping voltammetry (ASV), potentiometric stripping analysis (PSA),


                                         28
current stripping chronopotentiometry (CSP) and differential pulse voltammetry

(DPV). The advantages of these methods are that they are accurate, specific and

reproducible (Bontidean et al., 2004).



However, the methods described above have several disadvantages. They do not tell

anything about the toxicity of compounds to the organisms, very expensive, need

very skilled technician to operate, need sophisticated instrumentation, complicated

sample pre-treatment and sometimes a long measuring period.          It is also not

economical for large scale screening of toxic xenobiotics in the environment (Bitton

and Koopman, 1986).



Xenobiotic can be defined as components which are foreign to the metabolic network

of an organism and derived from the Greek xenos and bios (stranger to life)

(Vedagiri, 1998).    In other words, xenobiotics are man-made compounds with

chemical structure foreign to a given organism. Therefore, the need for a simple,

easy to handle and highly sensitive detection method is obvious.



2.7.1   Classical Bioassay/ Bioindicator.



The classical bioassay/ bioindicator method often used rat or mouse where IPR

(Intraperitoneal-Injection) was used. Daphnia magna and rainbow trout were also

used to test for toxic chemicals (Brown et al., 1982). Typically, chemicals are more

toxic to fish than animals. Therefore, the test using fish is more appropriate since

they are more sensitive to the toxic chemicals than animals but less sensitive

compared to the D. magna test. But the disadvantages of the test for toxicity using


                                         29
D. magna is that it is very expensive and require skilled breeding techniques and

maintenance (Botsford, 2000). It is also not applicable for large scale screening of

toxic xenobiotics in the environment where a sophisticated instrument is required to

analyse data.



2.7.2   Modern Bioassay/ Bioindicator of Heavy Metals.



Modern bioassays involve the use of microorganisms (Botsford et al., 1997),

antibodies (Mehraban et al., 1998) and enzymes (Christensen et al., 1982) to detect

the toxicity of xenobiotics such as heavy metals, pesticides and organic solvents.

Several bioassays have been designed to detect toxicity in the environment. Most of

them used the inhibition of the cell’s biochemical characteristics to quantify toxicity.



There have been many tests developed using various bacteria as the indicator

organism (Bullich, 1986). Microbial bioindicator/bioassay provides a simpler and

less expensive method than classical bioassay since no specialized equipment is

required (Zonneveld, 1983).      The use of bacteria or microbes (yeast, fungi) as

bioindicators is more effective than the classical method because microbes are easily

grown and can be kept for years by controlling the abiotic factors, such as pH and

temperature. There are also certain microbes such as yeast that can imitate the

biochemical physiology of complex organism such as rats. This enables us to run a

comparable bioassay with complex organism.



Microbes such as bacteria also exist at the lowest tropic level, so bacteria have the

ability to detect toxic compound before others do. Thus, bioindicators using bacteria


                                           30
has been commercialized such as the Lux-Fluoro (Baumstark-Khan et al., 2003), the

Polytox™ (Sun et al., 1994), the Deltatox™ and the Microtox™ assays (Hsieh et al.,

2004).



Protozoa bioassays use protozoa death as a bioindicator of toxicity, as visualized

under the microscope.      Mortality of the protozoa under the influence of the

xenobiotics forms the basis of xenobiotics toxicity (Botsford, 2001). Spirotox test is

one example of protozoa bioassay that used protozoa Spirostomum ambiguum to

estimate the toxicity of volatile compounds. This protozoa is a large protozoan used

for several years in toxicity tests in Poland (Jawecki and Sawicki, 1999). Another

example is Tetrahymena pyriformanis, ciliated protozoa which is a soil protozoa.

The physiological response of this ciliate was assessed in terms of mortality, growth

and grazing capacity after exposure to four toxicants: copper, zinc, cycloheximide

and Triton X-100 (Nicolau et al., 1999). Other soil protozoa that were used as

bioindicators were naked amoebae, testate amoebae, flagellates and sporozoans

(Foissner, 1999).



Other bacteria that have been used as an indicator is Rhizobium meliloti. The assay

is based on the ability of R. meliloti to reduce tetrazolium dye MTT rapidly from

light yellow colour to dark blue colour or increasing the absorbance reading value.

This reduction process is inhibited by several toxic chemicals. It means that the

inhibition of the reduction of the dye indicates that activities of living cells are

disturbed and causes the cells to die (Botsford, 1998). Lethal concentration 50%

(LC50) was used to measure the toxicity in the R. meliloti bioassay.




                                         31
Heavy metals such as Hg, Cd, Cu, Mn, Co, Ni, Se and Zn can inhibit the reduction

process in small amounts when using the R. meliloti assay. Hg and Cd are most toxic

while Pb, Fe, and lanthanum (La) are not toxic at concentrations approximately less

than 1200 mg/L. Sodium (Na) and potassium (K) are only toxic at concentrations of

more than 15,000 mg/L (Botsford, 1998). The LC50 of heavy metals which inhibited

R. meliloti is shown in Table 6.



Table 6: LC50 of heavy metals which inhibits the reduction of tetrazolium dye
MTT by R. meliloti (Botsford, 1998).

   Heavy metals                     LC50 (mg/L ± SD)                  Samples
Cadmium (Cd2+) ion                     0.791 ± 0.324                     12
               2+
Calcium (Ca ) ion                      5.65 ± 1.86                       13
              2+
Cobalt (Co ) ion                      12.3 ± 5.65                        11
              2+
Copper (Cu ) ion                       0.95 ± 0.181                      13
                    2+
Magnesium (Mg ) ion                   50.8 ± 10.1                         9
Manganese (Mn2+) ion                   1.44 ± 0.504                      11
Mercury (Hg2+) ion                     0.0159 ± 0.004                    10
Nickel (Ni2+) ion                     58.6 ± 6.45                        11
Selenium (Se2+) ion                 277 ± 108                             9
         2+
Zinc (Zn ) ion                         0.84 ± 0.059                      10




2.7.3   Microtox™ Bioluminescence Assay.



Beckman Instruments Inc. developed the Microtox™ bioassay system as a screening

instrument used for a variety of toxicity testing applications. Microtox™ bioassay

used freeze-dried bioluminescent marine bacterium such as Vibrio fischerii (Boluda

et al., 2002). The results could be obtained in only five minutes. However, the cost

of the kit which includes a luminator to measure the light produced by bacteria, a
                                        32
refrigerated 15°C water bath, a computer and computer program to analyse data is

quite expensive and need relatively high skills to handle (Botsford, 2000).



Measurement of toxicity in this bioassay used decrease of light emitted by the

bioluminescent microorganisms. They emitted light as a normal function of its

metabolic activities. Therefore the presence of toxicants in the sample reduced light

emission of the bioluminescent microorganisms (Shettlemore and Buddy, 2001).



Effective concentration 50% (EC50) was used to measure the toxicity in the

Microtox™ bioassay. EC50 represents the concentration of a compound where 50%

of its effect is observed. Lower EC50 values showed substances were more toxic

(Shettlemore and Buddy, 2001).



2.7.4   Polytox™ Bioassay.



Polytox™ is a method developed by the Polybac Corporation, United State to

determine toxicity. This bioassay uses an artificial consortium of 12 bacteria in

which the bacteria utilize oxygen. Utilization of oxygen by bacteria was measured

using the oxygen electrode or respirometer. The data from the respirometer or

electrodes was analysed using a sophisticated computer program (Botsford, 1998).



The toxicity of the sample was detected when the consumption of oxygen level by

the bacteria was reduced. However, the major problem in this bioassay was to

maintain the respirometers and electrodes. This is because oxygen electrodes are

expensive and easily damaged. Besides, the consortium of 12 bacteria could only be


                                          33
obtained from the vendor. They could not be duplicated in the laboratory (Botsford,

2000).



2.7.5    DeltaTox™ Bioassay.



DeltaTox™ is a bioassay using the same protocol as Microtox™.                It uses

bioluminescent marine bacteria such as Vibrio fischerii to detect toxicity.      The

difference between both methods is that the DeltaTox™ method measured the

percentage of light extinguished compared to a blank control, whereas, the

Microtox™ method measured EC50 values. The higher degree of light loss indicates

more toxic samples (Mowat and Bundy, 2001).



Usually DeltaTox™ is used as the preliminary screening tool for hotspot

identification, followed by more intensive investigation of pollution using

Microtox™ (Mowat and Bundy, 2001).



2.7.6    Lux-Fluoro Test Bioassay.



The Lux-Fluoro test is a bioassay which is a combination of two bioassays that

measures the cytotoxicity (LAC-Fluoro test) and the genotoxicity (SOS-Lux test) that

is potency of a given substance based on the receptor reporter principle. For testing

cytotoxic, Salmonella typhimurium TA 1535 cells were transformed with the

bacterial protein expression vector pGFPuv were employed. This plasmid controls

green fluorescent protein (GFP) expression by the lac promoter in TA 1535 cells.

Meanwhile, for genotoxicity testing, as a response to the presence of DNA-damaging


                                         34
agents, bioluminescense is brought about by the induction of the promoterless

luxCDABFE genes of Photobacterium leiognathi as reporter component.             As a

consequence of exposure to genotoxicity agents (mitomycin C, chloramphemicol,

cexorubin hydrochloride, bleomycin sulphate and hydrogen peroxide) the intensity of

the emitted light is proportional to the concentration of the compound (Baumstark-

Khan et al., 2001, 2003).



For the combined Lux-Fluoro test, the genotoxic potential of agents was determined

by an increased bioluminescence (SOS-Lux) and the cytotoxic potential by a

decreased fluorescence (GFPuv-Fluoro) in S. typhimurium TA 1535 cells. The light

and fluorescence transmissions of untreated and chemical-treated cells were

measured in a microtiter plate reader and luminescence induction factor (Fi) while

fluorescence deduction factors (Fd) were calculated for the genotoxic and cytotoxic

potential of the applied agents (Baumstark-Khan et al., 2001).



Therefore, this bacterial whole-cell bioassay was reported that can be applied for the

first screening steps for testing new substances during the development process in the

pharmaceutical research. It is also reported as fast, simple and inexpensive bioassay

for the combined determination of the genotoxic and cytotoxic potential of chemical

and mixtures of chemicals as they occur in environmental samples (Baumstark-Khan

et al., 2001).




                                         35
2.7.7   Enzyme Bioassay.



Enzyme bioassays are selective and sensitive.        These properties together with

simplicity, speed and ease of automation make the method highly promising to use to

indicate environmental pollution (Dalmanova et al., 1987; Rodriguez et al., 2004).

The inhibition of enzymatic reaction has been used to determine environmental

pollution through the complex of enzyme-inhibitor or (enzyme-substrate) - inhibitor

(Anna et al., 1984). The interaction of inhibitor with an enzyme could result in a

reduction of the enzyme activity (Rodriguez et al., 2004).



This method has already been used for the determination of Hg(II) by its inhibition

of enzymes such as xanthine oxidase, glucose oxidase, alchohol dehydrogenase and

β-fructofuranosidase. β-fructofuranosidase has also been used for the determination

of I-, S2-, CN- and Ag(I) and thiocarbamide. Other enzymes that have been used are

lactic dehydrogenase (EC 1.1.1.27), glutamate oxaloacetic transaminase (EC

2.6.1.1), carbonic anhydrase, pyrophosphohydrolase (EC 3.6.1.8), acethylcholine

esterase (EC 3.1.1.3), urease (EC 3.5.1.5) (Christensen et al., 1982), adenosine

triphosphatases, esterases, phosphatases and yeast invertase. The enzymes acetyl-

and butyrylcholinesterase (AChE and BChE) are known to have high sensitivity to

phosphororganic and carbamate pesticides while the enzyme urease is strongly

inhibited by most of the heavy metal ions (Starodub et al., 1999).




                                          36
2.7.8   Metabolic/ Microbial Bioassay.



Microbial bioassay differs from the enzyme assay in the conventional sense, where

metabolic activity is involved. In order to determine the bacterial metabolic activity,

the use of tetrazolium redox dyes has gained increased recognition in recent years.

Tetrazolium redox dyes scavenge electrons from microbial oxidation/ reduction

reactions and are intracellularly reduced to brightly coloured formazan precipitates

by the electron transport system (ETS) components or dehydrogenases in

metabolically-active microorganisms. These coloured precipitates can be accurately

detected within individual cells by direct microscopy or quantified in cell extracts by

spectrophotometry (Kaprelyants and Kell, 1993; Smith and McFeters, 1997).



These tetrazolium salts are first prepared in 1894 and have been used widely as

indicators in ecological and environmental studies (Altman, 1976). 3-(4,5-dimethyl-

thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is an example of tetrazolium

salts. MTT is a monotetrazolium salt which is one of the most frequently used

methods for measuring cell proliferation and cytotoxicity (Mosmann, 1983).



Other specific dyes that also can be used as indicators of electron transport system

(ETS) are methylene blue, triphenyltetrazolium chloride (TTC), tetrazolium blue,

resazurin, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT)

and          2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide

(XTT) where they act as artificial hydrogen acceptors and change colour upon

reduction.




                                          37
2.7.8.1 Application of Growing Microbial Cells for Metals Detection.



Bioassays using growth inhibition of microorganisms, inhibition of enzyme activity

and enzyme biosynthesis are simple, rapid, cost-effective and require only small

sample volumes. Therefore, some commercial test kits have been developed for this

purpose such as the toxichromotest based on the inhibition of the biosynthesis of β-

galactosidase (E.C.3.2.1.23) in the Gram-negative bacterium E. coli (Bitton and

Koopman, 1992; Codina et al., 1994). It is reported that bioassay based on β-

galactosidase activity is not sensitive to toxic organic compounds while being

relatively sensitive to heavy metals compared to α-amylase (E.C.3.2.1.1)

biosynthesis in the Gram-positive bacterium Bacillus subtilis       which was more

sensitive to heavy metals, particularly to mercury as low as 0.1 ppm (Guven et al.,

2003).



An important aspect of toxicity testing using bacteria or other organisms seems to be

the permeability of the cells to environmental toxicants, particularly those of

hydrophobic nature. The complex envelope structure of Gram-negative bacteria is

known to consist of a cytoplasmic membrane, a rigid peptidoglycan cell wall and an

outer membrane.     The outer membrane is an effective diffusion barrier against

hydrophobic substances. Gram-positive bacteria which lack an outer membrane may

be utilized for toxicity tests to determine organic compounds (Guven et al., 2003).



Gadd (1998) and Brierley (1990) have described the many ways in which bacteria,

fungi and algae can take up toxic metal ions. Heavy metal ions can be entrapped in




                                         38
the cellular structure and subsequently biosorbed onto the binding sites present in the

cellular structure.



There is also reported that a cell may develop metal resistance systems in an attempt

to protect sensitive cellular components. Limiting metal access or altering cellular

components decreases their sensitivity to metals. Several factors determine the level

of resistance in a microorganism such as the type and number of mechanisms for

metal uptake, the role each metal plays in normal metabolism and the presence of

genes located on plasmids, chromosomes or transposons that control metal resistance

(Bruins et al., 2000). Six mechanisms are postulated to be involved in resistance to

metals.    They are exclusion by permeability barrier, intra- and extra-cellular

sequestrations, active transport efflux pumps, enzymatic detoxification and reduction

in the sensitivity of cellular targets to metal ions (Rouch et al., 1995).

Microorganisms can have one or a combination of several resistance mechanisms.



The most well-known example is Hg(II) resistance coded for by the mer (mercury)

operon. Hg is highly toxic because of its affinity for thiols. It inactivates thiols that

are part of enzymes and other essential cellular proteins. Some bacteria have adapted

to the presence of Hg(II) by evolving a set of genes that form a resistance operon.

The gene products of this operon not only detoxify Hg(II) but are also involved in

transport and self-regulation (Ni’bhriain et al., 1983; Liverelli et al., 1993; Utsching,

1995; Wiess et al., 1997).




                                           39
Therefore, in order to control the production of toxic compounds, there is always a

need to develop fast and simple microbiological screening tests to determine the

toxicity in soils and waters.



2.7.8.2 MTT Assay.



MTT assay is based on the reduction of a tetrazolium dye 3-(4,5-dimethyl-thiazol-2-

yl)-2,5-diphenyltetrazolium bromide (MTT) by bacteria. This tetrazolium salts are

dissolved and colourless in their native form but form nonwater, soluble, coloured

salts as they are reduced.      After reduction, the colour of the respective MTT-

formazan derivative is purple-blue. The reduction is inhibited by toxic compounds or

chemicals thought to be toxic that damage living organisms. This assay can be used

to determine the IC50 values of xenobiotics. The reduction of MTT can be followed

with a simple spectrophotometer. The absorbance at 550 nm increases if the dye is

reduced. The MTT dye has molecular weight of 414.32 and the chemical formula is

C18H16BrN5S. Figure 3 shows the molecular structure of MTT.




                          Figure 3: Molecular structure of MTT.



In this study, this MTT assay was carried out in microplates. It is because it allows

large amounts of data from numerous bacteria to be generated quickly in one assay

procedure (Eloff, 1998). The MTT assay is also simple but sensitive due to its ability

                                          40
to detect low active cell numbers. Furthermore, it is reliable because the absorbance

obtained usually correlates to the cell numbers. The increasing number of living

cells is associated with increase in the total metabolic activity in the bacterial sample.

