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Chinese Science Bulletin 2003 Vol. 48 No.9 862 868
Characteristic and
mechanism of inactivating
algae with O3 and ClO2
HU Wenrong, LIU Peiqi & PEI Haiyan
Reseach Center for Environment Science & Technology, Shandong
University, Jinan 250061, China
Correspondenced should be addressed to Hu Wenrong (e-mail: wenrongh
@sdu.edu.cn)
Abstract Both O3 and ClO 2 have a high effect on inactivating-algae in source water with no forming THMs which
do harm to human in producing drinking water, so they will
be favorably substituted for Cl2. In order to make certain of
the mechanism of inactivating algae with O3 and ClO 2, the
algal cell number change and its different characteristics of
figures and structures in treated and untreated water have
been studied by the microscopy and SEM and the mode of
inactivating algae has been inferred. The results show that
the mechanism of inactivating algae by O3 is not completely
identical with that by ClO 2. The actual reaction process and
efficiency have been controlled by many factors, such as the
different characteristics of oxidants and algal cells.
Keywords: ozone, chlorine dioxide, inactivating algae, mechanism.
DOI: 10.1360/02wb0159
Algae blooms have serious influence on operation of
most waterworks with surface water as source water.
Over-multiplication of algae can jam the filter tank,
shorten operation cycles of the tank, increase back flushing frequency, water consumption and running cost.
Moreover, algae are the main origin of natural organic
matters (NOM) in source water, which have been regarded as the predecessors of by-products in producing drinking water when using Cl2 as a disinfectant [1]. It is very
important to study and overcome over-multiplication of
algae in source water.
Most waterworks in China adopt Cl2 as algaecide to
alleviate the negative effects of algae on producing
drinking water. Cl2 can effectively inactivate the algae, but
it also reacts with the organic substances in source water,
forming some deleterious by-products of mutagenicity[2] .
Many studies have shown that both O 3 and ClO 2 had a
high effect on inactivating algae[3,4], with no forming Trihalomethanes (THMs)[ 4 — 6] , and Ames tests also showed
that the mutagenicity in the treated water was not increased when O3 and ClO2 had been used as disinfectants
in producing drinking water1). Therefore, both are favorably substituted for Cl 2. So it is significant to study the
technology of inactivating algae and make certain of its
mechanism.
1 Materials and methods
( ) Water samples. The source water with algae
was sampled from a man-made lake. The algae in the
water sample were used in the tests after magnification
culture. The main algae species included Chlorella, Pediastrum, Ulothrix, Scenedesmus, Navicula and so on. In the
cultured water, Chlorella was the dominant species, accounting for about 80% of the total algae in number.
( ) Methods. The continuous running process was
applied in the test.
(1) The test on inactivating algae with O 3. The
apparatus included a reaction cylinder and an ozonizer
system. The cylinder was made of glass, with 1500 mm
height and 3.0 L cubage. The type of ozonizer was
FDHK-2, with oxygen as air source.
Ozonization gas from the ozonizer passed through
the aerating device made from silica sand at the bottom of
the reaction cylinder and became tiny gas bubbles. The
gas bubble inversely contacted with the down flow water
and inactivated the algae in water.
(2) The test on inactivating algae with ClO2. The
apparatus included the oxidant doser system and mixreaction vessel. ClO2 was added in the vessel through
peristaltic pump after the stable form activated by hydrochloric acid. The dimension of the mix-reaction vessel
was 160 mm 80 mm 120 mm. Its available capacity
was 1.0 L. In the middle of the vessel the cross wall was
set to split the vessel into the mixing zone and the reaction
zone.
The activated ClO2 intensely mixed with influent
water in the mix zone. Then the mixture came into the
reaction zone through the holes under the cross wall,
where algae were inactivated.
During the test, the microscopy, chlorophyll-a (Chla)
measurement and examining with a scanning electron
microscope (SEM) were employed to analyze the changes
of algae cells after being treated in order to make sure of
the mechanism of inactivating algae with O 3 and ClO2.
2 Results and discussion
( ) Microscopy results. In order to determine the
acting character and modes of O3 and ClO2 on algal cells,
the number, genus and the changes of the cells were analyzed by the microscopy. The results of microscopy and
the Chla changes are shown in Tables 1 and 2. The reaction time of O3 and ClO2 to algae in this test was 30 min.
As shown in Table 1, the cell number and the Chla in
water decreased with O3 dose increasing. But the decreasing amplitude between the number and the Chla was different. And the removal rate of Chla was markedly higher
than that of cell number. When 10.0 mg/L O 3 dose w as
1) Pei Haiyan, Study on algae remove in the polluting source water by oxidization methods, Master degree paper, Shandong University, Jinan,
2001, 11.
