)
Doped with H2O2 against staphylococcus aureus
Human pathogen in aqueos solution.
Hawraa Whab Azize
College of Science for women, Babylon University.
hwazize@gmail.com
Abstract
The effects of parameters such as amount of TiO2, presence of H2O2, irradiation time, dark and
light condition were studied and investigated their acted against S.aureus human pathogen in aqueous
solution. The results show that the number of S.aureus was greatly reduced after treatment with a
visible light and Tio2 doping H2o2 better than after treatment with visible light only and visible light
with Tio2 only. In the treatment with Tio2 and light the number of bacteria was decreased with survival
ratio at 40% . The best result has been obtained at 0.33mg/ml Tio2 concentration which is equal to
survival ratio 30%. Two dark/H2O2 treatments have been done in presence and absence of Tio2,
in
adding 10ppm H2o2 the bacterial number decreases (survival ratio 45%) and in adding 0.33mg/ml of
Tio2 with 10 ppm H2O2 in dark the survival ratio 40%. Two light/H2O2 treatments have been done in
presence and absence of Tio2. Survival ratio in presence of light /H2O2 was 23%, the survival ratio of S.
aureus in presence of light/H2O2/Tio2 was 11%. This study suggests that H2o2 doped Tio2 can be used
as disinfectant in water phase.
Keywords: photocatalytic killing of S. aureus, TiO2 photocatalyst, H2O2, sterilization, water phase,
light, dark.
الخلاصة:
تم دراسة تاثير عدد من العوامل مثل كمية Tio2 ،وجود H2O2 .وقت التشعيع ،وجود او عدم وجود الضوء على بكتريا
S.aureus الممرضة للانسان في الطور المائي .بينت النتائج ان اعداد البكتريا اختزلت بشكل كبير بعد المعاملة مع Tio2/H2O2
بوجود الضوء افضل من المعاملة بالضوء فقط او المعاملة بالضوء و Tio2 فقط . عند المعاملة Tio2 والضوء انخفضت اعـداد
البكتريا بنسبة بقاء 40% . كانت افضل النتائج المستحصلة عند تركيز mg/ml 0.33 من Tio2 اذ كانت نسبة البقاء 30% . تم
اجراء تجربتين بظروف الظلام وH2O2 بوجود او عدم وجود Tio2 ، عند اضافة ppm 10 من H2O2 والظلام انخفضت اعداد
البكتريا (نسبة البقاء 45%) وعند اضافة Tio2 كانت نسبة البقاء للبكتريا 40%. تم اجراء تجربتين بظروف الضـوء و H2O2
بوجود او عدم وجود Tio2 ، عند عدم وجود Tio2 كانت نسبة بقاء البكتريا 23% في حين عند وجود Tio2 بهذه الظروف كانت
نسبة بقاء البكتريا 11%. توصي هذه الدراسة باستخدام H2o2 وTio2 كمعقم في الطور المائي .
الكلمات المفتاحية : القتل الضوئي ، بكتريا ,aureus, TiO H2O2 ، التعقيم ، الضوء المائي ، الضلام
Introduction:
Staphylococcus aureus are capable of prolonged survival on a variety of
environmental surfaces, it can be alive in distilled water and in all parts of hospital
and it is resistant to chemical disinfectants and many of conventional antibiotics
(Carson etal, 1988).
It is the most virulent of the many staphylococcus species and it is responsible
for infections ranging from superficial skin to soft tissue infections (Kasper etal,
2005), and its exotoxin producing pathogen, can cause food-borne disease in human
(Salyers and Whitt, 1994) it has the highest pathogenic effect in human and it is the
first pathogen agent in hospitals (Gacesa and Russell, 1990).
The Bacteria can be destroyed by a number of different techniques including heat,
radiation and chemical oxidizing agents such us H2O2,Which are used to treat
microbial pollution of waters ,but they are not completely efficient on some of
resisting microorganism such us S.aureus (Mills and Lettunte ,1997).
The wide spread use of antibiotics led to the emergence of more resistant and
virulent strains of bacteria and this caused an urgent need to develop alternative
sterilization technologies to disinfect water and waste water from hospital such us
using photocatalytic effect of Tio2,this method is feasible and inexpensive to act as
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(23): 2015
618
disinfectants (Russell, 2004 and Manes etal 1999 and Aiello and Larson ,2003 and
Block, etal 1997 ) and powerful biocide process due to production of redox reaction
species from Tio2, when irradiation Tio2 particles are indirect contact with or close to
microbes to initial oxidative attack (Daneshvar etal, 2007 and Lee etal, 2005) and
photokilling action was associated with the reduction in the level of intracellular coA
through photo oxidation (Mills & Lettunte 1997).
The oxidation processes such us Tio2/Uv and H2o2/Uv are useful in water
purification and surgical suites (Coates etal, 2007 , Julian etal, 2007 and Cho
etal,2002).
Since 1981 many papers have been published about semiconductor, the most
studies in this field have been done on bacteria especially E.coli and S. aureus and
other bacteria. Mastsunaga and coworkers published first report about photocatalytic
disinfection in 1985 (Mills & Lettunte 1997; Aiello & Larson 2003; Julain etal 2007;
Hemraj etal,2014).