This increase is directly correlated to the amount of purple formazan crystals formed

as monitored by the absorbance readings (Tengerdy et al., 1967; Eloff, 1998).



MTT has been used to estimate the survival of animal cells (Marshall et al., 1995)

and is used extensively to test for chemotherapeutic compounds.             It is widely

assumed that MTT is reduced by active mitochondria in living cells. Although this

finding has often been taken as evidence, definitive proof for the association of

mitochondria with MTT reduction in intact cells has been lacking because many non-

mitochondrial dehydogenases or flavin oxidases can also reduce MTT (Altman,

1976; Burdon et al., 1993).



It also has been proposed that the tetrazolium dyes are reduced by the cytochromes

(Altman, 1976). In prokaryotes, the electron transport system is associated with the

cytoplasmic membrane.         Hydrophobic toxic chemicals will interact with the

membrane, affecting cytochrome activity.         The dye can also be reduced by a

NAD(P)H reductase (Sowberry & Ottaway, 1965; Berridge and Tan, 1993). Other

than that, Berridge and Tan (1993) found that NADH and NADPH were much better

substrates than succinate in supporting MTT reduction by subcellular components

after fractionation and that the mitochondrial electron transport chain inhibitor

rotenone failed to affect MTT reduction by intact cells. Figure 4 shows the principle

of NADH assay with a tetrazolium salt and phenazinium salt (PMS) as an electron

carrier where reduction occurred to form a formazan.


                                           41
    Figure 4: NADH assay principle with a tetrazolium salt and PMS
    (phenozine methosulfate) as an electron carrier (Marshall et al., 1995).


Without a clear understanding of the site and the enzymatic system involved in

cellular MTT reduction, it has been difficult to explain the discrepancies between the

MTT assay and other measures of cell growth and viability (Berridge and Tan,

1993).



In the presence of xenobiotics and heavy metals in the electron transport system, the

system will be interrupted where production of NADH will be inhibited. In other

words, this process will eventually lower the MTT assay results. The inhibition

could be in any enzymes at any stages of NADH production. MTT uses the many

features on enzyme of the metabolic pathway as a test for xenobiotics which are

involved in glycolysis, Kreb’s cycle and lastly electron transport chain. Figure 5


                                         42
shows the diagram of electron transport system pathway from glycolysis and Kreb’s

cycle of cellular respiration where NADH is produced.




Figure 5: Diagram the flow of NADH production in the glycolysis and Kreb’s
cycle to the electron transport chain. This pathway represents the metabolic
pathway in cellular respiration (Malik, 2004).




Therefore in this study, the MTT assay was carried out using Botsford’s modified

MTT assay. However, the results obtained from this bioassay have to be modified

by adding Ca2+ ion and Mg2+ ion. This is because in Microtox™ and Polytox™

bioassay, these two divalent cations were not examined for their toxicity in inhibiting

the reduction of the dye. Therefore, it was suggested by adding chelator EDTA or

EGTA in this assay will take care of the problem as in the Botsford’s assay.


                                          43
EDTA is a powerful chelating agent that complexes many divalent cationic metals

(USEPA, 1989b), thereby making them biologically unavailable. As an example,

this chelator will bind to the divalent cations Ca2+ and Mg2+ and eliminate them.

This will lessen the sensitivity of the bioassay. In addition, EDTA also affects the

permeability of the outermost layer of Gram-negative bacteria and exposure to high

concentrations of this chelator decreases cell viability (Ayoub et al., 1995).

Therefore, the chelator was not used in this study. The influence of interfering

compounds on the proposed method was also investigated with several pesticides and

xenobiotics tested.



In order to determine which growth indicator is the most sensitive and rapid to

measure bacterial growth, resazurin dye was used for comparison in this study.

Resazurin is a redox indicator dye that can be added directly to cells in culture. The

indicator is particularly useful when cells have a tendency to adhere together or

where other additives affect the colour of the growth medium (Eloff, 1998).



The cells will convert the dark blue oxidized form of the dye (resazurin) into a red

reduced form (resorufin). The system is specific for viable cells since nonviable

cells rapidly lose their metabolic capacity. The ability of different cell types to

reduce resazurin to resorufin varies depending on their metabolic capacity. The

maximum absorbance of resazurin and resorufin are at 605 nm and 573 nm,

respectively (Eloff, 1998).




                                         44
2.7.8.3 The Advantages of Microbial Bioassay of Heavy Metals.



There are many advantages of using microbial bioassay for testing toxicity of heavy

metals using the MTT assay. Microorganisms are easy to culture, besides providing

rapid results. The generation time of bacteria is very rapid and so their response

times to organic enrichment or to toxic substances are likely to be quite rapid. They

can also be maintained under known, controlled conditions in large numbers.



Microorganisms also have evolved various measures to respond to heavy metal stress

via processes such as transport across the cell membrane, biosorption to cell walls

and entrapment in extracellular capsules, precipitation, complexation and oxidation-

reduction reactions (Veglio et al., 1997). They have proven capability to take up

heavy metals from aqueous solutions especially when the metal concentrations in the

effluent range from less than 1 to about 20 mg/L (, 1990).



2.7.9   Bioassay Using Antibodies

2.7.9.1 Immunoassay



Immunoassay is an in vitro antibody-based assay to detect heavy metals, pesticides,

herbicides including industrial pollutants. This assay is quick, inexpensive, simple to

perform and sufficiently portable to be used at the site where the sample is taken.

The principle in immunoassay is that once toxic chemicals are present in the sample,

specific antibodies will detect the toxic chemicals and reduction will occur to form

green colour resulting from the activity of the coupled enzymes in the system.

Inhibitory concentration, 50% (IC50) values are determined to quantify the toxicity in


                                          45
the samples (Mehraban et al., 1998). IC50 is the concentration of a chemical that is

inhibited by 50 % of a test population.



In 1991, an antibody-based immunoassay for ionic Hg has been described that is the

basis for the only metal ion immunoassay presently available commercially. This

assay captured soluble ionic Hg on a reactive sulfhydryl surface and utilized a Hg-

specific monoclonal antibody to bind the Hg-sulfhydyl complex. Meanwhile, an

immunoassay for detecting Cd was also developed where it utilizes a monoclonal

antibody that binds tightly to a cadmium-ethylenediaminetetraacetic acid (EDTA)

complex but not to metal-free EDTA (Mehraban et al., 1998).



2.7.10 Biosensors for Heavy Metals Detection.



Biosensors combining a biological recognition element and a suitable transducer are

useful analytical instruments. Several biosensor configurations have been developed

in the past by using a variety of recognition elements such as enzymes (Amine et al.,

1995), whole cells (Rasmussen et al., 2000) and coupled to various types of

transducers (Bontidean et al., 2004).



In the last 10 to 15 years it was known that a number of biosensors were developed

for the quantification of different toxic compounds. There are two different types of

biosensors such as monosensors and multibiosensors. Monosensors are developed

from ion selective and pH-sensitive glass and metal electrodes. The sensor is based

on pH-sensitive field-effect transistor and conductometric electrodes. The problem is

that only one toxin can be determined by such monosensor. It is an uneconomic


                                          46
system (Arkhypova et al., 2001). Therefore, multibiosensor was developed to solve

the monosensor problem. The multibiosensor consists of several transducers with

different bioselective membranes that enable simultaneous determination of various

toxic substances (Arkhypova et al., 2001).



Widely used multibiosensors are known to have used many specific enzymes to

quantify heavy metals       and   pesticides.   Acethylcholinesterase   (AchE)   and

butyrylcholinesterase (BChE) enzymes for example were used because they are

highly sensitive to phosphororganic and carbamate pesticides. Whereas, urease and

oxidase were highly sensitive to heavy metal ions (Starodub et al., 1999). Even so,

the drawbacks of biosensors are that it involves expensive equipments that are not

economical for daily tests and large numbers of samples.




                                         47
                                    CHAPTER 3



                          MATERIALS AND METHODS



3.1     Chemicals and Equipments.



All reagents were of analytical reagent grade unless otherwise stated. The MTT dye

was purchased from Sigma. All the plastics and glassware were cleaned by soaking

in dilute HNO3 (10%) to remove all traces of metal ions and were rinsed with an

appropriate amount of deionized water prior to use. All chemicals and equipments

used are listed in Appendix II.



3.2     Preparation of Solutions.

3.2.1   MTT Dye Stock Solution.



MTT dye at 10 mM stock solution was prepared by dissolving 0.2072 g of MTT

powder in 50 mL sterilized (autoclaved) 10 mM PBS, pH 7.5 in a sterile bottle and

wrapped with aluminium foil to prevent exposure to direct light because it is

photosensitive. The solution was then stored at 4 ºC. This MTT dye was then added

into the reaction mixture to a final concentration of 1 mM.



3.2.2   Heavy Metals Stock Solutions.



Heavy metals such as manganese (i) (MnSO4.H2O), borate (ii) (H3BO3), tin (iii)

(SrCl2.6H2O), selenium (iv) (Na2SeO4), zinc (v) (ZnSO4 anhydrous), tungsten (vi)


                                         48
(Na2WO4.2H2O), potassium (vii) (KCl), cesium (viii) (CsCl) were prepared from

commercial salts by dissolving in deionized water with a few drops of concentrated

nitric acid (90%) to solubilise the heavy metals and stored in acid-washed

polypropylene containers. All of these heavy metals stock solutions were prepared in

concentration of 100 mg/L. Mercury [Hg(NO3)2], arsenic (As2O5), cadmium

[Cd(NO3)2], lead [Pb(NO3)2], copper [Cu(NO3)2], silver (AgNO3), magnesium

[Mg(NO3)2.6H2O] and calcium (CaNO3) are Atomic Absorption Spectrometry

standard solutions from MERCK, 1 g/L (Merck, Darmstadt, Germany). Working

solutions were prepared from these stock solutions by diluting in deionized water to

the required concentrations. The chosen metal concentrations were calculated as

mg/L of each metal.



3.2.3   Pesticides and Miscellaneous Xenobiotics Stock Solutions.



Pesticides with chemical purities of >99%, (Ehrenstorfer, Augsburg, Germany and

Pestanal®, Riedel de Häen, Germany) such as metolachlor, glyphosate, diazinon,

endosulfan sulphate, coumaphos, imidacloprid, dicamba and diuron were prepared

by dissolving the pesticides in the appropriate solvents or used directly from the

liquid solutions.   The final concentration of all these pesticides in the reaction

mixture was 4 mg/L.



The xenobiotics tested are as follows; acetone (MERCK), n-hexane (MERCK),

ethylene glycol (MERCK), ethyl acetate (MERCK), ethanol (BDH), isopropanol

(BDH), methanol (BDH), pyridine (SIGMA), diethylamine (SIGMA), Triton-X-100

(SIGMA) and SDS (SIGMA). These xenobiotics were prepared as 2% (v/v) solution


                                         49
in deionized water and added into the reaction mixture to a final concentration of

0.4% (v/v).



The concentration of pesticides and xenobiotics chosen in this study is generally

much higher than normally found in natural water and also limited to the solubility of

pesticide and xenobiotics in water.



3.3    Sample Collection.



Soil and water samples were collected from 10 different locations in Peninsular

Malaysia. They are Tanjung Karang, Selangor, Tanjung Blanja, Perak, a parking Lot

at Mutiara Hotel, Johor Bahru, grounds at the Biochemistry and Microbiology

Department, University Putra Malaysia, Bukit Ekspo, University Putra Malaysia,

Taman Tasik Taiping, Perak, Sungai Congkak, Selangor, Taman Sri Serdang,

Selangor, Sri Serdang Lake, Selangor and Masjid Abidin Taman Skudai, Skudai,

Johor. Whenever possible, the exact location of the samples was recorded by noting

down the map coordinates provided by a global positioning system (GPS) locator.

The soil and water samples were collected from a depth of 5-8 cm from the soil and

water surface. The pH of the samples was determined in the laboratory using pH

meter. The soil samples were placed in sterilized plastic bags and stored in ice

during transfer from the sites to the laboratory.




                                           50
3.4    Isolation and Culture of Bacteria.



All of the subsequent laboratory works were done in a disinfected laminar flow

cabinet. The purpose was to prepare the soil for further analysis. The pH of the

samples was measured. The soil samples (about 10 g) were weighed and diluted

with 100 mL sterilized water. One mL of the sample was pipetted into a universal

bottle containing 9 mL of nutrient broth which had been diluted 100 times. Serial

dilution technique was performed in order to dilute the concentration of the bacteria.

Shaking the diluents and soil mixture is essential to disperse clumping cells and to

facilitate mixing.



This was followed by the pour plate and streak plate technique in order to obtain pure

cultures. For preservation of cultures, the bacteria were inoculated in slant agar and

kept in the fridge at 4 ºC. For long term preservation, stock cultures were stored in

the inoculated cryovial at -70 ºC for extended storage.



In order to confirm the purity of the bacterial isolates, observation of the bacterial

morphology was carried out. The morphology of the bacteria isolated was observed

using macroscopic and microscopic techniques. The characteristic of the colonies

were observed and each of the different colonies with different morphologies were

picked and streaked on nutrient agar for 24 hours at room temperature to isolate a

single colony. The colony growth was examined by its colour, size, shape, margin

and elevation. The colony was observed under the light microscope at 100 X, 400 X

and 1000 x magnification.




                                          51
3.5     MTT Assay of Bacterial Inhibition Studies.



An assay using tetrazolium salts such as MTT is a colorimetric assay system, which

measures the reduction of the tetrazolium component and was chosen to screen

bacteria as bioindicators for heavy metals. It has been used as a growth indicator

since 1940s (Gabrielson et al., 2002).        The MTT was added to facilitate the

measurement of cell growth (Bitton and Dutka, 1986; Botsford et al., 1997) and was

measured as an increase in either absorbance (Gellert, 2000; Schrader et al., 1997) or

bioluminescence (Gellert and Stommel, 1999).          In this study, the qualitative

observations as well as the quantification was made by reading the absorbance at 550

nm using a 96-well microplate reader. According to Gabrielson et al. (2002), this

absorbance reading was chosen as it is the optimum absorbance of the MTT dye.

The assay procedure consists of the preliminary screening of isolates with divalent

cations and heavy metals and then isolates selection was narrowed down by

secondary screening with different stages of microbial growth with the heavy metals.



3.5.1   Preliminary Screening of Bacterial Respiration Inhibited by Divalent
        Cations and Heavy Metals.


Preliminary screening based on the MTT assay in the presence of common divalent

cations such as calcium (Ca2+) and magnesium (Mg2+) at the highest final

concentration of 25 mg/L and 50 mg/L respectively was carried out to select isolates

that were not inhibited by these cations as well as the selected heavy metals in this

study. Six toxic heavy metals were selected in this study. They were silver (Ag),

mercury (Hg), cadmium (Cd), arsenic (As), lead (Pb) and copper (Cu). The final

concentrations in the reaction mixture used were 10 mg/L for Ag, Hg, Cd, As and Pb


                                         52
and 5 mg/L for Cu. Two hundred and fifty bacterial isolates were screened for their

sensitivity to these divalent cations and the heavy metals that were performed

separately according to different tested heavy metals.



Prior to start of the assay, bacteria were grown in 10 mL of nutrient broth (NB) for

18 hours in a 15 mL conical flask. The bacteria were incubated at room temperature

in a rotary incubator shaker at 150 rpm for growth. Bacterial samples of 1 mL were

centrifuged at 10 000 x g for 10 min in an eppendorf tube at room temperature. The

supernatant was discarded while the pellets were washed once with 10 mM

phosphate buffer saline (PBS), pH 7.5 and resuspended in the same buffer by mixing

vigorously with vortex. The purpose of resuspending the pellet in the buffer was to

dilute the pellets. The cells were ready to use for subsequent assays.



The MTT assay was carried out by combining 50 L of 10 mM PBS, pH 7.5 at the

final concentration in a 250 L total reaction mixture followed by 75 L aliquot of

1% (v/v) bacterial suspensions, 25 L of each two divalent cations, Ca2+ and Mg2+

and finally 50 L of tested heavy metals in a flat bottomed 96-well microplate. As a

control, the tested heavy metals were replaced with deionized water in the first well.

The reaction mixture was pre-incubated for an hour at room temperature by covering

the microplate with aluminium foil as a precaution before adding 25 L of MTT (10

mM stock) to allow the reaction to proceed.



Pre-incubation time of one hour was chosen as the optimum period for maximum

inhibition after several attempts were performed. The pre-incubation was performed

to allow the reaction between the bacteria and the heavy metals. The reaction


                                          53
mixture was mixed on a microplate reader with a shaking mode.                 Colour

development was visually observed after the incubation period. The results showed

that formazan production was saturated after 20 minutes and therefore this was

chosen as the incubation period for all further experiments. All the tests were

performed in triplicates.



The assay procedure was also repeated for resazurin dye in a similar manner. It was

found that resazurin was difficult to use since it shifts between three colours.

Resazurin is a coloured compound that changes colour (from blue to pink to

colourless) but does not precipitate upon reduction. Preliminary results showed that

a variation of colour was observed after incubation making it difficult to identify

inhibition effects of heavy metals. Since it displayed three different colours (blue-

pink-colourless) during transformation, the tested strains of bacteria could not be

easily read by the spectrophotometer at a single wavelength and also had the

drawbacks of being reduced by fewer strains of bacteria (Gabrielson et al., 2002).

