EFFECT OF TITANIUM DIOXIDE NANOPARTICLES AND NUTRIENT

CONCENTRATION ON THE PHYSIOLOGY OF Chlorella vulgaris

(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

11
th
JULY, 2014
1
INTRODUCTION
Microalgae:
important component of the aquatic ecosystem

have the ability to fix CO
2
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).



2
INTRODUCTION
Nanoparticles:

loosely defined as manufactured materials that are smaller than 100
nanometer 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 (TiO
2
)
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).



3
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.



4
Introduction Contd

Phosphorus is also an important nutrient element.
a component of nucleic acids governing protein synthesis and of the
adenosine 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.



5
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).


6
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).


7
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 use
of 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).

8
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.




9
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 vulgaris is 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 be
used to directly observe toxicity at the cellular level (Wong et al., 1997).


10
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)
11
AIM OF THE STUDY

to study the effect of titanium dioxide nanoparticles and nutrient concentrations on
the growth, biomass production, biochemical composition and antioxidant response
of Chlorella vulgaris.

12
OBJECTIVES OF THE STUDY
To determine:

i. the effect of titanium dioxide nanoparticles at different nutrient concentrations
on 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.

13
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.

14
MATERIALS AND METHODS

ALGAL CULTURE SPECIES
The microalgae Chlorella vulgaris strain was obtained from the freshwater
microalgae 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 2
o
C under continuous lighting at an
intensity of 1 20mol m
-2
s
-1
with white fluorescent lamps.

Algae suspension will be periodically shaken by manual means daily, to
prevent clumping (Wei et al., 2010).



15
MATERIALS AND METHODS CONTD

TITANIUM DIOXIDE TREATMENT
Dry Titanium (IV) oxide-anatase nano-powder will be purchased from
Sigma-Aldrich, USA; (CAS number 637254), particle size <25nm.

Preparation of nanoparticles dispersion

Titanium dioxide nanoparticles obtained will be used to produce
suspensions in OECD algal medium 201 (OECD, 2011).

Titanium dioxide nanoparticle stock solution will be prepared by
suspending titanium dioxide nanoparticles in OECD medium in a
concentration of 5000mgL
-1
(Chen et al., 2012).

This suspension will be kept at 5
o
c in the dark and will be sonicated
for 10 minutes prior to preparation of the test suspensions.



16
MATERIALS AND METHODS CONTD

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.

17
MATERIALS AND METHODS CONTD

Nutrient source

Nitrogen will be provided as NH
4
Cl at 1.110
-3
M (control), 2.910
-6
M,
1.110
-5
M, which represent limiting and environmentally replete
nitrogen concentrations in aquatic environments (Reynolds, 2006).

At each nitrogen concentration, n-TiO
2
will be added at the chosen n-TiO
2

concentrations after the EC50 determination, while the control will have
no n-TiO
2.


Phosphorus will be provided as KH
2
PO
4
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-TiO
2
will be added at the chosen n-
TiO
2
concentrations after the EC50 determination, while the control will
have no n-Tio
2



18
T
0
T
1
T
2
T
3
N
0
N
0
T
0
N
0
T
1
N
0
T
2
N
0
T
3
N- N-T
0
N-T
1
N-T
2
N-T
3
N+ N+T
0
N+T
1
N+T
2
N+T
3
T- Titanium dioxide nanoparticles + : Replete
N- Nitrogen - : Deficient
P- Phosphorus

MATERIALS AND METHODS CONTD

Table 1: Treatment combinations of titanium dioxide with nitrogen





19
MATERIALS AND METHODS CONTD

Table 2: Treatment combinations of titanium dioxide with phosphorus



T
0
T
1
T
2
T
3

P
0
P
0
T
0
P
0
T
1
P
0
T
2
P
0
T
3
P- P-T
0
P-T
1
P-T
2
P-T
3
P+ P+T
0
P+T
1
P+T
2
P+T
3
T- Titanium dioxide nanoparticles + : Replete
N- Nitrogen - : Deficient
P- Phosphorus

20
MATERIALS AND METHODS CONTD

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 60
o
C for 2 h.



21
MATERIALS AND METHODS CONTD

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)


22
MATERIALS AND METHODS CONTD

ANTIOXIDANT ENZYME EXTRATION AND 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)







23
MATERIALS AND METHODS CONTD

DATA ANALYSES
The EC
50
values (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).

24
Thank You for Listening
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