Physiological and biochemical responses of a Malaysian red alga, Gracilaria manilaensis treated with...

J. Environ. Res. Develop.

Journal of Environmental Research And Development Vol. 7 No. 3, January-March 2013

1246

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF

A MALAYSIAN RED ALGA, Gracilaria manilaensis

TREATED WITH COPPER, LEAD

AND MERCURY Zakeri Hazlina Ahamad* and Shuib Nor Shuhanija

Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia

Terengganu (UMT), Kuala Terengganu (MALAYSIA)

Received August 10, 2012 Accepted January 10, 2013

ABSTRACT

The relative effects of three heavy metals, copper (Cu), lead (Pb) and mercury (Hg) exposure on

the photosynthetic quantum yield (Fv/Fm), ion leakage and activity of two antioxidative enzymes,

catalase (CAT) and Ascorbate Peroxidase (APX) on a Malaysian red alga, Gracilaria

manilaensis were investigated. Of the three metals, Hg was observed to affect the alga the most.

There was about 89% reduction in the Fv/Fm and 63% increase in ion leakage of the alga in the

presence of Hg. In contrast, there was an increased in the activity of both the antioxidative

enzymes. CAT was increased to 11 U/mg Total Soluble Proteins (TSP) as compared to 0.4 U/mg

TSP in the control while APX was increased to 0.12 U/mg TSP as compared to 0.01 U/mg TSP

in the control. Both Cu and Pb did not show any significant changes in the ion leakage of the

alga. However, there was a 17% and 12% reduction in Fv/Fm of the alga in Cu and Pb,

respectively. CAT and APX in Cu were increased to 1.8 and 1.2 U/mg TSP as compared to 0.4 and 0.02 U/mg TSP in control, respectively. Pb on the other hand, increased the activity of CAT

from 0.5 U/mg TSP to 2.3 U/mg TSP and APX from 0.01 U/mg TSP to 0.3 U/mg TSP.

Key Words : Gracilaria manilaensis, Fv/Fm, ion leakage, Antioxidative enzymes, Heavy

metals effect

INTRODUCTION

Contamination by metal ions, including copper

(Cu2+

), lead (Pb2+

) and mercury (Hg2+

) has

become a major issue throughout many countries due to their possible toxic effects.

1 Metal toxicity

has high impact and relevance to plants and other

autotrophs and since these organisms are primary producers, it will consequently affect the whole

ecosystem. Some metals are required in small

amounts by the autotrophs to grow and develop

but accumulation or high amounts can have negative effects to the organisms.

Photosynthesis, an important metabolic process

for the autotrophs has been known to be very sensitive to heavy metals. Increased

concentration of Cu for instance, results in

chlorosis and reduced growth of algae.2 Pb

exposure can damage the structure and function of photosystem II (PSII).

3 Mercury on the other

hand, is able to alter the photosynthetic

machinery including the chloroplastic photosys-

tem I (PSI) reaction center subunit II, the

oxygen-evolving protein and the chloroplastic

ATP synthase β-subunit. 4

In this study,

chlorophyll (chl) fluorescence analysis was used

as a useful physiological tool to assess early

stages of change in photosynthetic performance

of algae in response to heavy metal pollution.5

This method has been shown to be rapid, non-

invasive and reliable for assessing photosynthetic

performance in a changing environment.6

Among the parameters of chl fluorescence, the

dark-adapted maximal quantum yield or Fv/Fm

has been widely used and is directly proportional

to the quantum efficiency of PSII photoche-

mistry.6

Toxic effects of metals appear to be partly related

to the production of Reactive Oxygen Species

(ROS) and the resulting unbalanced cellular

redox status.7 ROS that can be generated by the

metals include superoxide anion (O2-), hydrogen

peroxide (H2O2), singlet oxygen (1O2) and

hydroxyl radical (OH).

