11th July Power Point

8/11/2019 11th July Power Point

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EFFECT OF TITANIUM DIOXIDE NANOPARTICLES AND NUTRIENT

CONCENTRATION ON THE PHYSIOLOGY OF Chlorel la vulgar is

(Trebouxiophyceae, Chlorophyta)

A RESEARCH PROPOSAL

BY

SULEIMAN DAUDA

(M.Sc/SCIE/11233/2011-2012)

DEPARTMENT OF BIOLOGICAL SCIENCES,

AHMADU BELLO UNIVERSITY, ZARIA.

SUPERVISORS:

PROF. S. P. BAKO

DR. M. A. CHIA

11th

JULY, 2014 1


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INTRODUCTION

Microalgae:

important component of the aquatic ecosystem

have the ability to fix CO2 while capturing solar energy with

efficiency 10 to 50 times greater than that of terrestrial plants (Wang

et al., 2008).

higher biomass production compared to energy crops (Wang et al.,2008).

Chlorella:

unicellular, non-motile green microalga

found both in fresh and marine water.

cells are solitary, very small (2-12 m) and spherical, globular or

ellipsoidal in shape (Sharma, 1986).

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INTRODUCTIONNanoparticles:

loosely defined as manufactured materials that are smaller than 100nanometer in at least one dimension

Have been of scientific interest for several decades, but are now being

used in a wide range of commercial applications (Kulacki and

Cardinale, 2012).

Titanium dioxide (TiO2)

An example of manufactured nanosized materials that are already

widely used (Hartmann et al., 2010).

Also known as titanium (IV) oxide or Titania

the naturally occurring oxide of titanium, and titanium is the ninth

most abundant element in the world, it is five times less abundant

than iron but 100 times more abundant than copper (IARC, 2010).

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Introduction Contd

primarily used as a pigment because of its brightness, high refractive

index, and resistance to discolouration (Diebold, 2003).

The global production of titanium dioxide for all uses is in the

millions of tons per year. In the cosmetics industry the use of

nanosized particles.

Microalgae require nutrient for their growth

Nitrogen is an important element for algae growth, it is essential in protein

synthesis and in pigment construction.

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Introduction Contd

Phosphorus is also an important nutrient element.

a component of nucleic acids governing protein synthesis and of theadenosine phosphate transformations that power intracellular transport

Nitrogen and phosphorus are the primary nutrients of concern in relation to

water quality issues because they can stimulate primary productivity. (Dodds,

2002).

Nitrogen and phosphorus are often the primary limiting nutrients for aquatic

algal production (Lv, 2011) because they are frequently in short supply relative

to cellular growth requirements.

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Introduction Contd

Accrual of algal biomass, and thus overall ecosystem productivity, may be

controlled by the type and intensity of nutrient limitation (Dodds et al., 2002)

The term oxidative stress refers to the situation of serious imbalance between

production of reactive species and antioxidant defense.

According to Sies, 1991, it is a disturbance in the prooxidantantioxidant

balance in favour of the former, leading to potential damage.

Antioxidant defense mechanisms include enzymes that catalyze reactions of

ROS scavenging, such as catalase, ascorbate peroxidase, glutathione

peroxidase and superoxide dismutase (Pinto et al., 2003; Mallick, 2004).

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Introduction Contd

Oxidative stress also leads to cell death, which occurs by two mechanisms;

necrosis and apoptosis (Halliwell, 2001).

During evolution, living organisms have adapted to the presence of natural

nanoparticles in the environment. For synthetic nanoparticles, however, it is

recognized that their potential harmful properties on ecosystems have to be

evaluated (Handy et al., 2008; Nowack, 2009).

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STATEMENT OF THE RESEARCH PROBLEM

The potential harmful properties of synthetic nanoparticles on ecosystems have to

be evaluated (Handy et al., 2008; Nowack, 2009).

The increasing presence of nanoparticles in many products has made it all the

more likely that they will also be released into the aquatic environment.

The recent advances in nanotechnology and the corresponding increase in the useof nanomaterials in products in every sector of society have resulted in uncertainties

regarding environmental impacts (Klaine et al, 2008).

Titanium dioxide nanoparticles novel appearance in the environment and the lack

of protective mechanisms in the course of biological evolution in living organisms

(Valavanidis and Vlachogianni, 2010).

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STATEMENT OF THE RESEARCH PROBLEM CONTD

Nanoparticles act on algae, by influencing the bioavailability of particles and the

mechanisms of toxicity (Behra et al., 2009).

Mechanisms of nanomaterial toxicity include cellular damage due to oxidative

stress, physical damage to the cell surface, dissolution at the cell surface, and

impacts via bioaccumulation (Batley and McLaughlin, 2010)

Impacts of nanoparticles on nutrient availability in aquatic ecosystems have not

been assessed in detail yet. The effect of titanium dioxide nanoparticle on the

nutrient uptake by Chlorella vulgaris needs to be studied.

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JUSTIFICATION

Algae is one of normally used model organism for the toxicity examination of

toxicants and nanoparticles as well, (Ji et al, 2011)

Algae contribute approximately half of the global primary production as well as

atmospheric oxygen (Aruoja, 2011).

Algae contributes to the self-purification of polluted water.

Due to their importance, algal growth response to existing and potential new

environmental threats has to be clarified.

Chlorella vulgarisis distributed widely in freshwater and seawater and has a short

growth cycle and this makes it ideal for aquatic eco-toxicity studies and it can beused to directly observe toxicity at the cellular level (Wong et al., 1997).