The action of this technology in aqueous phase is in presence of Tio2 alone or
with H2o2 upon Uv light excitations ,the photo energy excites valenee band electron
and generate pairs of electrons and holes that diffuse and trapped on or near the Tio2
surface (Wong etal 2006; Cheng etal, 2009; John etal, 2014) and these pairs have
strong reducing and oxidizing oxygen to yield reactive species such us –OH &-O2
(Fujishima and Honda 1972) which are extremely reactive upon contact with organic
compounds and bacterial cell (Saleh, 2011) and complete oxidation to carbon dioxide
(Jacoby etal, 1998). These radicals operate in consent to attack poly unsaturated
phospholipids in bacteria (Wong etal, 2006) Another related study was carried out by
Hirakawa and coworkers (2004) which has shown that photo irradiation Tio2
catalyzed site-specific DNA damage via H2o2 .These findings suggested that Tio2
might exert antimicrobial effects similar to these of peroxygen disinfectant H2o2
(McDonnell and Russell, 1999).
The aim of this study is to investigate the effect of Uv visible light on the
antibacterial activity in presence of H2o2/Tio2 and using photo catalytic reaction for
disinfecting water and waste water instead of chemical material.
Materials and Methods
Bacterial Strains and Culture
Basic bacterial cultured methods have been done (Johnson and Case, 1995).
Clinical isolated S. aureus was collected from Babylon Hospital of pediatric and
maternity.
In this study, bacterial concentrations were determined by standard plating
count method (SPC).Afresh bacterial culture was diluted by factors 10-1
to 10-6
and
bacterial concentrations of these dilutions were determined using SPC. After this step
the right dilution has been selected to be used in these experiments and before every
experimental the number of bacteria reading and this number is used as astandard
number.
Chemicals
A:-Nutient agar was supplied from HIMDIA.
B:-H2O2was supplied from DDH at 30%.
C:-Titanium dioxide (Tio2) was supplied from Degussa P25(Cheng etal,
2009; Julain etal, 2007).
2-3 Instruments: photo catalysis cell.
A:-Source of irradiation: use low pressure mercury lamp type OSRAM (160W)
(306-750nm)
B: Reaction vessel: content photo cell (35 cm
3
) with quartz window (2cm
2
).
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(23): 2015
619
C: Regulator circulating thermostat (Desagafrigosta): using to control the
temperature.
D: Oxygen gas container was connected with flowmeter (Rato) to control the rate
of gas passing on the surface of aqueous solution.
E: Amagnetic stirrer (Abovolt) was used to homogenous suspension.
F: Centrifuge (Hettich) was used to remove Tio2 practical and the supernal liquid,
the instruments used in this work were previously described in detail (Gassim etal,
2004).
2-4 Photo Catalysis Experiments:-
In all photo catalytic experiments 30 ml of aqueous solution of S. aureus cell
suspensions were added to a known weight of Tio2 particles in photo cell quartiz
window and suspended by using magnetic stirrer the oxygen was passed on the
surface of aqueous suspension at the rate 10 ml/min. The temperature was controlled
at 25c
0 by using circulating thermostat. The suspension was irradiated for 40 min.
Other experiments have been done by adding 10 ppm of H2o2 S. aureus aqueous
solution in absence and in presence of Tio2 catalyst, dark and light condition. After
each 10(min) samples of irradiated mixture were drawn by using syringe with along
pliable needle and then centrifuged at 1000 rpm /5min in all experiments, 0.5 ml of
the suspension was immediately added to 20ml nutrient agar media in petri dish with
triplicates per each treatment. The culture was kept in the dark at 37c
0
for 24h .Colony
forming units (cfu) of S. aureus were counted by SPC (Saleh,2011).
The incident light intensity was measured by using parcker and Haut chard
method (Maness etal, 1999) this method consists of irradiated potassium ferrioxalate
actinometry k3fe (c2o4) 2.3 H2o2 for 3min after passing nitrogen gas for 15 min at
25c
0
. The average light intensity is 6.2×10-8 Einstein L-1
S
-1
(Gassin etal, 2009).
Results & Discussion:-
In this study the effects of parameters such as amount of TiO2, presence of
H2O2, irradiation time, dark and light condition were studied and investigated their
acted against S.aureus human pathogen. All experiments occurred in presence of
oxygen.
1-Determination of optimum conditions for photocatalytic reactions.
To determine the optimum condition, which led to high killing efficiency, four
primary experiments have been done .Figure(1) shows that ,in the first treatment with
dark only the number of bacterial cell was increased with time because the bacteria
have acclimatized to their new environment and synthesis the enzymes needed to
initialize the available materials (Johnson &case ,1995) and division by binary fission
this leads to increase the number of cells with time in aqueous solution(Kwaadsteniet
etal,2011).
In the treatment with Tio2 in dark (Figure-2), there s no effect on bacterial
number because Tio2 are biologically and chemically invert and its antibacterial
activity can be switched on and off or modulated by controlling the light intensity,
and this agrees well with other studies (wong etal,2006; cheng, etal,2009;
Kwaadsteniet etal,2011; Hemraj etal,2014).
In the treatment with light only, the number of bacteria is decreased, because
the light used in this work has wave length ranges from 306 to 750 nm which can
cause damage