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UNIVERSITI PUTRA MALAYSIA
THE DEVELOPMENT OF PHOTOSYSTEM II - HERBICIDE DETECTION METHOD USING
MICROALGAE AS A BIOSENSOR
SHAKINAZ DESA
FSAS 1997 19
THE DEVELOPMENT OF PHOTOSYSTEM II -
HERBICIDE DETECTION METHOD USING
MICROALGAE AS A BIOSENSOR
By
SHAKINAZ DESA
Thesis Submitted in Fulfillment of the Requirements for the Degree of Master of Science in the
Faculty of Science and Environmental Studies, Universiti Putra Malaysia
June 1997
� "'If � �Kd, 1Jatdt�, ((J aft IW9 � �
ACKNOWLEDGEMENTS
Tlns study was first suggested by Prof. Dr. Kamaruzaman bin
Ampon fonnerly of Department of Biochemistry and Microbiology in
UPM. His idea was supported by his wife, Assoc. Prof Dr. Ann Anton,
who was also an academic staff at Department of Biology, UPM. Both
are now serving at University Malaysia Sabah. This study is wisely
supervised by Assoc. Prof Dr. Nor Aripin Shamaan. I thank him very
much for giving me invaluable advice and encouragement at every step
of the way.
I wish to express my gratitude to Dr. Misri Kusnan of Department
of Biology for his brilliant ideas ,comments and tremendous support. I
am greatly indebted to Encik Ismail Omar of Department of
Biochemistry and Microbiology who has put in many long hours in
reviewing the thesis in order to produce a perfect text. My greatful
thanks also goes to Cik Rohaidah Ramli of Department of
Biochemistry and Microbiology and Puan Zaiton Basar of Department
of Biology for their assistance in the research work. Finally, I would
like to thank the staff of Graduate School, especially to Cik Fazlun for
her supportive ideas and efforts.
vi
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ............... ........................ ,................... iii LIST OF TABLES ....... .... ........ ............. ..... ...... ....... ....... ................. vii LIST OF FIGURES ......................................................................... viii LIST OF PLATES ......... ............. .......... ...... ..................... ......... ....... xi LIST OF ABBREVIATIONS ......................................................... xii ABSTRAC'r .................................................................................... xiii ABSTRAK ....................................................................................... xv
CHAPTER
I INTRODUCTION . .............................. . .. ....................... I
II LITERATURE REVIEW .................................... . . ........ 4 Herbicide . ....................................................................... 4
Introduction ......................................................... 4 Herbicide action in general ......................... .... .... 5 Tnazlne ............................................................... 7 Urea ............ ... .................. . . ..... ............................ 12
Herbicides in our environment ...... .. ....... . .... .................. 14 Risk of contamination ............. . .. ......... ..... .......... 17 Pollutant residence .. ......... ............ ................ ...... 20
Effects to the aquatic environment ... . . . ... . . ..... ......... ....... 22 Effects to humrul being .................. . ............................ .... 23 Herbicide action: inhibition ofPSII dependent electron flow .. ................. ...... ........ . .... , .......................................... 25 Herbicide binding site ....................... .............................. 27 Microalgae photosynthetic system . .................. .............. 31
Algal chlorophyll ................................................ 3] Photosynthesis: in general ....... . .......................... 33
Microalgae growth ..... . .... . ... ........... . .............................. 36 I..ag phase .............. ..... .............. . .......... .... ............ 36 Exponential phase ............................................... 36 Declining relative growth phase ....................... . . 37 Stationary phase .............................. ...... .............. 38 Deatll pllase ........ . ........................... ..................... 38 Nutrient replacing and batch culture ...... ...... .... . .. 39
iv
Fluorescence .................................................................... 40 The potential of algal chlorophyll fluorescence as biosensor ......................................................................... 42
Fluorescence potential ......................................... 45 Changes in fluorescence ...................................... 46 Requirement to develop monitoring system ....... 47
III MATERIALS AND METHODS .................................. 49 Preparation ofthe test organism .................................. 49
Selecting the test organism .................................. 57 Detennining the main test culture obligations .... 62 Culturing method ................................................ 62 Optimal age culturing ......................................... 65
Herbicide preparation .................................................. 65 Spectrofiuorometer ...................................................... 68 Satnple preparation ..................................................... 71 Statistical and data analysis ........................................ 74