Due to this, resazurin dye was not further used as an indicator of inhibition of

bacterial growth by heavy metals in this study.



3.6 Effects of Different Stages of Microbial Growth on Inhibitory Effect of
      Heavy Metals.


From the preliminary results, one bacterial isolate showed good inhibition by Hg, Cd

and Pb while four isolates showed good inhibition by Ag qualitatively.         These

isolates were designated as SC 27 as indicator for Hg, SC3 as indicator for Cd, SC41

as indicator for Pb and S8, S7, S1 and K104 as indicator for Ag. Therefore, these

isolates were used for the rest of the study.     In order to determine the lowest


                                         54
concentration of Hg and Ag that inhibit the reduction of the dye, six different growth

periods were tested using the same MTT assay procedure as previously described in

section 3.5.1. The final concentrations of the heavy metals that were used in the

reaction mixture ranged from 0.01 mg/L to 10 mg/L. Prior to start of the assay, one

mL aliquots of inoculated culture from 10 mL of NB were transferred to 1.5 mL

eppendorf tubes and incubated for 6, 8, 10, 12, 14 and 16 hours. Bacterial cells were

washed as previously described in section 3.5.1. After one hour of pre-incubation

time at room temperature, the absorbance at 550 nm was read. The absorbance was

read again after 20 min of incubation time with the MTT dye and set as a final

absorbance. All the tests were carried out in triplicates. The graph of absorbance at

550 nm versus the heavy metal concentrations (mg/L) was plotted by initially

subtracting the initial absorbance at time zero from the final absorbance. The data

was analysed using Graphpad Prism™ version 4.0 software using non-linear

regression analysis.



3.7 Determination of the IC50 Values of the Heavy Metals.



An experiment was set up to find the IC50 value for the heavy metals inhibition of the

microbial isolates as previously described in the section 3.6. The final concentrations

that were used in the reaction mixture ranged from 0.01 mg/L to 10 mg/L.

Therefore, in order to determine the inhibition concentrations at 50% (IC50) of each

sample, the data from section 3.6 was plotted using Graphpad Prism™ version 4.0

software. The data at 8 hours growth of isolate SC27 and 12 hours growth of isolate

S8 were selected. This is due to the ability of both the heavy metals to inhibit the

reduction of the MTT dye significantly using these two bacterial growths at the


                                          55
chosen time. The IC50 value obtained is dependent on the range of concentration

used. Regression curves of Hg and Ag were generated using the PRISM non-linear

regression analysis for four-parameter logistic equation software. Means and

standard errors were determined according to at least three independent experimental

replicates.



3.8 Effect of Different Buffers System.



This experiment was set up to improve the sensitivity of the microbial isolates to

heavy metals by manipulating the buffer systems. Jung et al. (1995) found out that

the sensitivity of urease to heavy metals was increased several fold if the buffer

system was changed from phosphate to Tris. In order to investigate the effects of

different buffers in this assay system, six buffers at pH 7.5 were selected.



All of the buffers used were commercial buffers purchased from Sigma-aldrich

except Tris-HCl which was prepared in the lab (Appendix III). The commercial

buffers used were Pipes, Imidazole, Hepes and Bicine at a final concentration of 16

mM while Tricine and Tris-HCl were used at a final concentration of 10 mM. The

MTT assay procedure performed as previously described in section 3.5.1 using 1%

(v/v) bacterial suspension of isolate SC27 and S8 at growth period 8 hours and 12

hours respectively. The choice of incubation times for these bacterial growths had

been selected from the previous screening assay in section 3.6 and was used for

further studies. The final concentrations of Hg and Ag in the reaction mixtures were

0.3 mg/L and 0.2 mg/L, respectively. The final concentration that was used had been

selected from the previous screening assay in section 3.6 which is the lowest


                                           56
concentration of the inhibition of the reduction of the dye. As a control, the heavy

metals were replaced with deionized water. The tests were carried out in triplicates.

Measurement of absorbance was read at 550 nm. The graph of absorbance at 550 nm

versus different buffers system was plotted as previously described in section 3.6.



3.9 Interfering Effects of Other Xenobiotics on Selective Bacteria.



Studies were carried out on the interfering effects of several other xenobiotics on the

selective bacteria. The MTT assay was performed as previously described in section

3.5.1 using 1% (v/v) bacterial suspension of isolate SC27 and S8 at 8 hours and 12

hours growth respectively. However, the assay was carried out slightly different

from the previous assay where the tested xenobiotic was added in the presence of the

selected heavy metals, Hg or Ag at the final concentration of 0.3 mg/L and 0.2 mg/L

respectively in the reaction mixture. This assay was performed separately according

to different tested xenobiotics. The tested xenobiotics consist of various heavy

metals, pesticides and detergents. The final concentration of heavy metals tested in

the wells was at 5 mg/L while the concentrations of xenobiotics tested were 4 mg/L.

As a control, the tested xenobiotics were replaced with deionized water. All the tests

were performed in triplicates. The graph of absorbance at 550 nm versus xenobiotics

was plotted by initially subtracting the initial absorbance at time zero from the final

absorbance.




                                          57
3.10   Preliminary Testing on Water Samples from Polluted Areas.



Water samples were obtained from four locations in Pulau Pinang. These were from

Sungai Pinang and Sungai Juru industrial outlets that were concentrated in the Prai

Industrial estate and Sungai Derhaka. These samples were designated as sample 1 to

sample 4 respectively. The estate bound by the Juru river is amongst the earliest

industrial complex built in the 1970’s and is notorious for high levels of heavy

metals concentration in the river (Mat et al., 1994). The high concentration of

industries especially electronics and foundries in the estate is a good testing site for

the newly developed microbial bioassay method. Water samples were stored in

polypropylene bottles with a few drops of concentrated nitric acid to solubilise the

available heavy metals. The determination of heavy metals in the samples was

carried out using Atomic Emission Spectrometer (AES) (Perkin Elmer Optima 3000)

to compare with the results obtained from this study. All experiments were

performed in triplicates.



To perform the assay, both of the isolates SC27 and S8 were used as indicators for

Hg and Ag respectively. The four water samples were centrifuged at 7000 x g for

five minutes to precipitate bigger particulate matter. Smaller particles were filtered

using syringe filters with a filter pore size of 0.45    m. These pre-treated water

samples were then kept in plastic tubes at room temperature. The assay tests were

carried out as a previously described unless otherwise stated where the heavy metal

was replaced with the water samples.        For control, samples were replaced by

deionized water.     All tests were performed in triplicates.      Changes in colour

development were observed after incubation time at 20 minutes. The measurement


                                          58
was made at 550 nm using a microplate reader. The graph of absorbance at 550 nm

versus water samples was plotted as previously described in section 3.6.



3.11   Identification of Bacteria.



Isolate SC27 and S8 which were chosen as the best indicator to detect Hg and Ag

were characterized and identified for their physiological properties. Bacteria

identification and confirmation were attempted using Gram staining method,

commercial identification systems such as Microbact™ Identification Kit 24E (12A

+ 12B) and molecular phylogenetics of the partial sequence of 16S rRNA.



3.11.1 Biochemical Test Using Microbact™ Kit.



The physiology of the bacteria can be characterized using a multitest system known

as Microbact™ Identification Kit 24E (Medvert Diagnostics). There are a number of

commercially available systems for the identification of various groups of bacteria,

especially of the family Enterobacteriacea. These are convenient and rapid systems,

which allow the simultaneous interpretation of a variety of biochemical tests using

mathematical probabilities. The system was created by Medvet Science Pty. Ltd.,

Adelaide, Australia. The system that was used was the Microbact™ System 12E and

24E for the identification of Enterobacteriacea which was used routinely in

pathology laboratories for identification of bacterial populations in the samples.



The kit contained 24 wells in which individual well performs different biochemical

tests. Strip 12A contained 12 designation tests. They were tests for lysine, ornitine,


                                          59
H2S, glucose, mannitol, xylose, ONPG, indole, urease, VP, citrate and TDA tests.

Strip 12B tests for gelatin, malonate, inositol, sorbitol, rhamnose, sucrose, lactose,

arabinose, adonitol, raffinose, salicin and arginine tests were performed. A pure

culture of the organism to be identified must be obtained first. An oxidase test on the

organism to be identified had been performed using oxidase reagent prior to using

the kit.



Pure cultures of the bacteria were grown on nutrient agar plates for 24 hours. A

single colony was emulsified in 0.85% (w/v) sterile saline. The mixture was inverted

thoroughly to prepare a homogenous suspension. This was then used to inoculate the

wells in the Microbact™ system kit using a micropipette. One hundred microliter of

the bacterial suspension was pipetted into each well in the set. The substrates

underlined on the holding tray were overlayed with sterile mineral oil.            The

inoculated rows were resealed with the adhesive seal and the identification number

was written on the tag with a marker pen. The kit was incubated overnight at 37 ºC.



The 12A and 12B strip should be read at 24 hours when identifying

enterobacteriaceae. All systems should be read within 48 hours for the identification

of miscellaneous Gram negative bacilli. After 24 hours, the 12A strip was removed

from the incubator. The reactions were then scored with references to a colour code

system and a unique four digit code number. For well no 8 in the 12A strip, 2 drops

of Indole (Kovacs) reagent was added and evaluated within 2 minutes of the addition

of the reagent before going to well no 10 (Vogues-Proskauer reaction). One drop of

each VP I and VP II reagent was added. The test was evaluated 15 to 30 minutes

after the addition of the reagents. Finally, for well no 12, one drop of TDA reagent


                                          60
was added. The test was evaluated immediately after the addition of the reagent. For

the 12B strip, the arginine reaction was interpreted differently at 24 hours and 48

hours incubation.     This was then referred to the Microbact Computer Aided

Identification Package where possible identification choices of bacteria were

displayed. The percentage figure shown against the organism name is the percentage

share of the probability for that organism as a part of the total probabilities for all

choices.



3.11.2     Partial Sequence of 16S rRNA for the Identification of Bacteria.



16S ribosomal RNA (rRNA) sequence was amplified via Polymerase Chain Reaction

(PCR) with two degenerate primers. PCR was invented in 1985 by Kary Mullis who

earned the Nobel Prize for Chemistry in 1993 (Mullis, 1990). The PCR is an in vitro

technique which allows the amplification of a specific deoxyribonucleic acid (DNA)

region that lies between two regions of known DNA sequence. Therefore,

sequencing of the 16S rRNA gene has served as an important tool for determining

phylogenetic relationships between bacteria. This gene is present in all bacteria, thus

it is a universal target for bacterial identification. Other than that, the use of the 16S

rRNA gene for identification of bacteria provides a faster, reliable and better ability

to accurately identify them in addition to contributing significantly in the discovery

of new species in microbiology laboratories and is highly acceptable in publications.



Identification using 16S rRNA is highly reliable as it is based on a large and

authentic database and the computerized Basic Local Alignment Search Tool

(BLAST) search. BLAST provides a method for rapid searching of nucleotide and


                                           61
protein databases. It can also assign the correct evolutionary and taxonomy position

of a species and the evolutionary conserved sequences detectable with a single base

pair alteration. This identification system involves the genomic DNA extraction of

the selected bacteria, the amplification of the genomic DNA using polymerase chain

reaction (PCR) and sequencing of the 16S genes.



3.11.2.1       Genomic DNA Extraction.



Cells from overnight 1.5 mL broth cultures were harvested by centrifugation at

16,000 x g for 3 minutes. The cellular genomic materials were extracted using

Wizard® Genomic DNA Purification KitTM (Promega, USA) according to the

manufacturer’s instructions. The DNA obtained at the end of the protocol was used

as template in the Polymerase Chain Reaction (PCR). The DNA was stored at 4 °C

and analysed with 1.0% (v/v) Tris-borate (TBE) agarose gel electrophoresis using

HindIII (Fermentas, USA) as marker to determine the size of the genomic DNA.



3.11.2.2       Quantification of Genomic DNA.



Two L aliquots of bacterial genomic DNA were diluted in 998 L (1:100) sterile

deionized distilled water. Absorbance of the DNA at 260 nm and 280 nm was

measured using Ultro Spec 2000 spectrophotometer (Pharmacia Biotech). The ratio

of A260 to A280 was calculated. The value of pure DNA sample should be in the

range of 1.8 and 2.0. A lower ratio is an indication of protein contamination.




                                          62
A DNA solution with A260 of 1 contains approximately 50              g/mL of DNA.

Therefore, the concentration of the DNA can be calculated according to the

following formula:



Concentration of DNA ( g/mL) = A260 x 50 g/mL x dilution factor (Mullis, 1990).



3.11.2.3       Agarose Gel Electrophoresis.



Four L of extracted DNA samples were mixed with appropriate loading dye and

resolved by electrophoresis in a 1% (w/v) agarose gel (Promega, USA). 1% agarose

in 1X TBE buffer (Promega, USA) was prepared by dissolving and melting 0.30 g of

agarose in 30 mL of 1X TBE buffer. Molten agarose was cooled to 50 ºC before

being poured in a sealed tray containing a comb. After the agarose gel solidified, the

comb was removed and the gel was ready to be used.



Samples were loaded into the wells and electrophoresis was carried out at room

temperature at 80 volts for about 45 min. HindIII marker (Fermentas, USA) was

used as a standard DNA molecular weight marker. The gel was then stained with

ethidium bromide (EtBr) at a concentration of 0.5         g/mL for 10 minutes and

destained with distilled water for 15 min. The agarose gel was observed and

photographed under UV light using Gel Doc™ 2000 (BioRad).




                                         63
3.11.2.4      Polymerase Chain Reaction (PCR).



The PCR employs two oligonucleotide primers, which flank the region of interest in

the target DNA. The reaction consists of template denaturation, primer annealing

and extension of the annealed primers by a thermostable DNA polymerase. The

DNA polymerase carries out the synthesis of complementary strand of DNA in the 5’

to 3’ direction, using a single-stranded template. The suitable primers are arranged

for primer extension reaction directing the synthesis of DNA towards the other. PCR

is stable in high temperatures (90-95 ºC) because the Taq polymerase used is heat

stable.



16S rRNA sequence was amplified via PCR with two degenerate universal primers

synthesized by 1st Base company: Forward: 5’-AGA GTT TGA TCA TGG CTC

AG-3’; and Reverse: 5’-ACG GTT ACC TTG TTA CGA CTT-3’. These primers

amplified the 1500-bp PCR product. PCR amplification were performed in 25 L of

reaction volumes in 0.5 mL thin-walled microcentrifuge tubes containing 10.8 L of

sterile deionized water, 2.5 L of 1X Taq buffer with NH4SO4 (Promega, USA), 2

 L of 25 mM MgCl2 (Promega, USA), 0.7 L of 10 mM dNTPs (Promega, USA),

1.5 L of 15 M forward and reverse primers, 5 L of template DNA and 1 of 5 unit

Taq DNA Polymerase (Promega, USA). The parameters were as follows: pre-

denaturation at 94 ºC for four minutes to completely denature template DNA and

contaminating enzymes; followed by 30 PCR cycles of denaturation at 94 ºC for 1

min, primer annealing at 58 ºC for 2 min and primer extension at 72 ºC for 2 min

were performed. This was followed by 1 cycle of 10 min at 72 ºC for complete

synthesis of elongating DNA molecules and hold at 4 ºC.          The reaction was


                                        64
amplified in a programmable thermal controller (MJ Research Inc., USA). The

amplified products were examined by gel electrophoresis.



3.11.2.5      Purification of Amplified PCR Product.



The amplified PCR product (25 L) was analysed using gel electrophoresis with 1%

(w/v) TBE agarose gel electrophoresis. One kb DNA ladder marker (Fermentas,

USA) was used as a marker to determine the size of the PCR product. The gel was

stained with ethidium bromide (0.5 g/mL), visualized and photographed under UV

light using Gel Doc 2000. The PCR product of ~ 1.5 kb was excised and purified

with Wizard® SV Gel and PCR Clean-Up System (Promega, USA) according to the

protocol in the manufacturer’s handbook. The purified PCR product was sent for

sequencing. The same forward PCR primers (5’-AGA GTT TGA TCA TGG CTC

AG-3’) and reverse primers (5’-ACG GTT ACC TTG TTA CGA CTT-3’)

synthesized by 1st Base company were used for sequencing.



3.11.2.6      16S rRNA Gene Sequence Analysis.



The sequences obtained were compared to known 16S rRNA gene sequences in the

composite non-reduntant database using the BLAST 2 sequences search program

(www.ncbi.nlm.nih.gov/BLAST/bl2seq/). The Pair-wise comparisons to assess the

level of homology between the two nucleotide sequences of the forward and the

reverse complement of the reverse primer sequences was determined using the

BLAST 2 sequences algorithm using the blastn option with the matrix turned off and




                                        65
default     parameters     available     from      the      server     at     NCBI

(http://www.ncbi.nlm.nih.gov/blast/) (Tatusova and Madden,1999).



Based on the overlapped region between the forward and reverse complement of the

reverse primer sequence, both of the sequences were combined and checked for

errors and omissions of bases especially at the overlapped region using the

CHROMAS software Version 1.45. The sequences were combined at bases giving

the least ambiguous characters and gap. The combined 16S rRNA gene sequence

was compared with the GenBank database using the BLAST server at NCBI

(http://www.ncbi.nlm.nih.gov/BLAST/).