7 These ROS are *Author for correspondence



1247

continuously produced during normal metabolic

processes but can be extremely harmful to organisms at high concentrations since they can

oxidize proteins, lipids and nucleic acids which

often leads to alterations in the cellular

structure.8 Therefore, the production and removal

of ROS must be controlled. To serve this

purpose, organisms have developed a wide range

of protective mechanisms such as production of enzymatic and nonenzymatic antioxidants.

7 This

study only focused on two main natural

antioxidative enzymes, catalase (CAT) and Ascorbate Peroxidase (APX). CAT [EC

1.11.1.6] catalyzes the dismutation of H2O2 into

O2 and H2O. The enzyme occurs in all aerobic

eukaryotes and its function is to remove the H2O2 generated in peroxisomes by oxidases involved

in β-oxidation of fatty acids, photorespiration,

purine catabolism and during oxidative stress. 9

APX [EC 1.11.1.11] on the other hand, uses

ascorbate as a hydrogen donor to break down

H2O2 to form H2O and monodehydroascorbate and performs this function in chloroplasts and

cytosol of plant cells.

Macroalgae (or seaweeds) play a major role in

marine ecosystems. As the first organism in marine food chains, they provide nutrients and

energy for animals. Moreover, beds of

macroalgae provide shelter and habitat for scores of coastal animals for all or part of their lives.

Macroalgae like any other plants require

inorganic nutrients for growth. The fast-growth

rate of some species of macroalgae can account for rapid nutrient removal from marine waters.

Most of them are able to immobilize the metals

to make them less toxic. 10

In addition, they have the ability to adsorb and metabolize trace metals

due to their large surface: volume ratios, the

presence of high-affinity, metal-binding groups on their cell surfaces and efficient metal uptake

and storage systems. 11

These characteristics

make them suitable for bioremediation process, a

process which uses organisms to return the natural environment altered by pollutants or

contaminants to its original state. 12

AIMS AND OBJECTIVES

To determine the effects of three most highly

found heavy metals pollutants in Malaysian

marine ecosystem, Cu, Pb and Hg on a red alga, Gracilaria manilaensis in terms of its dark-

adapted quantum yield, ion leakage and activity

of two antioxidative enzymes, CAT and APX.

This study is a preliminary study to determine the suitability of this alga as bioremediator and

bioindicator of metals-polluted marine waters.

MATERIAL AND METHODS

Algal materials

The alga, Gracilaria manilaensis (Rhodophyta)

was obtained from Kuala Muda, Kedah,

Malaysia and further cultivated in an open

system culture tanks at the Seawater Hatchery,

Universiti Malaysia Terengganu, Malaysia Prior

to treatment and the algae were cleaned to get rid

of unwanted parasites or particles.

Treatment of heavy metals

About 2 g of the algae were treated with 2 mg/L

of either copper (II) nitrate (Cu(NO3)2), lead (II)

nitrate (Pb(NO3)2) or mercury (II) nitrate

(Hg(NO3)2) in filtered seawater for 24 h under

0.34-0.48 klux of white light. The conditions for

untreated samples (i.e. control) were similar as

above but with no addition of metals. All

treatments and control were done in triplicates.

Chlorophyll (chl) a fluorescence determina-

tion

The maximal quantum yield (i.e. Fv/Fm) of the

samples was measured by a portable handheld

chl fluorometer, AquaPen-P AP-P 100 (Photon

Instruments System, Czech Republic). At the

start of the measurement, a short, red, actinic

pulse (~300 µmol m-2

s-1

at 655 nm) was

prompted for 5 s to ensure a stabilized

fluorescence emission during the following Fm

measurement. Then Fo was measured with a

pulsed, blue measuring light (~900 µmol m-2

s-1

,

455 nm) and Fm was determined with a

saturating white light pulse (~3000 µmol

m-2

s-1

). The maximal quantum yield was

calculated as (Fm-Fo)/Fm.

Ion leakage measurements

Ion leakage was measured as electrical

conductivity with a Ecoscience EC300 (YSI,

USA) conductivity meter at room temperature

using a method by Cordi et al.13

The tissue was

rinsed very quickly in 50 mL ultra-pure water

and transferred to 25 mL ultra-pure water

(sample 1). After 2 minutes, the sample was

drained quickly and transferred to a second

beaker containing 25 mL ultra-pure water and

boiled for 5 minutes (sample 2).