IV RESULTS ..................................................................... 75 Selection oftest organism .......................................... 75
Preliminary test .................................................. 75 Cell density ......................................................... 78 Growth curve ..... , ................................................ 80 Chlorophyll concentration .................................. 84
Basic C. vulgaris obligation detetmination ................. 90 Culturing method selection ................................. 90 Optimal age detennination ................................. 92
Experiment using Atrazine .......................................... 97 Experiment using Simazine ........................................ 100 Experiment using Vinron ................... ........................ 1 06 Comparative study of atrazine, simazine and diuron ...................................................................... 109
V DISCUSSION ............................................................ 112 Selection ofChlorelia vulgaris ........ .. .................... 112
Suitability of test organism .............................. 112 Efficiency ........................................................ 115 Stability ........................................................... 116
v
Reliability on the fluorescence method ................... 119 Herbicide concentration and variable fluorescence
detemunatlon ... ........................................................ 12 1 Teclmical advantages and disadvantages ................ 125
VI CONCLUSION .. .............................. . ......................... 128 Suggestion .... . . . . ... . ... ... . .. . . . . . . ......... . ......... ....... . . . . . . .... 129
REFERENCES ............................................................................. 132
APPENDIX A Bold Basal media 147
BIOGRAPHICAL SKETCH 148
vi
LIST OF TABLES
Table Page
] Some examples ofberbicide and their specific inhibition 6
2 Comparison of environmental zone ................ ..... .... ... ..... 21
3 Test organisms and origin ................................................ 51
4 Basic information on the test herbicide ............................ 67
5 The growth rate oftest organism ............ .... ..... .......... ...... 84
6 The chlorophyll concentration per cell (�glcell) ....... . . . . . . 87
7 The relative fluorescence for atrazine at day 21 and 28, and the statistic comparison with day 14 ........... .............. 99
8 The relative fluorescence for simazine at day 21 and 28, and the statistic comparison with day 14 .......... ..... .... ...... 104
9 TIle relative fluorescence for diUl'on at day 21 and 28, and the statistic comparison with day 14 ...... ................... 108
vii
LIST OF FIGURES
Figure Page
1 General structure of triazine .......................................... 8
2 Structure of diuron or DCMU ..... .......... ...... .... .............. 13
3 The basic process of how pesticides can enter a waterbody .. ............ .......... ..... .......... ..... ............ ...... ......... 16
4 The process of herbicide accumulation along a food chain ................................................................................ 24
5 Binding sites of herbicides to a DIID2 complex of the reaction centre of PSI I .......................................... ......... 29
6 Formula structure of Chi a ............................................. 32
7 Steps of filling P680+ electron hole ............................... 35
8 The characteristic pattern of growth .............. ................. 37
9 Summary of the process used in the determination of the fluorescence method .. .......... ................................. ..... 50
10 The process of culturing microalgae ............................... 56
1 1 The process to determine chlorophyll concentration ....... 6 1
12 The procedure of detecting herbicide ............................... 73
13 The relationship between relative fluorescence and atrazine concentration ...................................................... 76
14 The relationship between cell density and age of culture .................................. ............................................. 79
Vl11
Page
15 Growth curve of Chlorella vulgaris ........... ............ .... 8 1
16 Growth curve of Scenedesmus quadriqauda ... ...... ....... 82
17 Growth curve of Chlorococcum sp ...... .... ................. .... 83
18 The relationship between relative fluorescence and chlorophyll concentration of Hibiscus sp. leaves ........ 85
19 The relationship between chlorophyll concentration and age of culture .......................... .... ,. .... .......... ........... 86
20 The relationship between chlorophyll fluorescence and age of culture ... .............................. ................ ............ ... 89
2 1 Growth curve of Chlorella vulgariS in two culturing nleth.ods .......................................................................... 91
22 The relationship between relative fluorescence and chlorophyll content and age of culture ......... ......... ......... 93
23 The relationship between relative fluorescence with different chlorophyll concentration over atrazine concentration .. 95