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JUSTIFICATION CONTD

Bioavailability and toxicity of nanoparticles are largely unknown (Biswas and Wu,

2005)

Due to the increased use, titanium dioxide nanoparticles will inevitably reach the

aquatic environment, where they have so far been traced from urban applications

into receiving waters of urban runoff (Kaegi et al., 2008)

Algae and crustaceans were also the most sensitive environmentally relevant

species for synthetic nanoparticles (Kahru and Dubourguier, 2010)

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AIM OF THE STUDY

to study the effect of titanium dioxide nanoparticles and nutrient concentrations on

the growth, biomass production, biochemical composition and antioxidant responseof Chlorella vulgaris.

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OBJECTIVES OF THE STUDYTo determine:

i. the effect of titanium dioxide nanoparticles at different nutrient concentrationson the growth and biomass production of Chlorella vulgaris.

ii. the effect of titanium dioxide nanoparticles as a function of nutrient

concentrations on the biochemical composition of Chlorella vulgaris.

iii. the effect of titanium dioxide nanoparticles and nutrient concentrations on the

antioxidant response of Chlorella vulgaris.

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HYPOTHESES

i. There is no significant effect of titanium dioxide nanoparticles and nutrient

concentrations treatment on the growth and biomass of Chlorella vulgaris.

ii. There is no significant effect of titanium dioxide nanoparticles and nutrient

concentrations treatment on the biochemical composition of Chlorella vulgaris.

iii. There is no significant effect of titanium dioxide nanoparticles and nutrient

concentrations treatment on the antioxidant response of Chlorella vulgaris.

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MATERIALS AND METHODS

ALGAL CULTURE SPECIES

The microalgae Chlorella vulgarisstrain was obtained from the freshwatermicroalgae culture collection of the University of Texas, USA.

CULTURE MEDIA

The algal species will be cultured in the OECD medium (2011).

CULTURE CONDITIONS:

Culture will be maintained at 23 2oC under continuous lighting at an

intensity of 1 20mol m-2s-1with white fluorescent lamps.

Algae suspension will be periodically shaken by manual means daily, to

prevent clumping (Wei et al., 2010).

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MATERIALS AND METHODS CONTD

TITANIUM DIOXIDE TREATMENT

Dry Titanium (IV) oxide-anatase nano-powder will be purchased fromSigma-Aldrich, USA; (CAS number 637254), particle size


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Nanoparticles Treatment

5 ml of the different titanium dioxide nanoparticles concentrations will

be added to 35 ml of algal suspension (Wei et al., 2010) .

Treatment will be replicated thrice and will be for 96hrs.

Selection of these concentrations will be based on 96hrs EC50 of

titanium dioxide nanoparticles that will be obtained in preliminary

experiment on determining the acute toxicity.

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Nutrient source

Nitrogen will be provided as NH4Cl at 1.110-3M (control), 2.910-6M,

1.110-5M, which represent limiting and environmentally replete

nitrogen concentrations in aquatic environments (Reynolds, 2006).

At each nitrogen concentration, n-TiO2 will be added at the chosen n-TiO2concentrations after the EC50 determination, while the control will have

no n-TiO2.

Phosphorus will be provided as KH2PO4 at 0.02mg/l, 0.06mg/l, 0.2mg/l,

which represents phosphorus concentrations at different aquatic trophic

levels (Mainstone, 2002).

At each phosphorus concentration, n-TiO2will be added at the chosen n-

TiO2 concentrations after the EC50 determination, while the control will

have no n-Tio2

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T0 T1 T2 T3

N0 N0T0 N0T1 N0T2 N0T3

N- N-T0 N-T1 N-T2 N-T3

N+ N+T0 N+T1 N+T2 N+T3

T- Titanium dioxide nanoparticles + : Replete

N- Nitrogen - : Deficient

P- Phosphorus


Table 1: Treatment combinations of titanium dioxide with nitrogen

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Table 2: Treatment combinations of titanium dioxide with phosphorus

T0 T1 T2 T3

P0 P0T0 P0T1 P0T2 P0T3

P- P-T0 P-T1 P-T2 P-T3

P+ P+T0 P+T1 P+T2 P+T3

T- Titanium dioxide nanoparticles + : Replete

N- Nitrogen - : Deficient

P- Phosphorus

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DETERMINATION OF GROWTH AND BIOMASS

Biomass Determination

Spectrophotometry at optical density at 500 nm (Liu et al., 2008)

Direct cell counting using a Neubauer haemocytometer.

Dry Weight Measurement

Dry weight determination will be done using Whatman GF/C filters (5 cm

diameter) pre-dried at 60oC for 2 h.

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PHOTOSYNTHETIC PIGMENT DETERMINATION

Chlorophyll a, b and total chlorophyll (Nmeth, 1998)

Total carotenoids (Wellburn, 1994).

BIOCHEMICAL COMPOSITION DETERMINATION

Total proteins(Bradford, 1976)

Total carbohydrates(Liu et al., 1973)

Total lipids (Blight and Dyer 1956)

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ANTIOXIDANT ENZYME EXTRATIONAND ASSAYS

Assay of Superoxide Dismutase (SOD) (Misra and Fridovich ,1972)

Assay of catalase (Luck, 1974)

Assay of Peroxidase (Reddy et al., 1995)

Assay of Glutathione S-Transferase (Habig et al., 1979)

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DATA ANALYSES

The EC50values (titanium dioxide concentration required to cause a 50%reduction in growth) will be computed using USEPA Probit analysis

program version 2.0.

SPSS version 20 Release 20.0.0 (IBM 2011)

Analysis of Variance (ANOVA)

Tukeys post hoc test will be used for separation of means where

significant.

STATISCA version 10 (Stat Soft. Inc. (2011)) program will be used for data

analysis.

Correlation based Principal Component Analysis (PCA).

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Thank You for Listening

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11th July Power Point