3.11.2.7      Phylogenetic Tree Analysis.



A multiple alignment of 19 16S rRNA gene sequences that closely matches isolate

SC27 and S8 were retrieved from GenBank and were aligned using clustal_W

(Higgins et al., 1994) with the PHYLIP output option. The alignment was checked

by eye for any obvious mis-alignments.       Alignment positions with gaps were

excluded from the calculations. A phylogenetic tree was constructed using PHYLIP,

version 3.573 (J. Q. Felsenstein, PHYLIP-phylogeny inference package, version

3.573, Department of Genetics, University of Washington, Seattle, WA.

[http://evolution.genetics.washington.edu/phylip.html]) with Escherichia coli strain

K12 as the outgroup in the cladogram.



Evolutionary distance matrices for the neighbour-joining/UPGMA method were

computed using the DNADIST algorithm program. The program reads in nucleotide


                                        66
sequences and writes an output file containing the distance matrix.      Available

enterobacterial   16S   rRNA     sequences    were    selected   from    GenBank

(http://www2.ncbi.nlm.nih.gov). When several sequences available within the same

species were compared, the one containing fewest ambiguous positions was selected.

The four models of nucleotide substitution are those of Holmquist et al. (1972),

Kimura (1980), the F84 model (Kishino and Hasegawa, 1989; Felsenstein, 1992) and

the model underlying the LogDet distance (Lockhart et al., 1994). Phylogenetic tree

was inferred using the neighbour-joining method of Saitou and Nei (1987). With

each algorithm, confidence levels for individual branches within the tree were

checked by repeating the PHYLIP analysis with 1000 bootstraps (Felsenstein, 1985)

by the SEQBOOT program in the PHYLIP package. Majority rule (50%) consensus

trees were constructed for the topologies found using a family of consensus tree

methods called the MI methods using the CONSENSE program and the tree was

viewed using TreeView (Page, 1996).




                                        67
                                    CHAPTER 4



                          RESULTS AND DISCUSSION



4.1    Sample Collection.



A total of 250 bacterial isolates with different morphologies were successfully

isolated from soil sampling of 10 locations. The selections of the locations were

random in Peninsular Malaysia where the soil sampling was carried out without

concerns of the locations either were polluted or unpolluted. Bacteria with different

morphologies were chosen, numbered and sampling locations were recorded (Table

7).   Soil from each sampling locations gave different pH readings.            The pH

differences of the soil were influenced by the soil type and moisture. The range of

pH of the soil samples collected in this study was in the range of 4.5 to 8 (Table 7).

The temperature was not stated because temperature does not influence the

conditions in this study. The soil samples were taken at a depth of 5-8 cm from the

ground surface to avoid getting anaerobic bacteria (Ramanand et al., 1988). Aerobe

or facultative anaerobes were chosen in this study as these species are easier to

handle in the laboratory. The total isolates of the bacteria varies for each soil sample

(Table 7).




                                          68
Table 7: Location of sample collection, pH of the soil, sample type, GPS location
and total number of isolates from each location.

Locations                      pH       Sample     GPS location Total number
                                         type                    of isolates
Tanjung Blanja, Parit,        6.05        Soil          -               22
Perak.
____________________________________________________________________
Tanjung Karang, Selangor      5.01  Soil    N 03°00.129’       24
                                            E 101°42.423’
____________________________________________________________________
Parking Lot at Mutiara Hotel, 6.50  Soil         -             24
Johor Bahru.
____________________________________________________________________
Biochemistry and Microbiology 5.78  Soil     N 03°00.187’      20
Department, UPM, Serdang,                    E 101°42.415’
Selangor.
____________________________________________________________________
Bukit Ekspo UPM, Serdang,     6.80  Soil     N 03°00.159’      21
Selangor.                                    E 101°42.414’
____________________________________________________________________
Taman Tasik Taiping, Perak.   5.55  Soil           -           24

____________________________________________________________________
Sungai Congkak, Selangor.    4.50   Soil      N 03º13.265’     33
                                             E 101º50.424’
                             6.12   Soil      N 03º13.264’     10
                                             E 101º50.423’
                             4.50   Soil      N 03º13.249’     23
                                             E 101º50.422’
____________________________________________________________________
Sri Serdang Lake, Selangor.  7.90   Soil     N 03°00. 240’      6
                                              E 101° 42.863’
____________________________________________________________________
Taman Sri Serdang, Selangor. 7.80   Water    N 03°00.500’       6
                                              E 101°42.883’
                             7.39   Soil     N 03°00. 373’      3
                                              E 101°42.604’
____________________________________________________________________
Masjid Abidin Taman Skudai,  6.32   Soil             -         24
Skudai, Johor.
____________________________________________________________________
Total                                                          250




                                       69
4.2     MTT Assay of Bacterial Inhibition Studies.

4.2.1   Preliminary Screening of Bacterial Respiration Inhibited by Divalent
        Cations and Heavy Metals.


Out of total 250 isolates screened, only seven isolates were observed inhibited by six

tested heavy metals in the presence of two divalent cations, Mg2+ and Ca2+ at

concentrations of 50 mg/L and 25 mg/L respectively. The results were obtained by

visual observation. This can be seen where the original colour of the MTT dye

remained yellow. Seven bacterial isolates which were sensitive to the presence of

heavy metals are isolate S7, S1, S8, K104, SC27, SC3 and SC41. All of these

bacterial isolates were only sensitive to four heavy metals out of six heavy metals

tested at a final concentration of 10 mg/L. They are Ag, Hg, Pb and Cd while As and

Cu showed no inhibition of the reduction of the MTT dye. This means that As and

Cu were non-toxic to all of the bacterial isolates tested although the concentration of

heavy metals used was relatively high. According to Botsford (1998), R. meloliti

assay is more sensitive to these two heavy metals than in this study where the

concentration used was much lower to inhibit the reduction of the MTT dye. The

observation of colour development was done after incubation at 20 minutes.



The MTT dye was taken up by the cell and is reduced inside the cell; the reduced dye

precipitates inside the cells. Transport of the MTT dye could be the critical step and

toxic heavy metals could inhibit this transport. In detection of Hg, only bacterial

isolate SC27 showed susceptibility to Hg. On the other hand, four bacterial isolates

namely S7, S8, S1 and K104 were inhibited by Ag while isolate SC3 and SC41 were

inhibited by Cd and Pb respectively. All of the selected isolates were then screened

again with different concentration of heavy metals, ranging from 0.01 to 10 mg/L in


                                          70
order to determine the lowest concentration that can inhibit their MTT-reducing

ability.



In this preliminary screening, the addition of Mg2+ and Ca2+ in the assay is to take

into account their toxicity that will interfere with the ability of bacteria to reduce the

MTT dye, giving false positive results. This was previously observed by Bostford

(2000) in which these ions were commonly encountered in soil and water.

Therefore, in order to analyse water and soil samples for toxic heavy metals, a

method resistant to this inhibition by metal was sought. This has not been examined

and not taken into account with the Microtox™ assay. Reports have shown that Hg,

Cd, Cu and Zn were shown to be toxic using the Microtox™ assay system.

However, there were no reports if Ca and Mg are toxic using the Microtox™ assay.

Therefore, this system may not provide accurate data with water samples.



The concentrations of the ions that were used in the assay were based on the

concentrations used in the R. meliloti assay which were toxic to the bacteria; only the

concentration of Ca2+ was increased up to 5 times from the original concentration of

5 mg/L. Therefore, the system used in the present study is resistant to the effects of

both Ca and Mg when determining the toxicity of the heavy metals using the

bacterial isolates.



EDTA was also used in the R. meliloti assay to eliminate these divalent cations. It

was found that EDTA, a compound used routinely in biochemistry to chelate divalent

cations and its also has the ability to eliminate toxicity of divalent cations. At the

same time, the addition of EDTA will also alter the apparent toxicity of some heavy


                                           71
metals and organic chemicals. Since the selected bacteria were resistant to Mg2+ and

Ca2+, EDTA was not added in the reaction mixture of the MTT assay.



The MTT dye is an alternative assay for quantification of viable cells. The bacteria

used in the present study were cultured in 96-wells microplates and the observations

were made after 20 minutes. The 20 minutes incubation time was chosen as the

reduction of the dye was essentially complete at this time (Botsford, 1998). It was

observed that no more inhibition of reduction would appear with longer incubation.



The MTT dye chosen as an indicator is particularly useful when cells have a

tendency to adhere together or where other additives affect the colour of the growth

medium (Eloff, 1998). They detect oxidative enzyme systems (Liu, 1981; Glenner

and Stommel, 1961) by acting as electron acceptors. Using this assay, bacterial

growth can be easily measured in microplates when the colour changed from yellow

to dark blue. It is desirable to minimize the indicator concentrations since the

indicator may interfere both with bacterial growth and with other chemicals added to

the reaction mixture.



On the other hand, a certain excess of growth indicator in the solution is required so

that the amount of indicator does not constitute a limiting factor to the pellet/colour

of the solution. During the assay, the lowest possible concentration of the MTT dye

of 1 mM in the reaction mixture was used based on the literature which was in the

range of (1-10 mM) (~0.05-0.5% w/v).           If the concentration is below 0.1 mM

(~0.005%), the reading of the dye was unreliable as the pellet size diminished with

decreasing dye concentration. Besides that, non-dissolved bacteria pellets were used


                                          72
throughout the assay due to better reproducibility with non-dissolved pellets

compared to dissolve pellets for a majority of the tested bacteria. This step was

taken into account in this study since it would both reduce the amount of work in the

assay and the risk of contamination when adding another component to the bacterial

suspension (Gabrielson et al., 2002).



Based on the results, the ability of isolated bacteria to act as an indicator to detect the

selected heavy metals was limited. Only Hg and Ag were successfully detected by

the bacterial isolates after several assay attempts were performed. This is probably

because many microorganisms demonstrate resistance to metals in water, soil and

industrial wastes.    Metal resistance systems may have developed shortly after

prokaryotic life started and are present in nearly all bacterial types (Ji and Silver,

1995).   They arose because bacteria exist in an environment that has always

contained metals. Toxic metals interact with essential cellular components through

covalent and ionic bonding. At high levels, they can damage cell membranes, alter

enzyme specificity, disrupt cellular functions and damage the structure of DNA.

Microorganisms have adapted to the presence of both essential and nonessential

metals by developing a variety of resistance mechanisms (Bruins et al., 2000).



Some authors suggest the addition of glucose to the suspension to increase the

bacterial sensitivity to heavy metals (Girotti et al., 2002). However, this is not

supported in this study. Addition of 1% of glucose and acetate in the reaction

mixture did not result in any changes of sensitivity of the bacteria to the selected

heavy metals. For this reason, the addition of these two nutrients in the subsequent

assays was not carried out.


                                            73
4.3    Effect of Different Stages of Microbial Growth on Inhibitory Effect of
       Heavy Metals.


From the preliminary screening results, only Hg and Ag showed high inhibition to

the bacterial respiration in the presence of low concentration of heavy metals using

selected period of bacterial growth.     Therefore, bacterial isolates SC 27 which

indicated high inhibition in the presence of Hg was selected for further study while

bacterial isolates S8, S7, S1 and K104 were chosen for further screening with Ag to

select the most sensitive bacteria.



In order to determine which stages of bacterial growth is most sensitive to the lowest

concentration of selected Hg and Ag, the effect of different stages of microbial

growth on the inhibitory effect of heavy metals was investigated. This is important

to find out the minimum period of bacterial growth which can detect the lowest

concentration of the heavy metals as the previous results indicate that four bacterial

isolates can detect Ag at the same concentration.          In this case, the growth

optimization becomes important to find out which stage of bacterial growth is most

stable and sensitive to detect the heavy metals. A ‘higher sensitivity’ means that the

reduction inhibition of specific bacteria is detected at a lower concentration of the

tested heavy metals (Gabrielson et al., 2002).



The assay was performed using the same MTT assay procedure as previously

described but this time, five different periods of bacterial growth at 6, 8, 10, 14 and

16 hours were used. The results were observed after 20 minutes of incubation.

Based on visual observation, both isolates SC3 and SC41 which were isolated from

Sungai Congkak that were inhibited by Cd and Pb previously showed no inhibition


                                          74
although tested with all periods of bacterial growth. Thus, both of these isolates

were not selected to be as indicator. In contrast, the respiration of isolate SC27 was

inhibited at eight hours by Hg at a concentration of 0.3 mg/L as shown by the

inhibition of reduction of the MTT dye. The qualitative results can be seen in Table

8. This bacterium was also isolated from Sungai Congkak. Table 8 shows the

inhibition effect of isolate SC27, SC41 and SC3 at selected periods of bacterial

growth by Hg, Pb and Cd at different concentrations based on the intensity of

inhibition colour of the MTT dye reduction.



Based on the results in Table 8, the comparison between the different periods of

bacterial growth with the lowest concentration of heavy metals was obtained. Hence,

isolate SC27 at eight hours of growth was used in subsequent tests. This was

supported by quantitative data as shown in Figure 6 where the inhibition of the

reduction of the MTT occurred significantly at eight hours of the bacterial growth

compared to other periods of bacterial growth. The differences between absorbance

at 0 mg/L of Hg and absorbance at 0.3 mg/L of Hg was plotted with the period of

bacterial growth to determine selected periods of bacterial growth based on the

higher absorbance reading (Appendix IV).       Data from the results were used to

determine the IC50 value of Hg for the isolate SC27.




                                         75
Table 8: The inhibition of MTT dye reduction at different periods of bacterial
growth by different concentrations of heavy metal.

Isolate                    Heavy metals concentration (mg/L)
                 0 0.01 0.05 0.10 0.20 0.30 0.50 1.00 2.50 5.00 7.50 10.00

SC27 (Hg)
6 hours          x   x     x     x    xx       xx    xxxx xxxx xxxx xxxx xxxx xxxx
8 hours          x   x     x     x    xx       xxx   xxxx xxxx xxxx xxxx xxxx xxxx
10 hours         x   x     x     x     x        x    xx xxx xxxx xxxx xxxx xxxx
14 hours         x   x     x     x     x        x    xx xxx xxxx xxxx xxxx xxxx
16 hours         x   x     x     x     x        x    xx xxx xxx xxx xxx xxx


SC41 (Pb)
6 hours          x   x     x     x    x         x    x    x     x    x    x     x
8 hours          x   x     x     x    x         x    x    x     x    x    x     x
10 hours         x   x     x     x    x         x    xx   xx    xx   xx   xx    xx
14 hours         x   x     x     x    x        xx    xx   xx    xx   xx   xx    xx
16 hours         x   x     x     x    x         x    x     xx   xx   xx   xx    xx


SC3 (Cd)
6 hours          x   x     x     x    x        x     x     x    x    x     x    x
8 hours          x   x     x     x    x        x     x     x    x    x     x    x
10 hours         x   x     x     x    x        x     x     x    x    x     x    x
14 hours         x   x     x     x    x        x     x     x    x    x     x    x
16 hours         x   x     x     x    x        x     x     xx   xx   xx    xx   xx


Note: The star indicates the intensity of inhibition colour of the reduction of the
      MTT dye.
      x      : Low inhibition/reduction occurred.
      xx     : Medium inhibition.
      xxx : Good inhibition.
      xxxx : High inhibition.




                                          76
                                                                   6 hours
                        0.35                                       8 hours
                                                                   10 hours
                                                                   14 hours
                        0.30                                       16 hours


                        0.25
  Absorbance (550 nm)




                        0.20


                        0.15


                        0.10


                        0.05


                        0.00
                               0   0.1   0.2        0.3     0.4    0.5        0.6
                                         Hg concentration (mg/L)


Figure 6: The inhibition of the reduction of MTT dye by isolate SC27 in
mercury using five different growth periods. Data collection begun after
initially subtracting the initial absorbance from the final absorbance at 20
minutes. Data represent mean ± SEM, n=3.




                                               77
As stated earlier, there are four bacterial isolates that were sensitive to Ag at a final

concentration of 0.5 mg/L. They are isolate S8, S7, S1 and K104. Based on the

results in Figure 7, isolate S8 showed significant inhibition of respiration at 12 hours

of growth at a concentration of 0.2 mg/L where the inhibition slightly drops and

became consistent or linear with increasing concentrations where the Ag inhibited

completely the respiration of the bacterial cells S8. Otherwise, the inhibition of

isolate S8 respiration at 12 hours of growth always gave consistent results after the

assay was repeated several times in triplicate compared to other periods of growth as

well as other selected bacterial isolates. However, the differences between

absorbance at 0 mg/L of Ag and absorbance at 0.2 mg/L of Ag was plotted with the

period of bacterial growth to determine selected periods of bacterial growth based on

the higher absorbance reading (Appendix IV). The inhibition of bacterial growth and

the sensitivity of other bacterial isolates to Ag were shown in Figure 8, 9 and 10.



As can be seen in Figure 8 and 9, the inhibition of isolate S7 and S1 respiration at 12

hours of growth are seemed similar with the inhibition of isolate S8. It is not

selected since it did not give consistent results after the assay was repeated several

times.   In addition, this was supported by determine the differences between

absorbance at 0 mg/L of Ag and absorbance at 0.2 mg/L of Ag with the period of

bacterial growth which is low absorbance reading was obtained (Appendix IV).