24 The relationship between relative fluorescence of atrazine treated sample and chlorophyll concentration .............. 96
25 The relationship between relative fluorescence and atrazine concentration ................................................................ 98
26 The relationship between relative fluorescence and pure atrazine ............... ................... ..................... ........... ....... 101
27 The relationship between relative fluorescence and simazine concentration ............ ............................................. ....... 102
28 The relationship between relative fluorescence and pure sitnazine .................... . .................. . . ............................... 105
ix
Page
29 The relationship between relative fluorescence and diuron concentration ................ ,............................................. 107
30 The relationship between relative fluorescence and pure diuron .................. ...................... ,',............................... 110
31 The relationship between relative fluorescence and herbicide concentration ................................................... ............ 111
x
LIST OF PLATES
Plate
1 A fish pond located below the palm oil plantation
Page
at Semenyih, Selangor ..... ... . .................. ........................ 18
2 A corn plantation situated above the fishing pond level at UPM's pond area .... ................................................... 19
3 Chlorella vulgaris with cup shaped chloroplast .......... . 53
4 Scenedesmus quadriqauda in form of colony spesies 54
5 Selenastrum sp. in cresent moon shaped, while Chlorococcum sp. shows a shape chloroplast ...... ......... 55
6 A haemocytometer is used to count cells ... .... . ............. .. 58
7 A batch culture allow the culture to grow without replenishing with fresh nutrient .......... ............................ 63
8 A nutrient replaced culture ............................................ 64
9 PSII inhibitor herbicide: atrazine, simazine and diuron. 66
lOA spectrofluorometer connected to a personal computer with Perkin-Elmer Fluorescence Data Manager prograr.nme ..................................................................... 69
xi
LIST OF ABBREVIATIONS
BBM Bold Basal media
ChI Chlorophyll
EPA Environmental Protection Agency
GC Gas chromatography
PSII Photosystem II
Q qumone
UPM Universiti Putra Malaysia
xii
Abstract of thesis submitted to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.
THE DEVELOPMENT OF PHOTOSYSTEM II - HERBICIDE DETECTION METHOD USING MICROALGAE AS A
BIOSENSOR
BY
SHAKINAZ BT DESA
JUNE 1997
Chairman Assoc. Prof. Dr. Nor Aripin bin Shamaan
Faculty Science and Environmental Studies
Herbicides have been found to be significant pollutants of water
bodies. In order to control and monitor the pollution, a convenient,
sensitive and reliable method is needed. The main objective of this
study is to develop a new method to measure the fluorescence yield of
microalgae in order to detect photosystem II (PSII) specific herbicides
in polluted water. TIle system was based on a simple mixing of
microalgae with PSII specific herbicides. The measured signal was in
the form of fluorescence intensity. The study involved the screening of
test organisms, detennining the best condition of the culture and
obtaining a standard curve for each of the PSII herbicides. Only three
herbicides were studied; atrazine, simazine and diuron. Chlorella
vulgaris was found as the most suitable test organism compared with
Chlorococcum sp., Scenedesmus quadriqauda and Selenastrum sp ..
Nutrient replacement culturing method was selected as the best
condition to grow C. vulgaris. A standard curve was determined for
each of the herbicides. C. vulgaris was able to signal atrazine's
concentration above O.04/lM according to the standard curve equation.
Simazine can be signalled at above O.05flM from its equation, while
diuron can be signalled at above than O.09/lM. However, -this basic
study needs more infonnation and improvement to be done in the
future, so that its full potential can be realised.
XlV
Abstrak tesis diserahkan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan Ijazah Master Sains.
PEMBENTUKAN KAEDAH MENGESAN RACUN RUMPAI FOTOSISTEM II MENGGUNAKAN MIKROALGA SEBAGAI
BIOSENSOR
Oleh
SHAKINAZ BT DESA
JUN 1997
Pengerusi Prof. Madya Dr. Nor Aripin bin Shamaan
Fakulti Sains dan Pengajian Alam Sekitar
Racun rumpai merupakan salah satu bahan cemar utama di dalam
badan air. Bagi mengawal dan mengesan pencemaran, kaedah yang
mudall, sensitif dan dipercayai adalah diperlukan. Objektif utama
kajian ialah untuk membentuk kaedah bam bagi mengukur floresen
mikroaIga dalam mengesan racun mmpai fotosistem II (FSII) yang
spesiftk di dalam air tercemar. Sistem illi berasaskan pencampuran
mikroalga dan racun rumpai FSII spesifik. Isyarat diukur di dalam
xv
bentuk keamatan floresen. Kajian ini melibatkan pemilihan organisma
ujian, kajian pengkulturan terbaik dan mendapatkan keluk piawai bagi
setiap faeun rumpai ujian. Tiga raeun rumpai yang dikaji; atrazin,
simazin dan diuron. Chlorella vulgaris didapati merupakan organisma
ujian yang paling sesuai berbanding Chlorococcum sp., Scenedesmus
quadriqauda dan Selenastrum sp .. Kaedah penggantian nutrien dipilih
sebagai kaedah pengkulturan terbaik bagi menumbuhkan C. vulgaris.