1248

The conductivity of both samples was measured

at room temperature and the health index was calculated on the basis of the ion loss as sample

2/(sample1 + sample 2), expressed as percentage.

A decrease in health index indicates a high ion

loss or leakage has occurred.

Total Soluble Protein (TSP) extraction and

determination

For extraction of TSP, algal sample was first

ground to fine powder in liquid nitrogen. Then,

50 mM potassium phosphate (pH 7.0) containing 0.4% protease inhibitors was added to the ground

sample and centrifuged at 16,000 rpm and 4°C

for 15 minutes. The supernatant (i.e. crude

extract) was collected and used for TSP concentration determination and enzymes assay.

The concentration of TSP was measured

spectrometrically at 595 nm according to Bradford method using 1 mg/L Bovine Serum

Albumin (BSA) as standard.

Antioxidative enzymes assay

Two antioxidative enzymes, catalase (CAT) and

Ascorbate Peroxidase (APX), activity was

measured in this study. The assays were done according to Aguilera et al.

14 Each enzymes

assay was done in triplicates and at 25°C.

Reaction mixtures were incubated in the spectrophotometer for 3 minutes prior to the start

of reaction to allow for temperature equilibration.

All enzymes activity was expressed as U/mg TSP. For CAT assay, 40 µL of crude extract of

the sample was added to 50 mM potassium

phosphate buffer (pH 7.0). The reaction was

started by adding 150 µL H2O2 followed by monitoring the decrease in absorbance at 240 nm

for 2 minutes. CAT activity was calculated by

using extinction coefficient for H2O2 of 0.0398 mM

-1 cm

-1. For APX assay, the reaction was

started by adding 30 µL of extract to a reaction

mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2O2 and 0.5 mM

ascorbate. The reaction was monitored for 1

minute by a decrease in absorbance at 290 nm.

APX activity was calculated using extinction coefficient for ascorbate of 2.8 mM

-1 cm

-1.

Statistical analysis

Values of Fv/Fm and health index parameters

tested were related to 100% of alga at time 0 h

values for better comparison. Mean values and standard deviation were determined from three

replicates of each treatment. The statistical

significance of differences among means was

calculated using the Student’s t-test. In each tests, a probability level of p<0.05 was applied.

RESULTS AND DISCUSSION

Fv/Fm. Fv/Fm were used to indicate the influence of metals on the photosynthetic activity of G. manilaensis. The Fv/Fm of the untreated algae was observed to be increased after 24 h under the experimental conditions (Fig. 1). However, in the presence of heavy metals, the Fv/Fm decreased significantly. There was about 34% and 22% reduction in Fv/Fm of the alga in Cu and Pb, respectively. Comparatively, the alga responded very adversely to Hg by reducing its Fv/Fm to 11%. In fact, according to Lobban and Harrison

15,

Hg is the most toxic metal to algae followed by Cu. At the physiological level, the measurement of Fv/Fm is an effective parameter to assess the photosynthetic status particularly the PSII of the alga under stress in which a reduction in this parameter indicates that the alga has been exposed to stress.

6

Measurements of Fv/Fm provide a first insight into changes of the photosynthetic apparatus upon the action of the metals

16 and can reveal

the mechanisms involved in metals toxicity. 17

It is known that heavy metals could seriously affect the photosynthetic apparatus by irreversibly binding the components of photosynthetic electron transport chain. For instance, Cu and Pb can substitute Mg in the center of chl molecule leading to termination of photosynthesis activity by forming nonfluorescent inactive metals substituted chl.

18

Cu can reduce or inactivate the rate of photosynthetic electron transport of algae either by destructing the photosynthetic carbon reduction cycle

19or by modifying the structure of

oxygen-evolving complex of PSII.20

A decrease in electron transfer within PSII has also been observed by Connan and Stengel

2. Pb, on the

other hand, can decrease photosynthetic rate by distorting chloroplast ultrastructure, diminishing chl synthesis, obstructing electron transport, and inhibiting activities of Calvin cycle enzymes.