In conclusion, isolate S8 that was isolated from Sri Serdang Lake, Selangor was

sensitive in detection of Ag at a minimum concentration of 0.2 mg/L. Therefore,

isolate S8 at 12 hours growth was used for subsequent tests. Data from the results

was used to determine the IC50 value of Ag using the isolate S8.


                                           78
                        1.00

                        0.90                                         8 hours
                                                                     10 hours
                        0.80
                                                                     12 hours
                                                                     14 hours
                        0.70
                                                                     16 hours
  Absorbance (550 nm)




                        0.60

                        0.50

                        0.40

                        0.30

                        0.20

                        0.10

                        0.00
                               0.0   0.2    0.4        0.6   0.8     1.0        1.2
                                           Ag concentration (mg/L)

Figure 7: The inhibition of the reduction of MTT dye by bacterial isolate S8 in
silver using five different growth periods. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                                  79
                       0.90


                       0.80                                       8 hours
                                                                  10 hours
                       0.70                                       12 hours
                                                                  14 hours
                                                                  16 hours
 Absorbance (550 nm)




                       0.60


                       0.50


                       0.40


                       0.30


                       0.20


                       0.10


                       0.00
                              0   0.2    0.4        0.6    0.8      1        1.2
                                        Ag concentration (mg/L)


Figure 8: The inhibition of the reduction of MTT dye by bacterial isolate S7 in
silver using five different growth periods. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                               80
                        0.90


                        0.80                                       8 hours
                                                                   10 hours
                        0.70                                       12 hours
                                                                   14 hours
                                                                   16 hours
  Absorbance (550 nm)




                        0.60


                        0.50


                        0.40


                        0.30


                        0.20


                        0.10


                        0.00
                               0   0.2    0.4        0.6   0.8     1     1.2
                                         Ag concentration (mg/L)


Figure 9: The inhibition of the reduction of MTT dye by bacterial isolate S1 in
silver using five different growth periods. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                                81
                        0.70

                                                                    8 hours
                        0.60                                        10 hours
                                                                    12 hours
                                                                    14 hours
                        0.50
  Absorbance (550 nm)




                                                                    16 hours


                        0.40



                        0.30



                        0.20



                        0.10



                        0.00
                               0   0.2   0.4        0.6   0.8       1       1.2
                                          Ag concentration (mg/L)


Figure 10: The inhibition of the reduction of MTT dye by bacterial isolate K104
in silver using five different growth periods. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                               82
4.4    Determination of the IC50 Value of Mercury and Silver.



The IC50 and its 95% confidence interval value for Hg and Ag were determined using

the non-linear regression curves for one site binding hyperbola generated using the

Graphpad Prism™ version 4.0 software. The IC50 values are dependent on the

chosen testing concentration range. The data were obtained from section 4.3 in order

to determine the IC50 value for Hg and Ag respectively.



Table 9 shows the percent of inhibition of the MTT dye reduction by Hg and Ag on

isolate SC27 and S8, respectively. The degree of inhibition was determined on the

basis of measured absorbance values, with the control representing 0% inhibition. It

can be seen that isolate SC27 was inhibited at 0.3 mg/L of Hg with 63.40%

inhibition, while 90.53% inhibition of isolate S8 is seen at 0.2 mg/L of Ag. These

supported the previous results in section 4.3 where the lowest concentration of Hg

and Ag detected were 0.3 mg/L and 0.2 mg/L, respectively. Although Ag at 0.1

mg/L showed an inhibition of 54.33%, this was not the lowest concentration to

inhibit the reduction of the MTT dye. This is because from the inhibition graph

(Figure 7) in section 4.3, the concentrations of 0.2 mg/L showed more significant

inhibition by Ag on isolate S8 where the gap between final and initial absorbance

was high. In addition, isolate SC27 is seemed almost inhibited at 0.5 mg/L of Hg,

while 94.21% inhibition of isolate S8 is seen also at 0.5 mg/L.




                                          83
Table 9: The percent of inhibition of the MTT dye reduction by mercury (Hg)
and silver (Ag) on isolate SC27 and S8 respectively.

       Test toxicant              Hg (%±SEM)                    Ag (%±SEM)
       0.01 mg/L                 5.42±0.016                      6.63±0.001
       0.05 mg/L                11.45±0.003                     27.39±0.021
       0.10 mg/L                18.52±0.012                     54.33±0.008
       0.20 mg/L                52.26±0.004                     90.53±0.005
       0.30 mg/L                63.40±0.003                     91.95±0.007
       0.50 mg/L                83.13±0.004                     94.21±0.004

Note: The inhibition % were calculated as follows: [(Initial control absorbance-final
absorbance)/ Initial control absorbance] x 100. Each entry in the table represents the
mean (±SEM) of at least three determinations.




As reported in other literature such as with α-amylase biosynthesis in B. subtilis and

β-galactosidase biosynthesis in E. coli assay, the data was presented as percent of

inhibition. It was reported by Guven et al. (2003) that α-amylase biosynthesis in B.

subtilis is more sensitive to heavy metals compared to β-galactosidase biosynthesis in

E. coli. It seems that Hg is by far the strongest inhibitor of α-amylase system among

the heavy metals tested. The effect of Hg in E. coli β-galactosidase test system is not

detected at 0.1 mg/L, whereas 53.45% inhibition is seen at the same concentration

for the α-amylase system.      In addition, β-galactosidase biosynthesis is totally

inhibited at 25 mg/L of Hg, while 100% inhibition of α-amylase enzyme is seen only

at 5 mg/L. These indicate that isolate SC27 in this study is potentially sensitive to

detect Hg at a concentration of 0.2 mg/L with 52.26% inhibition and almost inhibited

at 0.5 mg/L with 83.13% inhibition. Isolate S8 is potentially sensitive to detect Ag at

a concentration of 0.2 mg/L with 90.53% inhibition where there is no precious data

presented in this bioassays based on enzyme biosynthesis in E. coli and B. subtilis

that can detect Ag.




                                          84
In contrast, inhibition on R. meloliti by Hg is in the concentration of 0.0159 mg/L

while there is no data reported about Ag detection in this R. meloliti bioassay. The

comparative IC50 data for both metals, Hg and Ag in different toxicity tests are

shown in Table 10. The results for metals from this study were also compared with

fish (rainbow trout) assay, Daphnids (Daphnia magna), immobilized urease, free

urease and Microtox™ toxicity data. The IC50 values for Hg and Ag in this study are

0.2698 mg/L and 0.0732 mg/L respectively. The IC50 value of immobilized urease

was also included since the ubiquitous presence of high level of background

ammonia in samples usually prevent environmental analysis, hence there is a need to

immobilize the urease (Jung et al., 1995). As shown in Table 10, the Microtox™

toxicity test was the most sensitive assay in the detection of Hg and Ag.




Table 10: The sensitivity of isolate SC27 and S8 to mercury and silver
respectively in comparison to immobilized urease, free urease, Microtox™,
Daphnia magna and fish bioassays (rainbow trout).

                                    IC50 or EC50 (mg/L)
Metals            Immobilized Free         Microtox™ b 48 hours      96 hours
                  ureaseb     ureaseb       IC50        Dapnia      Rainbow
                  IC50         IC50                     magnaa       troutb
                                                        EC50         IC50
Hg                 0.33±0.021 0.008±0.002 0.003±0.002 0.0052-0.21 0.033-0.21
(0.2698±0.004)
____________________________________________________________________
Ag             n.d.       n.d.       0.008±0.001 1.930(LC5096)a 0.05
(0.0732±0.002)

a
    Rodgers et al., 1997.
b
    Jung et al., 1995.




                                          85
In this study, kinetic analysis was not carried out since it has been reported in the

literature that does not provide any useful information concerning the nature of the

reduction of the MTT dye and its inhibition by toxic chemicals (Botsford, 2000).

This is because the data showed that the kinetics of the inhibition was “mixed”. The

inhibition does not appear to be competitive where the dye and the toxic chemical do

not simply compete for electrons in the reduction. The inhibition is also not non-

competitive when the toxic chemical does not bind to the component responsible for

reduction at a site distinct from the active site but influencing the reaction. The

inhibition is not uncompetitive either which is the toxic chemical does not simply

damage the component so it can’t reduce the dye as effectively.



In conclusion, the isolate SC27 and S8 are only sensitive to Hg and Ag respectively

with the IC50 value of 0.2698 mg/L (Figure 11) and 0.0732 mg/L (Figure 12)

respectively. For the results reported here, nearly all the values presented with the

correlation coefficient was greater than 0.9. Results are expressed as mg/L as are

customary among toxicologists (Botsford, 1998).




                                         86
                         0.25



                         0.20
   Absorbance (550 nm)




                         0.15



                         0.10



                         0.05



                         0.00
                                0   0.1   0.2        0.3     0.4   0.5       0.6
                                           Concentration (mg/L)


Figure 11: Inhibition of isolate SC27 by mercury at eight hours bacterial growth
as measured using the MTT assay. The IC50 value of mercury is 0.2698 mg/L as
generated by Graphpad Prism. Data represent mean ± SEM, n=3. Correlation
coefficient value, R2 = 0.9842.




                                                87
                        1.00



                        0.80
  Absorbance (550 nm)




                        0.60



                        0.40



                        0.20



                        0.00
                               0   0.2   0.4        0.6     0.8   1       1.2
                                          Concentration (mg/L)


Figure 12: Inhibition of isolate S8 by silver at 12 hours bacterial growth as
measured using the MTT assay. The IC50 value of silver is 0.0732 mg/L as data
was generated by Graphpad Prism. Data represent mean ± SEM, n=3.
Correlation coefficient value, R2 = 0.9831.




                                               88
4.5    Effect of Different Buffers System.



The use of several different buffer substances revealed the influence of the latter on

the intensity of heavy metal inhibition. This opens the path to both the selective

analysis of heavy metals via pattern recognition and to the improvement of detection

sensitivity. The assay was performed by replacing the PBS buffer, pH 7.5 that was

used previously in this study with these six different buffers.      They are Pipes,

Imidazole, Hepes, Bicine, Tricine and Tris-HCl at the same pH. The subsequent

method of the assay is the same. Figure 13 and Figure 14 shows the interaction of

Hg and Ag respectively with the buffer substance influences the intensity of

inhibition.



The present results shows that 10 mM of PBS, pH 7.5 is the most stable buffer

system compared to other buffers that were used in the inhibition on both isolates,

SC27 and S8 by Hg and Ag respectively. As can be seen in Figure 13 and Figure 14,

the other six buffers used; Pipes, Imidazole, Hepes, Bicine, Tricine and Tris-HCl did

not stabilize the system but instead inhibited the system even in the control. There

was no reduction of the MTT dye in all buffers used except in the PBS buffer.

Therefore, the selection of suitable chemically inert buffer systems improves the

sensitivity of the detection method where the PBS buffer was selected as the best

buffer system in this study.




                                         89
                       1.20
                                                                                            Control
                                                                                            Buffer
                       1.00
 Absorbance (550 nm)




                       0.80


                       0.60


                       0.40


                       0.20


                       0.00




                                                                                                 l
                                                               e


                                                                       ne
                                            e
                               s




                                                   s




                                                                                    S



                                                                                             C
                                                            in
                                         ol
                              pe




                                                  pe




                                                                                PB



                                                                                            -H
                                                                        i
                                                          c



                                                                     ic
                                      az
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                                                He



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                                                                                          is
                                                                   Tr



                                                                                M
                                     id




                                                                                        Tr
                         M




                                                       M




                                                                               m
                                              M
                                   Im




                                                                  M
                        m




                                                      m




                                                                                        M
                                             m




                                                                            10
                                                                 m
                                  M




                                                                                       m
                       16




                                                   16
                                          16




                                                              10
                                 m




                                                                                    10
                              16




Figure 13: Effect of different buffers system on inhibition of MTT reduction
isolate SC27 by Hg at the final concentration of 0.3 mg/L. The control for each
buffers were carried out without Hg added. Data represent mean ± SEM, n=3.




                                                       90
                        2.00                                                 Control
                        1.80                                                 Buffer

                        1.60
  Absorbance (550 nm)




                        1.40

                        1.20

                        1.00
                        0.80

                        0.60

                        0.40
                        0.20

                        0.00
                                                         e
                                     s




                                                         e
                                                         s



                                                       ne




                                                                   S



                                                                                   l
                        -0.20




                                                                              C
                                                       ol
                                  pe




                                                      pe




                                                      in



                                                                 PB



                                                                              -H
                                                     ci
                                                    az




                                                    ic
                                Pi




                                                  He



                                                  Bi




                                                                            is
                                                 Tr



                                                                 M
                                                 id
                           M




                                                                         Tr
                                                                m
                                              Im



                                               M



                                               M



                                              M
                          m




                                                             10
                                             m



                                             m




                                                                         M
                                            m
                                       M
                        16




                                          16



                                          16




                                                                        m
                                         10
                                      m




                                                                     10
                                   16




Figure 14: Effect of different buffers system inhibition of MTT reduction on
isolate S8 by Ag at the final concentration of 0.2 mg/L. The control for each
buffers were carried out without Ag added. Data represent mean ± SEM, n=3.




                                                 91
4.6     Interfering Effect of Other Xenobiotics on the MTT Assay by Isolate
        SC27 and S8.


The effects of the inhibition of reduction of the MTT dye on the isolate SC27 and S8

by xenobiotics were investigated in order to rule out the interference effects except

inhibition by the tested heavy metal in the assay. The assay was carried out as

previously described in the presence of xenobiotics.         As a control, the tested

xenobiotics were replaced with deionized water. The observation and the absorbance

measurement were done after 20 minutes of incubation after the MTT dye was added

in the reaction mixture.



4.6.1   Interfering Effects of Xenobiotics on Isolate SC27.



The results of the interfering effect of 8 heavy metals tested on isolate SC27 are

shown in Figure 15. As can be seen, all of these tested heavy metals on isolate SC27

indicate no significant inhibition of the MTT dye reduction at the concentration of 5

mg/L when compared with the control. In comparison with other tests available such

as R. meloliti bioassay, it was found that the IC50 values of the heavy metals tested in

this bioassay was much lower than in this study. As reported earlier by Botsford,

1998, the IC50 values of Cu is 0.95 mg/L while IC50 of Cd, Ca, Mn and Zn are 0.791

mg/L, 5.65 mg/L, 1.44 mg/L and 0.84 mg/L respectively. Meanwhile, IC50 values of

heavy metals such as Co (cobalt), Mg, Ni (nickel) and Se (selenium) were much

higher than in this study.



Figure 16 showed 10 different xenobiotics that consisted of solvents and organic

chemicals at the concentration of 0.4%. From the results, it was found that there is


                                          92
also no significant inhibition of the reduction of the MTT dye on isolate SC27 by

tested xenobiotics as well as eight tested pesticides at the concentration of 4 mg/L as

shown in Figure 17.            Therefore, all these tested xenobiotics did not show any

interference effect on this assay using bacterial isolate SC27 for Hg detection.




                        0.60



                        0.50



                        0.40
  Absorbance (550 nm)




                        0.30



                        0.20



                        0.10



                        0.00
                                                               nc
                                     um




                                       m
                                        e
                                       ol




                                                                               t

                                                                            en
                                     um
                                      se




                                                                             al
                                     at

                                   iu
                                    tr




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                                  ne




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                                                                          st
                                  ni


                                  or




                                  si
                                 on




                                 ss




                                                                       ng
                               ga


                               ae
                               le




                                                                    C
                               B
                         C




                              ta
                            Se




                                                                    Tu
                             C
                            an
                           Po

                          M




Figure 15: Effects of inhibition of isolate SC27 respiration by heavy metals at
the final concentration of 5 mg/L using MTT assay. Data collection begun after
initially subtracting the initial absorbance from the final absorbance at 20
minutes. Data represent mean ± SEM, n=3.

                                              93
                       0.80


                       0.70


                       0.60
 Absorbance (550 nm)




                       0.50


                       0.40


                       0.30


                       0.20


                       0.10


                       0.00



                                             0
                                            te




                                   P y ol



                                it o ine
                                 ne ne




                                             e
                                 n - co l
                                            ol




                                             l
                                           ne




                                             l
                                          no
                                          no




                                          10
                                          in
                                          n
                                         ta
                                         tr

                                        to



                                        xa




                                      rid

                                      am
                                       ha
                                        y




                                      pa
                                      ha




                                       x-
                                      on




                                      ce
                                      gl
                          hy ce



                                    he




                                    et
                                   Et

                                   ro




                                    n
                                   la
                       C




                                   yl
                                   A




                                 M
                               op




                               th
                              hy
                              le




                             Tr
                             ie
                            Is
                           Et




                           D
                        Et




Figure 16: Effects of inhibition of isolate SC27 respiration by xenobiotics at the
final concentration of 0.4% using MTT assay. Data collection begun after
initially subtracting the initial absorbance from the final absorbance at 20
minutes. Data represent mean ± SEM, n=3.