Satu keluk piawai diperolehi untuk setiap raeun rumpai setiap satu. C.
vulgaris berupaya mengesan kepekatan atrazin di atas O.04J.lM
mengikut persamaan keluk piawai. Simazin boleh dikesan pada
kepekatan di atas O.05J.lM daripada persamaannya. Manakala diuron
dapat dikesan di atas O.09�lM. Bagaimanapu� kajian asas ini
memerlukan lebih banyak maklumat dan pembaikan di masa hadapan
bagi membolehkan penggunaan potensi sepenuhnya.
xvi
CHAPTER I
INTRODUCTION
Current methodologies for pesticide detection, particularly herbicides, are
not ideally suited for large scale monitoring programme. These include gas
chromatography (GC) and high penomumce liquid chromatography (HPLC).
These techniques need large sample volumes, extensive extraction and cle�Ul
up procedures for analyses. lbe chemical analysis of a water sample is
expensive and very time-consuming. Thus, the number of analyzed samples is
limited.
Other techniques include flow cytogenic analysis usmg whole cell
clastogenicity (Biradar and Rayburn, 1 995); photoacoustic spectroscopy on
excised leaf (Fuks et aI., 1992); leaf disc buoyancy (LDB) method (Saltzman
and Heuer, 1985); oxygen electrod method (Saka and Chisaka, 1 982);
treatment of activated carbon� reverse osmosis and deionisation (Delavaud et
aI., 1992); measuring aJJeviation of phytotoxic injury (Yanase et aI., 1990)
and soil extraction method by enzyme immunoassay and gas chromatography
(Del Valle and Nelson, 1994).
A good analytical method must be accurate, precise, sensitive and
selective. Therefore, we need a method that is capable to measure low
concentrations of herbicides, low cost, fast result, easy handling and able to
monitor large scale progranune. The purpose of this study is to develop a
method that utilises natural phenomenon as an early warning system.
The method should be able to detect herbicide at below the level of
pennissible concentration.
In tins work, the basic principle is based on the measurement of changes
in the yield of variable chlorophyll a - fluorescence of PSIL The method
involves a test organism and a spectrofluorometer. Aquatic organisms are
used for toxicology tests because they experience the changes in the aquatic
environment.
The changes of yield of the in vivo chlorophyll a - fluorescence of
photosystem II are correlated quantitatively with the concentration of
herbicides. Therefore, it can be used to set up a low price monitoring system
and to be a promising basis for a detection device. Other studies that share the
same principle were done by Jansen et a1. (1993), Yanase and Andoh (1992),
Duke et at. (1991), Voss et al. (1984), Richard et al. (1983) and Bohme et al.
(1981).
2
As microalgae grow in aquatic environment, they are exposed to
environmental changes. These disturbance, however, are adapted by
microalgae in various way. For example, some increase the cell density
(Trifonova, 1993), produce spore or resting stage (Nixdorf and Hoeg, 1993)
or by migrating (Vincent, 1990). However, there are microalgae that could
not survive with the changes. But, these microalgae can serve as a simple
plant cell model in the herbicidal sites test systems (Fedtke, 1993).
The inhibition of herbicide towards the photosynthetic electron transfer
can be detected by measurement of chlorophyll fluorescence (Gleiter and
Renger, 1993; Percival and Baker, 1991; Habash et aI., 1985). Fluorescence
is a reradiated light energy fraction which cannot be used to drive
photosynthetic electron transfer. The inhibition results in an increased
fluorescence intensity at constant excitation intensity (Schrotter et aI., 1994).
There are three objectives in this study. Firstly, to select the best test
organism. Secondly, to determine the selected test organism obligation and
thirdly to obtain a relationship between the fluorescence yield and herbicide
concentration.