21

An increase in Hg content induces a significant increase in the proportion of the QB-non-reducing PSII reaction centers which is formed when the electron transfer from QA

- to QB is inhibited.

5 Lu

et al. 5

also suggested that PSII reaction centers were the sites for Hg-induced damage. This



1249

suggestion was further supported by a study of Kukarskikh et al.

22 which observed an

increase in the steady-state level of P700 photo-

oxidation indicating a disturbance in electron transfers between photosystems as well as an increase in fraction of closed reaction centers

Fig. 1 : Maximal quantum yield (Fv/Fm) of Gracilaria manilaensis after 24 h treatment with different metals (grey bars) compared to untreated alga (black bars). Asterisks above bars indicate statistically

significant difference between treated and untreated algae of similar metals at p<0.05

leading to reduction in non-photochemical

quenching process.

Ion leakage

In addition to photosynthetic apparatus of algae,

cell membranes are also key targets of metals

toxicity. 21

To measure cellular damage, ion

leakage is used as a useful indicator. 23

According

to Cordi et al., 13

the decreased health index (as

the parameter used to measure ion leakage in this

study) indicates damage to plasmalemma and

high concentrations of ions are leaking from the

cells. However, from the results obtained, no

significant changes in the health index was found

for Cu and Pb (Fig. 2) indicating that membrane

damage is not one of the response in toxicity

effect of either Cu or Pb for G. manilaensis in

this study. Even though the concentration is high

(i.e. 2 mg/L), exposure to this concentration does

not affect the functional integrity of the plasma

membrane. This result is in contrast of that found

by Brown and Newman24

in a study of Cu effects

on a similar genus, Gracilaria longissima. They

observed that ion leakage is significant at Cu

concentration of 500 µg/L. This concentration is

much lower than that used in this study.

Nevertheless, in a review by Bertrand and

Poirier25

, they stated that excess of metals can be

treated differently inside the cell according to the

biological species or the genotypes of the same species.

Comparatively, health index of G. manilaensis in

this study was affected by the presence of Hg

which was reduced to 37% (Fig. 2). The damage by Hg may be caused by Reactive Oxygen

Species (ROS) which are known to initiate lipid

peroxidation resulting in lots of membrane rigidity, integrity and permeability.

26 Further -

more, Hg has been proven to induce production

of ROS.27,28

In a study by Elbaz et al. 27

with

Chlamydomonas reinhardtii, level of lipid peroxides was enhanced with increasing

concentrations of Hg, indicating lipid

peroxidation has occurred in the cell. Mercuric ions has been observed to induce K

+ leakage in

test plants of a macrophyte, Potamogeton crispus

L. after 24 hr which was increased with increasing Hg concentrations.

29

Antioxidative enzymes

Previous studies indicated that ROS were produced when plants or algae were exposed to

heavy metals. 27,30

Build up of ROS in cells

initiate signaling response to induce gene expression of antioxidative enzymes.

31 For

example, expression of the genes for CAT and

APX was observed to be activated and increased in green algae, Ulva fasciata

32

and C. reinhardtii. 32

The response of these

Metals

% o

f F

v/F

m



1250

antioxidant enzymes to metal stress, however,

varies among plant species and the metals involved.

33 Fig. 3 shows the activity of CAT

and APX in the alga after 24 h treatment with

the three metals. It was observed that there was

a significant increase in concentrations of both the enzymes. Hg triggered the highest activity

of CAT in the alga (Fig. 3(a)) while Cu

triggered the highest activity of APX (Fig. 3(b)). In addition, a higher increase in

APX compared to CAT was observed with Cu

(p=0.02) and Pb (p=0.01) while a higher

increase in CAT compared to APX was observed with Hg (p=0.003).