                                       94
                        0.60



                        0.50
  Absorbance (550 nm)




                        0.40



                        0.30



                        0.20



                        0.10



                        0.00
                                             r




                                             d




                                             e
                                          ba
                                            ol


                                            n




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                                       ac




                                      os
                                      ro
                                     on


                                      iu




                                    su




                                    ap
                                     zi
                                    al




                                   lo




                                  ph
                                   D




                                   ic
                                  ia
                         C




                                do




                                 m
                                 et




                                oc


                                 D
                                D




                               ly

                              ou
                              M

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                             id




                             G

                            C
                          Im




Figure 17: Effects of inhibition of isolate SC27 respiration by pesticides at the
final concentration of 4 mg/L using MTT assay. Data collection begun after
initially subtracting the initial absorbance from the final absorbance at 20
minutes. Data represent mean ± SEM, n=3.




                                       95
4.6.2   Interfering Effects of Xenobiotics on Isolate S8.



The influence of foreign species on the proposed method on the use of isolate S8 for

Ag detection was investigated. The interference effects on inhibition of isolate S8

respiration by eight selected heavy metals is shown in Figure 18. Based on the

results, all of eight heavy metals used at the concentration of 5 mg/L showed no

significant inhibition of the MTT dye reduction and the blue colour of the reduction

of MTT dye also can be observed.



Based on the results shown in Figure 19 and 20, it was also found that the other

xenobiotics consisting of solvents, organic chemicals and pesticides did not show

significant inhibition of the MTT dye reduction showing that inhibition of isolate S8

respiration was not interfered. All the xenobiotics tested in this study were selected

because they exist commonly in the environment due to high usage and is found in

the effluent of industrial discharge.



Other bioassays, such as the R. meliloti bioassay showed that the concentration of

heavy metals that were used to inhibit the reduction of the dye were much higher

than from this study. Botsford (1998) reported that the IC50 values for acetone,

ethanol and methanol are 68 000 mg/L, 73 362 mg/L and 66 027 mg/L respectively

when using the R. meloliti bioassay. In contrast, the effect of an organochlorine

insecticide called endosulfan on α-amylase biosynthesis was much higher than other

bioassays where lower concentrations were used. At 0.048 mg/L endosulfan, the

percent of inhibition was 22, while 95% inhibition was observed at 2.4 mg/L (Guven

et al., 2003). This showed that α-amylase biosynthesis is more sensitive in detection


                                         96
of endosulfan compared to this bioassay in which the inhibition of the reduction by

endosulfan was low.




                        1.00

                        0.90

                        0.80

                        0.70
  Absorbance (550 nm)




                        0.60

                        0.50

                        0.40

                        0.30

                        0.20

                        0.10

                        0.00
                                                         nc
                                       m
                                     um




                                                                  lt
                                        e
                                       ol




                                     um




                                                                            en
                                      se
                                     at




                                                                 a
                                   iu
                                    tr




                                                       Zi

                                                              ob


                                                                          st
                                  ne
                                  ni


                                  or




                                  si
                                 on




                                 ss




                                                                       ng
                               ga


                               ae
                               le




                                                              C
                               B
                         C




                              ta
                            Se




                                                                  Tu
                             C
                            an
                           Po

                          M




Figure 18: Effects of inhibition of isolate S8 respiration by heavy metals at the
final concentration of 5 mg/L using MTT assay. Data collection begun after
initially subtracting the initial absorbance from the final absorbance at 20
minutes. Data represent mean ± SEM, n=3.




                                        97
                         1.00

                         0.90

                         0.80

                         0.70
   Absorbance (550 nm)




                         0.60

                         0.50

                         0.40

                         0.30

                         0.20

                         0.10

                         0.00



                                               0
                                              te




                                     Py ol



                                  it o ine
                                   n e ne




                                               e
                                              ol




                                      he l




                                               l
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                                               l
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                                            no
                                            no




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                                            in
                                            n
                                           ta
                                           tr



                                          yc
                                          to




                                          xa




                                        rid

                                        am
                                         ha
                                        pa
                                        ha




                                         x-
                                        on




                                        ce
                                       gl
                                       ce




                                      et
                                     Et

                                     ro




                                      n
                                     la
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                                     yl
                                     A



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                                 op




                                  th
                                hy
                                le




                               Tr
                               ie
                              Is
                             Et
                            hy




                             D
                          Et




Figure 19: Effects of inhibition isolate S8 respiration by xenobiotics at the final
concentration of 0.4% using MTT assay. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                        98
                        0.90


                        0.80


                        0.70
  Absorbance (550 nm)




                        0.60


                        0.50


                        0.40


                        0.30


                        0.20


                        0.10


                        0.00
                                             r




                                             d




                                             e
                                          ba
                                            ol


                                            n




                                            n


                                            n




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                                          lo




                                          at
                                          pi




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                                         ro




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                                        no
                                         tr




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                                     on


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                                D




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Figure 20: Effects of inhibition isolate S8 respiration by pesticides at the final
concentration of 4 mg/L using MTT assay. Data collection begun after initially
subtracting the initial absorbance from the final absorbance at 20 minutes.
Data represent mean ± SEM, n=3.




                                       99
4.7    Preliminary Testing on Water Samples from Polluted Areas.



Preliminary field trials were set up in various locations in the Pulau Pinang industrial

estate, a site well known to harbour many metal-related industries. The purpose of

this field trial was to determine the sensitivity of this bioassay in detecting heavy

metals in particular Hg and Ag using both isolates SC27 and S8. The content of

heavy metals in water samples as determined by this microbial bioassay was

compared to readings obtained using AES. Results were expressed in mg/L. Four

sites were chosen based on their vicinity to industrial effluent discharge locations and

were identified by GPS as shown in Table 11. Water samples from these sites were

filtered and directly applied to the bioassay reaction mixture.



Table 11: Locations of water samples tested taken from Pulau Pinang river.

 Locations                            Sample                    GPS location
Sungai Pinang                           1                 N 05° 21.09’, E 100° 24.39’
Sungai Juru                              2                N 05° 21.06’, E 100° 24.21’
Prai industrial estate                   3                N 05° 21.07’, E 100° 24.30’
Sungai Derhaka                           4                N 05° 21.09’, E 100° 24.17’



As can be seen in Figure 21, the results showed that none of these four water samples

indicate any inhibition of the MTT dye reduction by isolate S8 compared to control.

This indicates silver was not present in these four pre-treated water samples or

present in a low concentration. The presence of Ag would be accompanied by

inhibition of the MTT dye reduction by isolate 8 as S8 is sensitive to Ag (Figure 22).

AES results as presented in Figure 22 also showed Ag was not present or present in

very low concentration.



                                          100
In contrast, Hg was detected in sample 4 with the reduction of the MTT dye showing

appreciable inhibition compared to control (Figure 21). The lowering of absorbance

is significant (R< 0.05). Meanwhile, other pre-treated water samples showed no

inhibition suggesting Hg could not be detected in the tested water samples. This

maybe because in the form of Hg is not soluble or accessible to the microbe. The

results showed that isolate SC27 has the potential to detect the presence of Hg in this

preliminary testing of pre-treated water samples. Analysis using AES also shows

that highest concentration of Hg is detected in sample 4.



In conclusion, this microbial bioassay technique for the detection of at least Hg could

be used in the future as the isolates tested were able to detect Hg in environmental

sample.




                                         101
                        1.0
                                                                         Iso SC27
                                                                         Iso S8
                        0.9


                        0.8


                        0.7
  Absorbance (550 nm)




                        0.6


                        0.5


                        0.4


                        0.3


                        0.2


                        0.1


                        0.0
                              Control   Sample 1 Sample 2 Sample 3 Sample 4


Figure 21: The inhibition studies on pre-treated water samples from polluted
areas by isolate SC27 and S8 for the detection of Hg and Ag respectively using
MTT assay. Data collection begun after initially subtracting the initial
absorbance from the final absorbance at 20 minutes. Data represent mean ±
SEM, n=3.




                                                  102
                          22

                          20
                                                                       Hg
                          18                                           Cu
                                                                       Zn
                          16                                           Fe
                                                                       Cd
                          14
  Concentrations (mg/L)




                                                                       Ag
                          12                                           Pb
                                                                       As
                          10

                           8

                           6

                           4

                           2

                           0
                               1   2            3              4
                          -2
                                       Sample


Figure 22: Concentrations of heavy metals taken from Pulau Pinang as
determined using AES (Perkin Elmer Optima 3000). Results were expressed in
mg/mL. Sample: 1= Sungai Pinang, 2= Sungai Juru, 3= Prai industrial estate,
4= Sungai Derhaka. Data represented were in the mean of triplicates.




                                       103
4.8     Identification of Bacteria.



Isolate SC27 and S8 were taxonomically characterized for their physiological and

biochemical characteristics and also for their 16S rRNA sequence similarities. Each

isolate was examined physiologically by the characteristics of its colony and their

type was differentiating by microscopic observation through Gram staining. Further

analysis of their biochemical characteristics was carried out using the Microbact™

kit while 16S rRNA sequence identification was carried out to confirm their identity.

The identification was performed up to the genus level only.



4.8.1   Colony Examination of Isolate SC27 and S8.



Colonies of isolate SC27 grown on nutrient agar appeared to be circular, smooth,

convex and yellowish in colour while colonies of isolate S8 were usually

homogenous for the first day or two and then became differentiated into a convex,

pigmented and with a relatively opaque centre and an effuse, colourless, almost

transparent periphery with an irregular crenated edge. The colonial characters of

bacteria are susceptible to considerable variation: colonies may vary in size, shape,

opacity, surface and consistency. There is likewise great variation in their ability to

produce pigment.



Meanwhile, Gram staining was carried out to differentiate the bacterial isolates.

Bacteria are subdivided by their reaction to this stain where the colour retained was

termed as Gram-positive and those which are decolourized was termed as Gram-

negative. A single colony from 24 hours culture was used for Gram staining as


                                         104
young culture reduces ambiguous results. The stained culture was observed under

light microscope at 100, 400 and 1000 times magnification. Morphological studies

showed that both the bacteria are Gram-negative rods which occurred singly and in

pairs. The microscopic and macroscopic observation of isolate SC27 and S8 are

summarized in Table 12.



Table 12: Microscopic and macroscopic observation of isolate SC27 and S8.

 Isolate        Gram          Morphology    Morphology           Oxidase
               staining         cell           colony             test
  SC27      Gram negative             Circular, smooth, convex
                              Small rod                          Negative
                                      and yellowish colour.
____________________________________________________________________
  S8      Gram negative   Rod         Convex, opaque centre      Negative
                                      and an effuse, colourless,
                                      irregular crenated edge.



The main difference between Gram-positive and Gram-negative organisms is seen as

whether they can be eluted. This may be due either to it being bound to specific

organism components or to differences in permeability of the cell wall of Gram-

positive and Gram-negative organisms. Figure 23 shows the photomicrograph of

isolate SC27 that are Gram-negative (red) rod, usually 0.6 to 1.0 m in width and 1.2

to 3.0 m in length while Figure 24 shows the photomicrograph of isolate S8 which

are Gram-negative (red) rod, usually 0.5 to 0.8 m in diameter and 0.9 to 2.0 m in

length.




                                        105
Figure 23: Photomicrograph of Gram-negative (red) rod isolate SC27 by
observation under light microscope 1000 x magnification (Olympus BX40.F4,
Japan) with immersion oil. This photomicrograph was obtained using Olympus
PM-C35DX, Japan camera after undergoing Gram staining procedures.




Figure 24: Photomicrograph of Gram-negative (red) rod isolate S8 by
observation under light microscope 1000 x magnification (Olympus BX40.F4,
Japan) with immersion oil. This photomicrograph was obtained using Olympus
PM-C35DX, Japan camera after undergoing Gram staining procedures.




                                   106
4.8.2   Biochemical Test Using Microbact™ kit.

4.8.2.1 Identification of Isolate SC27 and S8.



Tests using Microbact™ kit was conducted in order to identify isolates SC27 and S8.

Results indicate that isolate SC27 and isolate S8 showed typical characteristics of the

species Enterobacter cloacae and Serratia marcescens with 97.27% and 90.79%

similarity respectively based on The Microbact Computer Aided Identification

Package. The summary of results of the biochemical tests using Microbact™ 24E

(12A+12B) kit for isolate SC27 and S8 are shown in Table 13 and Table 14

respectively.



Table 13: Biochemical test result using Microbact™ 24E (12A+12B) kit for
isolate SC27.

        Reaction              Result                 Reaction               Result

        Lysine                  -                    Gelatin                   -
        Ornithine               +                    Malonate                  +
        H2S                     -                    Inositol                  -
        Glucose                 +                    Sorbitol                  +
        Mannitol                +                    Rhamnose                  +
        Xylose                  +                    Sucrose                   +
        ONPG                    +                    Lactose                   -
        Indole                  -                    Arabinose                 +
        Urease                  +                    Adonitol                  -
        V.P.                    +                    Raffinose                 +
        Citrate                 +                    Salicin                   +
        TDA                     -                    Arginine                  +

Note: + positive result
      – negative result




                                         107
Isolate SC27 is facultatively anaerobic. Enterobacter ferments mannitol, glucose,

sucrose, xylose, inositol, sorbitol and salicin with the production of acid and

sometimes small bubbles of gas. It showed negative results with indole and methyl-

red and gave positive results with Voges-Proskauer reaction and was grown in

Koser’s citrate medium. Isolate SC27 is lysine decarboxylase negative, arginine

dihydrolase positive and ornithine decarboxylase positive. H2S was not produced;

urea was hydrolyzed while gelatin was not hydrolyzed and malonate was utilized.

Lactose was not acidified or was acidified late. The determination of β-galactosidase

was identified with ONPG test while rhamnose was fermented.



An Enterobacter cloacae is a Gram-negative bacillus belonging to the

Enterobacteriaceae family.     Enterobacteriaceae are the most frequent bacterial

isolates recovered from clinical specimens. This species rarely cause disease in an

otherwise healthy individual. This opportunistic pathogen, similar to other members

of the family Enterobacteriaceae, produces an endotoxin known to play a major role

in the pathophysiology of sepsis and its complications. Lipid-A, also known as

endotoxin, is the major stimulus for the release of cytokines, which are the mediators

of systemic inflammation and its complications. This component is contained in an

outer membrane which is from lipopolysaccharides (Cunha et al., 2000).



In the microbiology laboratory, colonies of Enterobacteriaceae appear large, dull-

gray, and dry or mucoid on sheep blood agar and also able to grow in aerobic and

anaerobic atmospheres. MacConkey agar is a lactose-containing medium that is

selective for nonfastidious Gram-negative bacilli such as Enterobacteriaceae. Using

the enzymes beta-galactosidase and beta-galactoside permeases, the most frequently


                                         108
encountered species of Enterobacter and the majority of E. coli and Klebsiella strains

will activate the pH indicator (neutral red) included in MacConkey agar, giving a red

stain to the growing colonies (Cunha et al., 2000). Klebsiella and Enterobacter can

be readily differentiated by a few specific tests.       For example, Enterobacter

organisms are motile, usually ornithine decarboxylase-positive, and urease-negative

in contrast to Klebsiella.



Table 14: Biochemical test result using Microbact™ 24E (12A+12B) kit for
isolate S8.

       Reaction               Result                Reaction               Result

       Lysine                    +                  Gelatin                    -
       Ornithine                 +                  Malonate                   -
       H2S                       -                  Inositol                   +
       Glucose                   +                  Sorbitol                   -
       Mannitol                  +                  Rhamnose                   -
       Xylose                    +                  Sucrose                    +
       ONPG                      +                  Lactose                    -
       Indole                    -                  Arabinose                  -
       Urease                    -                  Adonitol                   -
       V.P.                      +                  Raffinose                  -
       Citrate                   +                  Salicin                    +
       TDA                       -                  Arginine                   -

Note: + positive result
      – negative result




Isolate S8 is also facultatively anaerobic.    Serratia ferments mannitol, glucose,

sucrose, xylose, inositol and salicin with the production of acid and sometimes small

bubble of gas. It showed negative result with indole, gave a negative methyl-red and

positive Voges-Proskauer reaction and was grown in Koser’s citrate medium. Isolate

S8 is lysine decarboxylase positive, arginine dihydrolase negative and ornithine

decarboxylase positive. H2S was not produced; urea and gelatin was not hydrolyzed


                                         109
and malonate was not utilized. Lactose was not acidified or was acidified late. The

determination of β-galactosidase was identified with ONPG test while rhamnose was

never fermented.



Serratia marcescens is on the whole, smaller than the average coliform bacillus. The

size of organism is, however subject to considerable variation and even on the same

type of medium a single strain may at one time give rise to cocco-bacilli and at

another to rods indistinguishable from other coliform organisms. Capsules are not

ordinarily formed but capsular material was formed on a well aerated medium poor

in nitrogen and phosphate. Most strains are motile and flagellation is peritrichate.

The flagella are usually best seen in cultures grown at temperatures below 37 ºC

(Wilson and Miles, 1975).



Serratia marcescens is widely distributed in nature. Pigmented strains have at times

caused alarm by giving rise to red colours in various foods by simulating the

appearance of blood in the sputum or by producing strains on babies napkins (Wilson

and Miles, 1975). Pigmented and non-pigmented strains are found from time to time

in the human respiratory tract and in faeces. The organism often appears to multiply

in solutions or in the moist parts of apparatus and some strains become endemically

established in hospitals. Only a small proportion usually less than 10 percent of the

strains responsible for infection are pigmented (Carpenter, 1961).