3
Introduction
CHAPTER II
IjITRRA TlJRE REVIEW
Herbicide
Herbicides are chemicals with the potential to kill and destroy
plants (Cobb, 1992). Herbicide can inhibit photosynthesis by
interfering with :
1 . the reproduction, development, structure, and integrity of chloroplasts,
2. biosynt11etic pat11ways that are involved in 11le production of output
products such as starch, amino acids synthesis,
3. photochemical induction pathways involved 10 the conversion of
radiation energy to chemical energy (Moreland and Hilton, 1976).
Herbicides may be classified in several ways, according to t11e
chemical compound, physiological characteristics or its selectivity
(Percival and Baker, 199 1). Herbicide can be classified into two major
groups : organic and inorganic. Examples of organic herbicides are
phenol and its derivatives, carboxylic acid and its derivatives,
4
pyridazine and pyrimidine, triazine, phenylurea, organophosphorus and
organoarsenic compounds . Examples of inorganic herbicides are
borate, sodium azide, sodium chlorate, copper sulfate and calcium
cyanamide.
Herbicide action bt general
Generally, the purpose of herbicides is to kill plant by inhibiting
photosynthetic electron transport (Palmeira et aI., 1995; Cobb, 1992).
Two biochemical mechanisms have been demonstrated to be of
primary importance in herbicidal action; they are firstly, inhibition of
PSII electron transport and secondly, diversion of electron transfer
through PSI (Fuerst and Norman, 1991). There are other mechanism of
herbicides actions in plants. They are summarized in Table 1.
There are also herbicides that enhance the development of auxin,
e.g., pheooxyalkanoic acid, benzoic acid, pyridine, and quinoline
carboxilic acid (Cobb, 1992). Excessive development of auxin may
cause tIle potassium ion accumulation in guard cells. Resulting in the
opening of the stomata and an increase in the photosynthesis rate.
Subsequently, cells die of excessive food production. Herhicide also
5
affect the increase of cell division and differentiation of cambium
tissues in higher plants.
Table 1
Some examples of herbicide and their specific hlhibition.
Inhibition
Photosyntllesis PSI
PS2
Pigment biosynthesis
Lipid syntllesis
Amino acid chain branching biosynthesis
Glutamine synthesis
Herbicide
Bipyridinium, Paraquat, Diquat Urea, Triazines, Triazinones, Uracil
Pyridazinone,
Metflurazone.
Cyclohexanadiones, Thiocarbamate
Sulphonilurea, Imidazolinone, Sulphonanilides.
Oxitin, Phosphinotrisine,
Reference
Dodge, 1988
Cobb, 1992
Britton et al. 1989.
Cobb, 1992
Dodge, 1988
Cobb, 1992
6
Most of the chemical compounds that inhibit photosynthesis
electron transport do so by binding to a set of overlapping sites on the
photo system II (PSII) reaction centre (Tietjen et aI., 1991; Sandmann
and Boger, 1986; Lavergne, 1982; Pfister et al., 1974). The phytotoxic
symptoms arise from the interaction of the herbicide molecule with
PSII. These chemical compounds include urea derivatives and
triazines.
Urea derivatives and triazines were introduced between 1950 and
1960. Diuron was the frrst herbicide known to specifically inhibit
photosynthetic electron flow (Wessels and Van der Veen, 1956). The
site of inhibition at the acceptor site of PSII was proposed, based on
the effect on chlorophyll fluorescence yield by Duysens and Sweers
(1963). Since then, diuron, atrazine and their photo affinity labelling
analogues have been used extensively in photosynthesis research.
Triazine
Triazine herbicides were first introduced in 1955 by Geigy.
Triazines are a commercially important class of herbicides. Triazine is
an extreme volatile crystalline solid which melts at 86°C and boils at
7
114 DC at 1 attn pressure. It is easily soluble in ether and ethanol at
Triazine is highly refracting rhombohedral crystals. The density is
approximately 1.38 glcm3• Triazine can be purified without
appreciable Joss by repeated distillation over metallic sodium (Smolin
and Rapoport, 1959).
Cyanuric chloric (2,4,6-trichloro-l ,3,5,-triazine) is the base
compound to foon all 1,3,5-triazine herbicide. General chemical
structure of triazine is shown in Figure 1.
Figure 1: General structure of triazine.
8