Fig. 2 : Percentage of health index of Gracilaria manilaensis after 24 h treatment with different

metals (grey bars) compared to untreated alga (black bars). Asterisks above bars indicate

statistically significant difference between treated and untreated algae of similar metals at p<0.05

Potential important sources of ROS in

photosynthetic cells include over-reduction of

PSII, the Mehler reaction and photorespiration.

34 Over-reduction of PSII occurs during

environmental stress due to repression of carbon

assimilation. PSII will become progressively

reduced which leads to oxidative stress through

the generation of 1O2 or O2

-. The Mehler

reaction as well as photorespiration can function

as alternative routes to de-energizing

photosystems and thus preventing the over-

reduction of PSII. In both the reactions, O2- is

converted to H2O2. The principal H2O2-

scavenging enzymes in plants are CAT, which

is located in peroxisomes and APX, which is

primarily found in the cytosol and

chloroplasts.35-37

In the Mehler reaction, O2 is

reduced first to O2- and then to H2O2. This

H2O2 is subsequently converted to H2O by

APX, thus generating a pseudocyclic electron

flow, in which electrons from the oxygen-

splitting complex pass through the

photosynthetic electron carriers back to O2.

Photorespiration, on the other hand, recycles

carbon that is used by oxygenation of ribulose-

1,5-bisphosphate and produces H2O2 in the

peroxisomes through the enzyme glycolate

oxidase. The subcellular distribution of these

enzymes suggests that chloroplastic APX

removes H2O2 produced during the Mehler

reaction and other chloroplastic processes,

whereas CAT scavenges photorespiratory H2O2.

Therefore, from the results obtained in this

study, an induction in both the antioxidative

enzymes observed when the alga was exposed

to all three metals can be correlated to an

increase in H2O2. Higher induction in APX

activity compared to CAT observed with Cu

and Pb may be due to a higher amount of H2O2

accumulated in the chloroplasts as compared to

peroxisomes generated through the Mahler

reaction. In the case of Hg, a different approach

was used. H2O2 generated by Hg was mainly

from the photo respiratory mechanism since

CAT activity was found to be higher than APX

activity. In addition, CAT can also scavenge

H2O2 generated during mitochondrial electron

transport as well as -oxidation of fatty acids. 9



1251

Thus, it can also be said that Hg may also has

an effect in respiratory as well lipid metabolic

processes of the alga while damage by Cu and

Pb may restrict only to the photosynthetic

processes. This may be true in this study since

Hg affects the membrane of the alga while Cu

and Pb do not (Fig. 2). However, more analyses

need to be done to prove this theory.

Fig. 3 : Activity of two antioxidative enzymes, catalase (CAT, a) and ascorbate peroxidase (APX,

b) of Gracilaria manilaensis after 24 h treatment with different metals (grey bars) compared to

untreated alga (black bars). Asterisks above bars indicate statistically significant difference between treated and untreated algae of similar metals at p<0.05

CONCLUSION

The alga G. manilaensis exhibited different

responses to Cu, Pb and Hg toxicity. Hg caused

the most adverse effects on the alga with the

highest reduction in the photosynthetic quantum

yield and disrupted the algal membrane

permeability as shown by increased in ion loss.

Contrastingly, Cu and Pb did not have an effect

on the membrane permeability of the alga but at

the same time reduced its photosynthetic

quantum yield. The metals induced the

production of ROS especially H2O2 in the algal

cells and in response to this, the alga increased

the production of antioxidative enzymes CAT

and APX. Hg induced the highest concentration

of CAT while Cu induced the highest

concentration of APX suggesting that different mechanisms were employed by the alga to treat

toxicity effects of the different metals. The

results also show that ion leakage is less sensitive to the toxicity of Cu and Pb than the Fv/Fm.

ACKNOWLEDGEMENT

This study is partially supported by the Malaysian Ministry of Higher Education under

the Fundamental Research Grant Scheme

(Phase: 2/2010) vot. no. 59221 managed by the

Research Management and Innovation Center, UMT, Malaysia.



1252

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Physiological and biochemical responses of a Malaysian red alga, Gracilaria manilaensis treated with...

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