862
Chinese Science Bulletin Vol. 48 No. 9 May 2003
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Table 1
Algae genus
O3 adding dose/reaction dose/mg L−1
0
5/4.1
10/7.9
15/11.2
20/14.5
30/20.6
2. 56
2. 32
2.00
1.12
1.04
0.8
4
0. 48
0. 48
0.32
0.24
0.16
0.08
104/mL)
3. 04
2. 80
2.32
1.36
1.20
0.88
22. 17
9.74
7.61
4.46
2.27
1.77
Chlorella ( 104/mL)
Others ( 10 /mL)
Total (
Chla/µg L
−1
Water flavo green, Turbidity and fishi- Chlorella reduced;
fishiness odor;
ness odor declined; Ulothrix broke and
Chlorella domi- Chlorella reduced reduced
nant
Phenomenon
Table 2
Algae genus
4
Chlorella ( 10 /mL)
4
The others ( 10 /mL)
Total (
Change of algae during O3 treatment process
4
10 /mL)
Chla/µg L
−1
Phenomenon
Chlorella reduced Chlorella reduced; Some Bacillariophyta
clearly; Bacillario- filiform segments shells broke down;
phyta shells appeared appeared
Scendesmus having no
obvious changes
Change of algae during ClO2 treatment process
ClO2 dose/mg L−1
0
2
4
6
14. 13
13.73
13.44
14.13
1. 33
1.07
1.00
0.80
15. 46
14.80
14.44
14.93
99. 70
Water flavo green; fishiness
odor; Chlorella dominant
13.92
4.39
2.82
Appeared white, feculent; smell
Color faded; smell increased Color completely faded and
still higer than raw water; cell
(partly from ClO2); intracellu- appeared white, feculent;
Bacillariophyta shells appeared number having no obvious
lar matter turned yellow
change
added, the cell number reduced from 3.04 104/mL to
2.32 104/mL (24% removal rate), while the removal rate
of Chla was 65.7%.
The microscopy indicated that O3 exerted different
acting efficiency on different algae. O3 had an ability to
inactivate and decompose Chlorella, Ulothrix and
Anabaenopsis. It could also inactivate Bacillariophyta by
destroying the cell organs. But O3 had a poor ability to
inactivate Scendesmus and Micractinium.
The cell number did not decrease markedly after
ClO2 was added, but the water chromaticity and the Chla
content decreased rapidly. When 2 mg/L ClO2 was applied,
the corresponding Chla removal rate reached 80%. It can
be seen that ClO2 could quickly invade the cell, oxidize
the intracellular Chla and inactivate the algal cells. But
ClO2 could not oxidize the cell structure and reduce the
cell number. The microscopy results showed that ClO2
could not inactivate the Scendesmus and Micractinium
effectively, whose cell color and structure have no obvious change.
( ) Acting characteristics of O3 and ClO2 on alga
cells. In this test, the SEM (Jeol JSM-T300) was used to
observe the changes of algae in the treated water with
adding 20.0 mg/L O3 and 6.0 mg/L ClO2 respectively. The
algae characteristics in both samples were contrasted with
those in untreated water. The observing results are shown
in Figs. 1 — 6.
Fig. 1 shows the changes of Pediastrum. Pediastrum
Chinese Science Bulletin Vol. 48 No. 9 May 2003
possessed sixteen cells which were made up of five or six
sides and appeared to be pentagonal or hexagonal. Pediastrum looked like an integral dish with regular holes.
There were many tiny granules on the surface wall (Fig.
1(a)). Pediastrum oxidized by O 3 twisted and distorted
seriously. The integral structure was destroyed and some
infielder cells were lost. The arrange- ment of outer field
cells was dislocated and the inside field cell’s groups were
destroyed (Fig. 1(b)). The surface of Pediastrum cells in
treated water with ClO2 was se- verely crimpled and cavitated. The cell inosculated and the boundary of each cell
was blurry. Some infielder cells were lost and tiny granules on the cell wall surface van- ished (Fig. 1(c)).
Scenedesmus in the untreated water sample was
made up of four plump cells arranged in a beeline. Two
cells in the middle looked like spindles, and the other two
liked flaxes. The cell wall of Scenedesmus was fairly
smooth (Fig. 2(a)). After being reacted with O 3, the cell
surface became crimpled and the middle cells twisted and
distorted. Some cracks or holes appeared in the cell wall
and the tips of spindles were breakaged (Fig. 2(b)). The
shape of Scenedesmus which had been treated by ClO 2
did not distinctly change, but the cell surface badly crimpled (Fig. 2(c)).
The changes of Chlorella are shown in Fig. 3. Each
cell in the untreated water appeared global or subglobose.
The cells got together and among them was thin and soft
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Fig. 1. Changes of Pediastrum.
Fig. 2. Changes of Scenedesmus.