On experimental inoculation into laboratory animals, organisms of this group prove

harmless except in very large doses. Culture filtrates of Serratia marcescens have

been used at times in the treatment of cancer but there is no reason to believe that the


                                          110
substance producing haemorrhage and necrosis in the tumour is other than the

endotoxin (Carpenter, 1961).



4.8.3          Partial Sequence of 16S rRNA Identification.



16S ribosomal RNA has been used for more than a decade to analyse evolutionary

relationships between organisms.       Ribosomal RNAs are ancient molecules,

functionally constant, universally distributed and moderately well conserved across

broad phylogenetic distances. rRNA sequence comparisons led to the construction

of a ‘universal tree of life’, dividing all the life on Earth into three equidistant

domains: Eukarya, Bacteria and Archaea (Woese, 1998).               The 16S rRNA

(approximately 1500 bp) contained several regions of highly conserved sequence

useful for obtaining proper sequence alignment and on the other hand contained

sufficient sequence variability in other regions of the molecule to serve as excellent

chronometers. The differences of sequence in the hypervariable regions reflect the

strain variations (Drancourt et al., 2000). Comparison of the sequences between

different species suggested a relatively earlier or later time in which they shared a

common ancestor.



4.8.3.1        Extraction of Genomic DNA.



Genomic DNA serves as repository for the information that is encoded within the

bases of the DNA. For genomic library construction, it is necessary to have intact

DNA fragments.      Fragmented DNA would generate an inefficient library that

decreased the chances of producing a positive recombinant clone.            Therefore


                                         111
precautions were taken to prevent DNA from shearing by mechanical force or

degraded by endogenous nuclease.



Genomic DNA extraction of isolates SC27 and S8 were successfully obtained using

Wizard® Genomic DNA Purification KitTM (Promega, USA) according to the

manufacturer’s procedure. For good gene amplification, it is necessary to have intact

DNA fragments during the extraction. Extracted DNA of two samples (isolates

SC27 and S8) were analysed with gel electrophoresis on 1% (w/v) agarose gel and

stained with ethidium bromide before being visualized under ultraviolet radiation.

Bands with high intensities indicated high concentration of DNA sample (Figure 25).

Besides that if the DNA was fragmented, smearing would occur.



After DNA extraction, the purity and concentration of the DNA was measured

spectrophotometrically. Pure DNA samples will have a A260/A280 ratio of between

1.7 and 2.0. DNA samples extracted were shown in Figure 25. The A260/A280 ratio

obtained lies between 1.8 and 1.9 which showed that contaminating proteins have

been adequately removed during the preparation.




                                        112
                                     Lane

           size (bp)    1      2       3      4       5




Figure 25: Extraction of genomic DNA from bacterial isolate SC27 and S8. The
extracted DNA was electrophoresed on 1% (w/v) agarose gel and stained with
ethidium bromide. Lane 1: Hind III marker, Lane 2-3: Genomic DNA of isolate
SC27, Lane 4-5: Genomic DNA of isolate S8.




                                    113
4.8.3.2       Polymerase Chain Reaction (PCR).



The genomic DNA of isolates SC27 and S8 obtained earlier were used as a template

in the amplification of the 16S gene.      The polymerase chain reaction (PCR)

technique was used to amplify the rRNA gene using synthetically produced

degenerate primers: Forward: 5’-AGAGTTTGATCATGGCTCAG-3’; and Reverse:

5’-ACGGTTACCTTGTTACGACTT-3’ which are synthesized by 1st Base. These

primers are highly conserved among prokaryotes and it amplified the whole region of

the rRNA gene which is 1500 bp and is the most commonly used (Acinas et al.,

1997). PCR products of the expected size of 1500 bp (Figure 26) were obtained

from the bacterial isolates SC27 and S8. The sequences of at least 1426 bp and 943

bp were obtained from each amplified products of both bacterial isolates

respectively. The amplified products were examined by electrophoresis.




                                       114
                               Lane

   size (bp)   1     2    3         4   5    6     7




Figure 26: 16S rRNA gene (~1500 bp) of bacterial isolate SC27 and S8
gene amplified via PCR. Lane 1: 1kb DNA ladder marker. Lane 2-4:
Amplified 16S rRNA PCR product of isolate SC27, Lane 5-7: Amplified
16S rRNA PCR product of isolate S8.




                              115
4.8.3.3        16S rRNA Gene Sequence Analysis.



By sequencing the 16S rRNA gene of isolates SC27 and S8, it was found that only

the forward and the reverse complement sequences of isolate SC27 were obtained

compared to isolate S8. Both of the forward and the reverse sequences of isolate S8

were unsuccessful to obtain which are the sequences were too short even several

attempts were performed. This is probably because the primers that were used for

sequence the bacterial isolate S8 was not suitable. Due to the limited time in this

study, further identification of isolate S8 was not carried out. Hence, only bacterial

isolate SC27 was used for further analysis to determine its identity. Those sequences

were aligned for homology using the BLAST 2 Sequences (Tatusova and Madden,

1999). The forward and the reverse complement 16S rRNA gene sequence of isolate

SC27 is shown in Appendix V. Figure 27 shows the region of homology between

the forward and the reverse complement sequences of isolate SC27. The Pair-wise

comparisons to assess the level of homology between the two nucleotide sequences

of the forward and the reverse complement of the reverse primer sequences was

determined using the BLAST 2 sequences algorithm using the blastn option with the

matrix turned off and default parameters available from the server at NCBI

(http://www.ncbi.nlm.nih.gov/blast/) (Tatusova and Madden, 1999).



Based on the overlapped region between the forward and the reverse complement of

the reverse primer sequence, both of the sequences were combined and checked for

errors and omissions of bases especially at the overlapped region using the

CHROMAS software Version 1.45 and the sequences were combined at bases giving

the least ambiguous characters and gap (Figure 27). The combined 16S rRNA gene


                                         116
sequence and the resultant 1426 bases were compared with the GenBank database

using the Blast server at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). Figure 28

shows the 16S rRNA sequence of isolate SC27 with the amplified product of 1426

bp. This analysis showed this sequence to be closely related to rrs from

Gammaproteobacteria. The results from 16S rRNA sequence analysis indicate that

isolate SC27 is assigned tentatively as Uncultured bacterium Strain Dr. Y13 after

deposited in GenBank under the following accession number DQ226214 (Figure 29).




Query: 508 gaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccggg 567
           |||||||||| |||| |||||| |||||| ||| ||||||||||||||||    ||||||
Sbjct: 187 gaattactggN-gtaaNgcgcacccaggcgttct-tcaagtcggatgtgaat--cccggg 242


Query: 568 ctcaacc-tgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaa 626
           ||||||| ||| ||||||||||||||||||||||||||| ||||||||||||||||||||
Sbjct: 243 ctcaaccNtggaaactgcattcgaaactggcaggctagactcttgtagaggggggtagaa 302


Query: 627 ttccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcc 686
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 303 ttccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcc 362


Query: 687 ccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagatacc 746
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 363 ccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagatacc 422


Query: 747 ctggtagtccacgccgtaaacgatgtcgatttggaggttgtt-ccttgaNgaatggcttN 805
           |||||||||||||||||||||||||||||||||||||||||| |||||| || ||||||
Sbjct: 423 ctggtagtccacgccgtaaacgatgtcgatttggaggttgttcccttgaggagtggcttc 482


Query: 806 cggaNcta--ccgttaaatcNacc-cctggggagtNcggcc-cNaggtt-aaactcaaat 860
           |||| |||   ||||||||| ||| |||||||||| ||||| | ||||| ||||||||||
Sbjct: 483 cggagctaacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaat 542


Query: 861 gaatt 865
           |||||
Sbjct: 543 gaatt 547




Figure 27: The region of homology between the forward and reverse
complement of isolate SC27. The sequences of overlapped 16S rRNA region
between the forward and reverse complement of isolate SC27 is in bold and
underlined.




                                        117
taacacatgcaagtcgagcggtaacacagggagcttgctcctgggtgacgagcggcggac     60

gggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagct     120

aataccgcataatgtcgcaagaccaaagagggggaccttcgggcctcttgccatcagatg     180

tgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagc     240

tggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggag     300

gcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatg     360

aagaaggccttcgggttgtaaagtactttcagcgaggaggaaggcattaaggttaataac     420

cttagtgattgacgttactcgcagaagaagcaccggctaactccgtgccagcagccgcgg     480

taatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtc     540

tgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcagg     600

ctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctg     660

gaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagc     720

gtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcgatttgg     780

aggttgttcccttgaggagtggcttccggagctaacgcgttaaatcgaccgcctggggag     840

tacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcat     900

gtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaactttc     960

cagagatggattggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagct     1020

cgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgcca     1080

gcggtccggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatg     1140

acgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatataca     1200

aagagaagcgacctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattgg     1260

agtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcagaatgctacg     1320

gtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaa     1380

agaagtaggtagcttaaccttcgggagggcgcttaccactttgaa                    1426



Figure 28: The 16S rRNA sequence of isolate SC27.      Results from DNA
sequencing revealed an amplified product of 1426 bp.




                                  118
DEFINITION   Uncultured bacterium Strain Dr.Y13 16S rRNA ribosomal gene, partial
             sequence.
ACCESSION    DQ226214
KEYWORDS     .
SOURCE       uncultured bacterium.
  ORGANISM   uncultured bacterium
             Bacteria; environmental samples.
REFERENCE    1 (bases 1 to 1426)
  AUTHORS    Shukor,Y. and Ahmad,F.
  TITLE      Environmental isolate
  JOURNAL    Unpublished
REFERENCE    2 (bases 1 to 1426)
  AUTHORS    Shukor,Y. and Ahmad,F.
  TITLE      Direct Submission
  JOURNAL    Submitted (29-SEP-2005) Biochemistry, University Putra Malaysia,
             Serdang, Selangor 43400, Malaysia
COMMENT      Bankit Comment: yunus.upm@gmail.com.
FEATURES              Location/Qualifiers
     source           1..1426
                      /organism="uncultured bacterium"
                      /strain="DR.Y13"
                      /db_xref="taxon:77133"
BASE COUNT       367 a    325 c     446 g    288 t
ORIGIN
         1 taacacatgc aagtcgagcg gtaacacagg gagcttgctc ctgggtgacg agcggcggac
        61 gggtgagtaa tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct
      121 aataccgcat aatgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcagatg
      181 tgcccagatg ggattagcta gtaggtgggg taacggctca cctaggcgac gatccctagc
      241 tggtctgaga ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag
      301 gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg
      361 aagaaggcct tcgggttgta aagtactttc agcgaggagg aaggcattaa ggttaataac
      421 cttagtgatt gacgttactc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg
      481 taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtc
      541 tgtcaagtcg gatgtgaaat ccccgggctc aacctgggaa ctgcattcga aactggcagg
      601 ctagactctt gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg
      661 gaggaatacc ggtggcgaag gcggccccct ggacaaagac tgacgctcag gtgcgaaagc
      721 gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgatttgg
      781 aggttgttcc cttgaggagt ggcttccgga gctaacgcgt taaatcgacc gcctggggag
      841 tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat
      901 gtggtttaat tcgatgcaac gcgaagaacc ttacctactc ttgacatcca gagaactttc
      961 cagagatgga ttggtgcctt cgggaactct gagacaggtg ctgcatggct gtcgtcagct
     1021 cgtgttgtga aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca
     1081 gcggtccggc cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg
     1141 acgtcaagtc atcatggccc ttacgagtag ggctacacac gtgctacaat ggcatataca
     1201 aagagaagcg acctcgcgag agcaagcgga cctcataaag tatgtcgtag tccggattgg
     1261 agtctgcaac tcgactccat gaagtcggaa tcgctagtaa tcgtagatca gaatgctacg
     1321 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa
     1381 agaagtaggt agcttaacct tcgggagggc gcttaccact ttggaa




Figure 29: The 16S rRNA sequence of isolate SC27 and its accession number as
deposited in GenBank.




                                         119
4.8.3.4        Phylogenetic Tree Analysis.



A multiple alignment of 19 (Appendix VII) 16S rRNA gene sequences closely

matches Uncultured bacterium strain Dr.Y13 were retrieved from GenBank and were

aligned using clustal_W (Higgins et al., 1994) with the PHYLIP (Phylogeny

Interference Package) output option. The alignment was checked by eye for any

obvious mis-alignments. Alignment positions with gaps were excluded from the

calculations. A phylogenetic tree was constructed by using PHYLIP, version 3.573

(Felsenstein, 1985) with Escherichia coli strain K12 as the outgroup in the

cladogram. Evolutionary distance matrices for the neighbour-joining or UPGMA

(Unweighted Pair Group Method with Arithmatic Mean) method were computed

using the DNADIST algorithm program. The program reads in nucleotide sequences

and writes an output file containing the distance matrix.



The four models of nucleotide substitution are those of Holmquist (1972), Kimura

(1980), the F84 model (Kishino and Hasegawa, 1989; Felsenstein, 1992), and the

model underlying the LogDet distance (Lockhart et al., 1994). Phylogenetic tree

(Figure 30) was inferred by using the neighbour-joining method of Saitou and Nei

(1987). With each algorithm, confidence levels for individual branches within the

tree were checked by repeating the PHYLIP analysis with 1000 bootstraps

(Felsenstein, 1985) by the SEQBOOT program in the PHYLIP package. Majority

rule (50%) consensus trees were constructed for the topologies found using a family

of consensus tree methods called the Ml methods using the CONSENSE program

and the tree was viewed using TreeView (Page, 1996).




                                         120
A moderately high bootstrap values (70.5%) links Strain DR.Y13 to uncultured

bacterium clone f6s1 indicating that the phylogenetic relationship is significant. The

strain is further grouped to albeit at a moderate bootstrap values (<40%) to many

Enterobacteria species such as Enterobacter amnigenus, Enterobacter asburiae

Enterobacter sp 253a, Enterobacter sp TUT1014, Klebsiella ornithinolytica strain,

Klebsiella planticola strain, Kluyvera ascorbata isolate 68, Kluyvera cryocrescens,

Pantoea agglomerans strain B1 and Pantoea sp 82353 implying that their

phylogenetic position can still be further modified in the future and it is difficult to

assigned a species name for (Figure 30).        For now, strain Dr.Y13 is assigned

tentatively as Uncultured bacterium strain Dr.Y13.



Hence, this 16S rRNA gene sequence results brought to the conclusion that the

bacterial isolate SC27 can be identified as Enterobacter species where it was found

closely related to each other based on to the phylogenetic tree analysis.          This

Enterobacter species is belongs to the Enterobacteriaceae family. This result give

the confirmation from the previous identification test using the Microbact™ kit

which is bacterial isolate SC27 was identified as Enterobacter species. In contrast to

the bacterial isolate S8, the bacteria could be identified as Serratia species based on

the identification test previously by using the Microbact™ kit with the similarity of

90.79% since no results were achieved from further identification using 16S rRNA

gene sequence.




                                          121
                                   Eschericia coli K12


                                   Enterobacter aeros AF395913]


                                   Pantoea sp 82353 [AF227851]


                                   Klebsiella planticola strain ATCC33531T [Y17659]


                            918    Uncultured bacterium clone 5s6[DQ068846]

                             521
                                   Klebsiella ornithinolytica strain 590681 [Y17662]
                                  889
                      504
                                   Uncultured bacterium clone 7s3 [DQ068860]


                                   Kluyvera ascorbata isolate 68 [AJ627201]
                                  969
   884          223
                                   Kluyvera cryocrescens [AF310218]


                                   Enterobacter sp 253a [AY082447]


                            472    Pantoea agglomerans strain B1 [DQ133596]

          236                670
                                   Uncultured gamma proteobacterium clone BIci26 [AJ318112]
                                  653
                                   Enterobacter amnigenus [AB004749]


                                   Enterobacter sp TUT1014 [AB098582]

    226                      548
                                   Uncultured bacterium clone f6s1 [DQ068815
                                  705
                                   Strain DR.Y13


                                   Endophyte bacterium SS06 [AY842147]


                            496    Uncultured bacterium clone s6s1 [DQ068932]

                             914
                                   Uncultured bacterium clone s4w18-5 [DQ068915]
                                  524
                                   Enterobacter asburiae [AB004744]




Figure 30: Phylogenetic tree of newly isolated bacteria for bioindicator of Hg.
Neighbour-joining method cladogram showing phylogenetic relationship
between Strain Dr.Y13 and other related reference microorganisms based on
the 16S rRNA gene sequence analysis. Species names are followed by the
accession numbers of their 16S rDNA sequences. The numbers at branching
points refer to bootstrap values based on 1000 re-samplings. The branch
lengths in the cladogram are not to scale. Eschericia coli Strain K12 is the
outgroup.

                                                         122
                                    CHAPTER 5



                                   CONCLUSION



Two hundred and fifty bacterial isolates as bioindicator for heavy metals were

successfully obtained from soil samples collected in 10 different locations in the

Malaysian Peninsular. After undergoing preliminary and secondary screenings based

on the inhibition of the reduction of MTT dye by six heavy metals in the presence of

common divalent cations such as Ca2+ and Mg2+ at the highest concentration of 25

mg/L and 50 mg/L respectively, an isolate designated as isolate SC27 which was

isolated from Sungai Congkak, Selangor ground was found to be sensitive to Hg at

the concentrations of 0.3 mg/L while isolate S8 which was isolated from Sri Serdang

Lake, Selangor was found to be sensitive to Ag at the concentration of 0.2 mg/L.