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Chinese Science Bulletin Vol. 48 No. 9 May 2003
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Fig. 3. Changes of Chlorella.
jelly. The cell wall was very thin and the cell inclusion
was in uniform distribution (Fig. 3(a)). After Chlorella
was oxidized by O 3, t he jelly among the cells was destroyed, cracks appeared on some cells’ surface and the
cell number decreased. (Fig. 3(b)). As Fig. 3(c) shows,
Chlorella cell was not acutely destroyed, but the cracks on
the cell surface appeared after being treated with ClO2.
In the untreated water, Ulothrix often br oke into
small fragments. The cell shape was columnar, with thick
cell wall and listric slots on the surface (Fig. 4(a)). After
being oxidized by O3, the cell outer surface was destroyed
and surface slots were damaged, the cell wall turned thin
and many irregular holes appeared, at the same time, some
Ulothrix twisted (Fig. 4(b), (c)). The Ulothrix treated by
ClO2 was still straight. Parts of some cells were destroyed
or ruptured and the cell inclusion ran down (Fig. 4(d)).
In the treated water with O3 or ClO2, changes of
Navicula were not obvious. In the untreated water, Navicula appeared rhombic or subacerose and the tip of them
was rostriform. On the surface, there were transverse and
lognitudinal stripes in cross (Fig. 5(a)). After being treated
by O3, the end of Navicula broke and the rostriform tip
was destroyed, and the crack appeared on some parts of
them (Fig. 5(b)).
Cyclotella was a genus of Bacillariophyta . The Cyclotella cell was drum- or disc-shaped, the central area
was smooth and possessed actinomorphic specks. There
Chinese Science Bulletin Vol. 48 No. 9 May 2003
were sphenoidal strip on the surface of the shell and the
strip became wide gradually from the center to the edge
(Fig. 6(a)). Cyclotella cell was badly destroyed and decomposed by O3. Cell inclusion ran off (Fig. 6(b)). There
were not obvious changes of algal cells in treated water
with ClO2 (Fig. 6(c)).
3 Mechanism analysis
( ) Reaction mode of O3. O3 is unsteady in water
and is easy to decompose into O2 and some strong-activity
secondary oxidants such as creative [O], hydroxide radical
[•OH] and so on[7]. It is generally accepted that two types
of reaction take place[8]when O3 dissolves in water. One is
the direct oxidation of O3 molecule, which is slow and of
high selectivity. The other is the chain reaction caused by
[•OH] from O3 decomposing. During this course many
reactive oxygen atoms [O] come into being, which can
oxidize and decompose organic substance and microorganisms in water[9].
In the acidic medium, molecular O3. is the main oxidant, and the direct reaction is dominant. When pH is
higher than 7, [•OH] in water can excit the chain reaction of
O3 and the free radical reaction will be dominant[10]. It is
difficult to make certain which type of the reaction could
be dominant due to the diversity of the algae’s chemical
composition and the complexity of water quality. But it
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Fig. 4. Changes of Ulothrix.
Fig. 5. Changes of Navicula.
can be supposed that algae inactivating by O3 should be an
extraordinarily complex course in the neutral condition
(pH 7). It is the result that O3 and the second oxidants
act on algae at the same time. On the one hand, O3 molecule could diffuse into the cell, destroy the intracellular
organelles and inactivate the algae. At the same time, O3
can attack the alga cellularity and make cell disintegrate.
On the other hand, the second oxidants derived from O3
decomposing can mainly react on the cell surface for its
high activity and poor selectivity. As Fig. 6(b) shows, the
cells of Cyclotella treated by O3 could be severely de866
stroyed, parts of them decomposed and the number of
Cyclotella decreased. This deduction is fully testified with
the results of SEM examining and the microscopy.
( ) Reaction mode of ClO 2. ClO2 is very easily
dissolved in water. Although the molecular structure of
ClO 2 is in the unsaturated state, it does not react with
water or exists as dimmers or polymers. ClO 2 is the neutral molecule in water, so it can diffuse onto the surface of
negatively charged cell and infiltrate into the cell by virtue
of its favorable adsorbability and penetrability. It can
oxidize some functional group of the cell, influence the
Chinese Science Bulletin Vol. 48 No. 9 May 2003
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t
Fig. 6. Changes of Cyclotella.
bination of protein, block the normal metaboliziton and
inactivate the algae finally. It was reported that[10], ClO2
could inactivate the enzyme with hydrosulfide group [-SH]
as active site and control the synthesize of algal protein
quickly through reacting with hydrosulfide group [-SH] of
the cysteine.
Chla in water drops down to low level in a few
minutes after adding ClO2 because ClO2 possesses good
adsorbability and penetrability to alga cells. However, the
change of cell number is not obvious. ClO2 can oxidize
the cell inclusions quickly, resulting in the breakage of the
skeletal cell structure and the collapse of the cell wall.
These can be proved by changes of Pediastrum and
Scenedesmus in Figs. 1 and 2.
( ) Factors influencing acting effect
( 1 ) Properties of oxidants. ClO2 transforms into
ClO2− under the test conditions and the actual oxidationreduction potential is only 0.95 V, thus the actual oxidizing power is weaker than that of O3. The acting effect of
O3 on algae is higher than that of ClO2. And O3 could
attack the alga cell domineeringly. Whereas the affinity,
that is adsorb ability and penetrability of ClO2 is higher
than that of O3, it can oxidize and remove the algal Chla
rapidly and thoroughly. The SEM graphs showed that the
breakage degree of algae reacted with O3 was stronger
Chinese Science Bulletin Vol. 48 No. 9 May 2003
than that of ClO2, and O3 can reduce the number of algae.
The water color was taken off rapidly after adding ClO2,
but the number of algae changed a little.
( 2 ) Structural characteristics of algal cells. The
structural characteristics of algal cells, such as the thickness of the cell wall, the characteristics of the cell surface
and so on, can influence the actual acting effect of ClO2
and O3 on algae. Usually, the alga whose cell wall is thin
and has the granules, slots and acanthus on the surface, or
has no jelly enwrapped is easily destroyed. For example,
the Chlorella and Ulothrix whose cell wall is very thin are
easily inactivated, but Scenedesmus are not easily inactivated because its cell wall is smooth, which makes the
oxidants cannot contact with the cell surface effectively.
As Fig. 4 shows, the Scenedesmus cells surface became
crimpled, but some of them only twisted and distorted
partly after being reacted with O 3. In contrast, the
Chlorella and Ulothrix were severely destroyed and the
alga number decreased.
( 3 ) Chemical composition of algal cells. Carbohydrates, lipids and protein are the major organic substances
of the algal cell. These organic substances are readily to
turn ageing or lytic under the strong oxidative condition.
The cell wall can protect the cell bioplasm and is the first
barrier to prevent the cell from the outer attack of oxidants.
The chemical composition of the cell wall restricts the
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actual oxidizing activity. The cell wall of algae is made up
of cellulose (β-1-4-D-polyglucose), hemicellulose and
pectic substances (polysaccharide), other than, SiO2,
CaCO3 and other inorganic matter are also important ingredients of some algal cell wall. For example, the cell
wall of the Bacillariophyceae is surely silicified and the
content of SiO2 is about 50% weight of the dry cell, which
enhance its intensity of cell wall, defend the outer attack
and protect the cell bioplasm. Although the stronger oxidants can enter the cell and discompose the inclusion, they
cannot oxidize and decompose the algal cell thoroughly,
so transparent shells of Navicula could be found in water
through the microscopy.
( 4 ) Other factors. Besides the kinds of algae influence on inactivating efficiency, there may be the difference between different algae individual of the same category. During the different growth phases, the same alga
possesses different antijamming ability because of the
difference of the cell wall thickness, cell strength and
tenacity. Under a certain condition, the algal cells during
stationary and decline phases can be readily oxidized and
inactivated, while those during the logarithmic phase have
the strong oxidation resistance[1].
factors including the oxidizing power of the oxidants, the
alga species, cell structural features and the chemical
composition. Under a certain condition, some special
configuration of algae can act as a protecting shield and
make the oxidant effect on the algae decrease.
Acknowledgements This work was supported by the Sino-Japan
Science Cooperative Program (Grant No. 003250103), Bonus Fund for
Excellent Young Scientists of Shandong Province (Grant No. 9934) and
Bonus Fund for Excellent Teachers of the Ministry of Education of
China ([2001]39).
References
1.
2.
3.
4.
4 Conclusions
Both ozone and chlorine dioxide can inactivate algae
by oxidation effect, but the mechanisms are incompletely
identical because of the different characteristics of the
oxidants and the algal cells.
Inactivating algae by O3 are an exceedingly complex
course, which is the result of O3 and its second oxidants
acting on algae at the same time. On the one hand, the
molecular ozone can disperse and penetrate into the cell,
destroy the cell organs and inactivate the algae. On the
other hand, some molecular ozone and its secondary oxidants, such as [•OH] and [O], can attack the intact cytoarchitecture including the cell wall, cause the cell to break
down, and decrease the cell number.
ClO2 has a good inactivating effect on algae because
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but its ability of breaking the cell down is faint. It can
diffuse onto the surface of negatively charged algal cells
and infiltrate into the cell rapidly, oxidize the cell function
organs, and inactivate the algae finally.
The reaction between the oxidants and the algal cells
is a very complex process and may be influenced by many
868
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(Received August 12, 2002; accepted March 20, 2003)
Chinese Science Bulletin Vol. 48 No. 9 May 2003