Investigations on effect of different stages of microbial growth on inhibitory effect of

heavy metals showed that isolate SC27 at 8 hours growth, seems to be more sensitive

to Hg while isolate S8 showed that the inhibition by Ag was highest at 12 hours of

growth. Therefore, the IC50 (50 % inhibitory concentration) of mercury for isolate

SC27 and silver for isolate S8 were determined respectively are 0.2698 mg/L and

0.073 mg/L after data was analysed using the Graphpad Prism version 4.0 software.

In the interference effect studies for both isolates, tests with other xenobiotics

showed no significant inhibition effects. For the stability of this bioassay system,

effects of different buffers system were investigated. It was found that the PBS

buffer system at pH 7.5 was selected as the most stable buffer in this bioassay.




                                          123
The species of these two isolates were identified using the Microbact™ kit system

and were confirmed using 16S rRNA gene analysis. Isolate SC27 was identified as

Uncultured bacterium strain Dr.Y13 (DQ 226214) which is related to Enterobacter

species while isolate S8 was identified as Serratia species with 90.79 % similarity

using only the Microbact™ kit. In conclusion, all objectives of this study were

achieved. This bioassay has the potential offers a good alternative to detect heavy

metals when measuring relative microbial/inhibition of respiration with MTT as

indicator in microplates. This may allow the development of inexpensive toxicity

assays that could be used much more widely than existing systems and could be used

at the sites. However, more research needs to be done in future to make this study to

be feasible such as optimization of bacterial growth and study the mechanisms of the

inhibition of the reduction of the MTT in bacterial cells in order to locate the

mechanisms site to optimize the sensitivity of the cells to heavy metals. Other than

that, study the enzymatic system that was involved in cellular MTT reduction.




                                        124
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                                         135
                                APPENDIX I



Parameter limits for heavy metals in effluents based on The Environmental
Quality (Sewage and Industrial Effluents) Regulations, 1979.



  Parameter              Units           Standard A      Standard B

  Mercury                mg/L              0.005            0.05
  Cadmium                mg/L              0.01             0.02
  Chromium hexavalent    mg/L              0.05             0.05
  Arsenic                mg/L              0.05             0.1
  Cyanide                mg/L              0.05             0.1
  Lead                   mg/L              0.1              0.5
  Chromium trivalent     mg/L              0.2              1.0
  Copper                 mg/L              0.2              1.0
  Manganese              mg/L              0.2              1.0
  Nickle                 mg/L              0.2              1.0
  Tin                    mg/L              0.2              1.0
  Zinc                   mg/L              1.0              1.0
  Boron                  mg/L              1.0              4.0
  Iron                   mg/L              1.0              5.0




                                   136
                                  APPENDIX II



                         List of Chemicals and Equipments



Chemicals                                              Manufacturer

λHindIII marker                                        Fermentas, USA
1 kb DNA ladder                                        Fermentas, USA
6X DNA Loading Dye                                     Fermentas
Etanol 95%                                             HamburghChemical
Forward primer                                         1st Base
Immersion oil, Grade A                                 Scharlau
Isopropanol 100%                                       Sigma
MTT                                                    Sigma Aldrich
Na2HPO4                                                Merck, Germany
NaH2PO4                                                Merck, Germany
Nutrient agar                                          Merck, Germany
Nutrient broth                                         Merck, Germany
Resazurin                                              Sigma Aldrich
Reverse primer                                         1st Base
Staining Gram                                          LabStain, LabChem
Taq polymerase buffer                                  Fermentas, USA
Tris                                                   Sigma Aldrich



Equipments                                             Supplier

Autoclave                                              Astell Model ASB
Balance                                                Sartorius
Bench Top Microcentrifuge                              Eppendorf
Centrifuge                                             Beckman, Coultier
Chiller                                                Memmert
Electronic Balance, AND ER-120A                        Sartorius
Global Positioning System Locator                      Garmin GPS III Plus
Incubator oven                                         Memmert
Light Microscope                                       Olympus
Magnetic stirrer                                       Favorit
Microplate reader, Spectrophotometer                   Shimadzu
Orbital Shaker, Gyromax™ 722                           Hotech Instruments Corp
pH Meter                                               Mettler Toledo 320
Syringe filter                                         Sartorius Minisar
Thermal cycler                                         MJ Research Inc., USA
UV mini 1240, UV-VIS, Spectrophotometer                Shimadzu
Vortexer                                               VM-300 Vortex Mixer
Water bath                                             B. BraunCertomat WR

                                       137
                                 APPENDIX III



Preparation of Tris-HCl, pH 7.5 Stock Solution



Tris-HCl, pH 7.5 at 1 M stock solution was prepared by dissolving 6.05 g of Tris-

pure powder in 30 mL sterilized deionized water in a bottle. The pH was adjusted to

pH 7.5 by adding 3 M HCl measured using pH meter. The deionized water was

added up to 50 mL. The solution was then stored at 4 °C after autoclaved. This Tris-

HCl was then added into reaction mixture to a final concentration of 10 mM.




                                        138
                                                       APPENDIX IV

The inhibition of the reduction effect of bacteria grown at different periods on
the absorbance by mercury and silver.


                                   0.20



             Absorbance (550 nm)   0.15



                                   0.10



                                   0.05



                                   0.00
                                          6   8          10         12         14        16   18

                                                               Time (hours)



The inhibition of the reduction of isolate SC27 by Hg (0.3 mg/L) at different growth
periods. Data collection begun after initially subtracting absorbance at 0.3 mg/L Hg
from the absorbance at 0 mg/L. Data represent mean ± SEM, n=3

                                   1.00

                                   0.90

                                   0.80
          Absorbance (550 nm)




                                   0.70

                                   0.60

                                   0.50

                                   0.40

                                   0.30

                                   0.20

                                   0.10

                                   0.00
                                          8       10          12          14        16        18

                                                               Time (hours)



The inhibition of the reduction of isolate S8 by Ag (0.2 mg/L) at different growth
periods. Data collection begun after initially subtracting absorbance at 0.2 mg/L Ag
from the absorbance at 0 mg/L. Data represent mean ± SEM, n=3




                                                              139
                                 0.80

                                 0.70

                                 0.60




          Absorbance (550 nm)
                                 0.50

                                 0.40

                                 0.30

                                 0.20

                                 0.10

                                 0.00
                                         8   10    12          14   16         18
                                 -0.10
                                                    Time (hours)




                                 0.80


                                 0.70


                                 0.60
           Absorbance (550 nm)




                                 0.50


                                 0.40


                                 0.30


                                 0.20


                                 0.10


                                 0.00
                                         8   10   12          14    16         18

                                                   Time (hours)



The inhibition of the reduction of isolates S7 (top) and S1 (bottom) by Ag (0.2 mg/L)
at different growth periods. Data collection begun after initially subtracting
absorbance at 0.2 mg/L Ag from the absorbance at 0 mg/L. Data represent mean ±
SEM, n=3




                                                  140
                                0.60



                                0.50

          Absorbance (550 nm)
                                0.40



                                0.30



                                0.20



                                0.10



                                0.00
                                       8   10   12          14    16          18

                                                 Time (hours)



The inhibition of the reduction of isolate K104 by Ag (0.2 mg/L) at different growth
periods. Data collection begun after initially subtracting absorbance at 0.2 mg/L Ag
from the absorbance at 0 mg/L. Data represent mean ± SEM, n=3




                                                141
                               APPENDIX V



Forward and reverse complement 16S rRNA sequence of isolate SC27. a) Forward
sequence of isolate SC27; b) Reverse complement sequence of isolate SC27. The
nucleotide sequences in bold and underlined marked the position of combined
sequence.


a)

TAACACATGCAAGTCGAGCGGTAACACAGGGAGCTTGCTCCTGGGTGACGAGCGGCGGACGGGTGAGT
AATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAATGTC
GCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAG
GTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAAC
TGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGAT
GCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCATT
AAGGTTAATAACCTTAGTGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCC
GCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTC
AAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGT
AGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAG
GCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT
GGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTTCCTTGANGAATGGCTTNCGGANCTACCG
TTAAATCNACCCCTGGGGAGTNCGGCCCNAGGTTAAACTCAAATGAATTANNGGGGCCCNCNNACNGG
NGANCTNNGGTTANTCNATNCACCNAAAAACTTCCTNCNTGNNTCCNAAAANTTCCNAAATGNTTGTG
CTNNGGAATTTNANAGTCNNAGNTTNCNACTNGTTTTAANTTGGTAANCCCACAGNACCTTNCTTTTC
NNNNCNCGNATNANGNNCCCNNNANNGTANGGGTNNCNNNNNGCTNNAGGGNCCCN



b)

NGNTTTGGGCCCNCCCNGNNNACCGTCCTTCNTGGNGCAANGNATTTCCAGGGCCACCTANCACCTCC
GGTTAAAANCCTNGGTTAAAGTNTTNAGGGNGAGGCTTAGGTAAAACCTGGGTTGCTTACTCCNGAAA
AGCNCGGTAANTCCGTCCACCAGCGNGGTAATCGGAGGTNCAACGTTATTGAATTACTGGNGTAANGC
GCACCCAGGCGTTCTTCAAGTCGGATGTGAATCCCGGGCTCAACCNTGGAAACTGCATTCGAAACTGG
CAGGCTAGACTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGG
AATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAA
CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGTTGTTCCCTTGAGGAGT
GGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGA
ATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCT
ACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGC
ATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTT
TGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATG
ACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAG
CGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACT
CCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTA
CACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGC
TTACCACTTTGGAA




                                    142
                               APPENDIX VI



The region of homology between the forword and reverse complement of isolate
SC27. a) Schematic representation and b) the sequences of overlapped 16S rRNA
region between the forward and reverse complement of isolate SC27 (in bold and
underlined).


a)




b)

Query: 508 gaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccggg 567
           |||||||||| |||| |||||| |||||| ||| ||||||||||||||||    ||||||
Sbjct: 187 gaattactggN-gtaaNgcgcacccaggcgttct-tcaagtcggatgtgaat--cccggg 242


Query: 568 ctcaacc-tgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaa 626
           ||||||| ||| ||||||||||||||||||||||||||| ||||||||||||||||||||
Sbjct: 243 ctcaaccNtggaaactgcattcgaaactggcaggctagactcttgtagaggggggtagaa 302


Query: 627 ttccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcc 686
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 303 ttccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcc 362


Query: 687 ccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagatacc 746
           ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct: 363 ccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagatacc 422


Query: 747 ctggtagtccacgccgtaaacgatgtcgatttggaggttgtt-ccttgaNgaatggcttN 805
           |||||||||||||||||||||||||||||||||||||||||| |||||| || ||||||
Sbjct: 423 ctggtagtccacgccgtaaacgatgtcgatttggaggttgttcccttgaggagtggcttc 482


Query: 806 cggaNcta--ccgttaaatcNacc-cctggggagtNcggcc-cNaggtt-aaactcaaat 860
           |||| |||   ||||||||| ||| |||||||||| ||||| | ||||| ||||||||||
Sbjct: 483 cggagctaacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaat 542


Query: 861 gaatt 865
           |||||
Sbjct: 543 gaatt 547




                                     143
                             APPENDIX VII


The accession number for the strains of bacteria used in the phylogenetic
comparisons.


Bacterial species                                          Accession
number

Escherichia coli Strain K12              NC_000913 REGION: 223771..225312
Endophyte bacterium SS06                                       AY842147
Enterobacter aeros                                             AF395913
Enterobacter amnigenus                                         AB004749
Enterobacter asburiae                                          AB004744
Enterobacter sp 253a                                           AY082447
Enterobacter sp TUT1014                                        AB098582
Klebsiella ornithinolytica strain 590681                         Y17662
Klebsiella planticola strain ATCC33531T                          Y17659
Kluyvera ascorbata isolate 68                                  AJ627201
Kluyvera cryocrescens                                          AF310218
Pantoea agglomerans strain B1                                  DQ133596
Pantoea sp 82353                                               AF227851
Uncultured bacterium clone 5s6                                 DQ068846
Uncultured bacterium clone 7s3                                 DQ068860
Uncultured bacterium clone f6s1                                DQ068815
Uncultured bacterium clone s4w18-5                             DQ068915
Uncultured bacterium clone s6s1                                DQ068932
Uncultured gamma proteobacterium clone BIci26                  AJ318112




                                   144
                             APPENDIX VIII

Lineage Report

Bacteria [eubacteria]
. uncultured bacterium -------------------- 2765 44 hits [eubacteria]
Uncultured bacterium clone f6s1
. Enterobacter sp. TUT1014 ................ 2757 1 hit [enterobacteria]
Enterobacter sp. TUT1014
. Pantoea sp. 82353 ....................... 2666 1 hit [enterobacteria]
Pantoea sp. 82353
. Pantoea agglomerans ..................... 2662 1 hit [enterobacteria]
Pantoea agglomerans strain B1
. Enterobacter aeros .................. 2662 3 hits [enterobacteria]
Enterobacter aeros
. endophyte bacterium SS06 ................ 2654 1 hit [eubacteria]
Endophyte bacterium SS06
. bacterium G2 ............................ 2654 1 hit [eubacteria]
Bacterium G2
. uncultured gamma proteobacterium ........ 2646 3 hits [g-proteobacteria]
Uncultured gamma proteobacterium clone BIci26
. Enterobacter asburiae ................... 2644 1 hit [enterobacteria]
Enterobacter asburiae
. Raoultella ornithinolytica .............. 2642 5 hits [enterobacteria]
Klebsiella ornithinolytica    , strain 590681, pa
. Enterobacter amnigenus .................. 2642 1 hit [enterobacteria]
Enterobacter amnigenus
. Enterobacter sp. 253a ................... 2639 1 hit [enterobacteria]
Enterobacter sp. 253a
. Kluyvera ascorbata ...................... 2639 3 hits [enterobacteria]
Kluyvera ascorbata    , isolate 6
. Kluyvera cryocrescens ................... 2639 1 hit [enterobacteria]
Kluyvera cryocrescens
. Raoultella planticola ................... 2631 2 hits [enterobacteria]
Klebsiella planticola    , strain ATCC33531T, par
. Klebsiella oxytoca ...................... 2627 13 hits [enterobacteria]
Klebsiella oxytoca    , strain SB73
. Klebsiella sp. R-21934 .................. 2627 1 hit [enterobacteria]
Klebsiella sp. R-21934     , isolate R-219
. gamma Proteobacterium BAL286 ............ 2627 1 hit [g-proteobacteria]
Gamma Proteobacterium BAL286     , partia
. Citrobacter freundii .................... 2625 1 hit [enterobacteria]
Citrobacter freundii strain GM1     , par
. Klebsiella pneumoniae ................... 2619 3 hits [enterobacteria]
Klebsiella pneumoniae     , seque
. Enterobacter sp. ........................ 2619 1 hit [enterobacteria]
Enterobacter sp.   ,
. bacterium SV6XVII ....................... 2615 1 hit [eubacteria]
Bacterium SV6XVII    ,
. Kluyvera sp. IAL9558/98 ................. 2615 1 hit [enterobacteria]
Kluyvera sp. IAL9558/98     , seq
. Kluyvera sp. IAL9557/98 ................. 2615 1 hit [enterobacteria]
Kluyvera sp. IAL9557/98     , seq
. Kluyvera sp. IAL9555/98 ................. 2615 1 hit [enterobacteria]
Kluyvera sp. IAL9555/98     , seq
. Klebsiella pneumoniae subsp. ozaenae .... 2615 2 hits [enterobacteria]
Klebsiella pneumoniae subsp. ozaenae    , strain
. Klebsiella pneumoniae subsp. pneumoniae . 2615 1 hit [enterobacteria]
Klebsiella pneumoniae subsp. pneumoniae     g
. Citrobacter sp. TSA-1 ................... 2611 1 hit [enterobacteria]
Citrobacter sp. TSA-1     , seque
. Citrobacter braakii ..................... 2611 1 hit [enterobacteria]
Citrobacter braakii     , sequence
. Enterobacter sp. pptphilum .............. 2607 1 hit [enterobacteria]
Enterobacter sp. pptphilum     ,
. Enterobacter cancerogenus ............... 2603 1 hit [enterobacteria]
Enterobacter cancerogenus LMG 2693

                                   145
                          BIODATA OF THE AUTHOR




The author, Fazuriana Binti Ahmad was born on 6th July 1981 in Alor Setar, Kedah.

She received her primary education at Sek. Ren. Keb. Sultan Ahmad Tajuddin, Jitra,

Kedah from 1988-1993. She then continued her study in the same province at Sek.

Men. Keb. Jitra, Kedah from 1994-1998, where she took her Penilaian Menengah

Rendah (PMR) and Sijil Pelajaran Malaysia (SPM). She completed her science

matriculation programme under Universiti Putra Malaysia at Kolej Matrikulasi

Kulim, Kedah in 2000 and then enrolled for a Bachelor of Science (Honours) in

Microbiology in the same year at Universiti Putra Malaysia, Serdang, Selangor. She

graduated in 2003 and further her studies by took up Master of Science in the field of

environmental biotechnology at the same university. Her research interests are in

microbiology, biochemistry, toxicology biochemistry, environmental biochemistry

and environmentally related fields.




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