Bioresource Technology 102 (2011) 5742–5748
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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Inhibition of the growth of two blue-green algae species (Microsystis aruginosa
and Anabaena spiroides) by acidification treatments using carbon dioxide
Xin Wang a, Chunbo Hao a, Feng Zhang a, Chuanping Feng a,⇑, Yingnan Yang b
a
b
School of Water Resource and Environment, China University of Geosciences (Beijing), Beijing 100083, PR China
Graduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e
i n f o
Article history:
Received 21 December 2010
Received in revised form 4 March 2011
Accepted 7 March 2011
Available online 12 March 2011
Keywords:
CO2
Blue-green algae
Acidification
pH
Inhibition
a b s t r a c t
The effect of pH adjusted by aeration with carbon dioxide (CO2) on the growth of two species of bluegreen algae, Microcystis aeruginosa and Anabaena spiroides, was investigated. Three conditions (pH 5.5,
6.0 and 6.5) were found to have significant inhibitory effects on the growth of the two algae species when
acidification treatment was conducted during the logarithmic phase. Differences in the inhibition effect
of acidification existed between the two species algae. The tolerance of M. aeruginosa to these conditions
was also investigated. The results indicated that M. aeruginosa was inhibited significantly, but not dead at
pH 6.5, whereas death occurred at pH 5.5 and 6.0. The greatest inhibitory effect of acidification treatment
conducted during the stable breeding phase of M. aeruginosa occurred at pH 5.5, while no inhibitory effect
was found at pH 6.5.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Eutrophication and algal blooms have resulted in widespread
deterioration of the quality of surface water, especially in lakes
and reservoirs. These conditions often arise from anthropogenic
pollution with nutrients, particularly the release of sewage effluents and agricultural run-off carrying fertilizers into natural
waters. The enhanced growth of choking aquatic vegetation or
phytoplankton in aquatic environments disrupts the normal function of the ecosystem, resulting in a myriad of problems such as
depletion of dissolved oxygen, decreased water transparency, degradation of biodiversity, changes in species composition and dominance, and toxic effects. Of particular concern are harmful algal
blooms (HABs) due to the natural toxins produced by some bluegreen algae. For example, Microcystis aeruginosa produces microcystin, which has been reported to be associated with acute liver
damage and possibly liver cancer in laboratory animals (Lora and
Fleming, 2002).
Several attempts have been made to address the issue of algal
blooms to date, including physical (e.g., ultrasonication, trapping
algae using nets, and centrifugal separation), chemical (e.g., use
of copper sulfate, hydrogen peroxide, and ozonization) and biological treatments (e.g., introduction of other types of plankton such
⇑ Corresponding author. Tel.: +86 13801205306; fax: +86 010 82321081.
E-mail
(C. Feng).
addresses:
[email protected],
[email protected]
0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.015
as Eichharnia crassipes) (Ferrier et al., 2005; Spiros et al., 2009;
Liang and Nan, 2009).
Although the approaches mentioned above can efficiently
remove blue-green algae from polluted surface water in the short
term, there are collateral ecosystem and economic limitations to
their long-time use (Hrudey et al., 1999; Oliveira-Filho et al.,
2004). Indeed, this is why these techniques are only used as
emergency solutions to algal blooms. Accordingly, additional
research is needed to develop a method of controlling algal blooms
with greater efficiency, lesser environmental impact and long-term
operation.
Surface water pH is an important environmental factor that can
drastically affect the growth of algal populations (Agrawal and
Singh, 2000; Dnailov and Ekelund, 2001). Algal species can only
grow well when the pH is within a certain range, without taking
into account other environmental factors. Under other conditions,
these organisms will die or their activity will be inhibited (Twist
et al., 1998). Blue-green algae, which is the most dominant and
dangerous species in summer lake blooms, usually prefer slightly
alkaline conditions (pH 7.7–9.4), which is close to the pH of natural
water (Wicks and Thief, 1990). Therefore, the pH of the water can
be adjusted to be slightly acidic to inhibit the growth of blue-green
algae to some extent. The potential for the use of low pH to control
the growth of algae has been recognized by several researchers.
Axelsson et al. (2000) found that high concentrations of heavy metals and low concentrations of soluble nutrients (e.g., phosphate) in
acidic environments had indirect effects on algal growth, whereas
high concentrations of hydrogen ion damaging species had direct
X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
effects. However, although acidification treatment may be a useful
method of controlling the excessive growth of algae and therefore
reducing the damage caused by summer blooms, little research has
been conducted in surface water bodies. This is because of the disadvantages that exist when common acid reagents such as sulfuric
acid and acetic acid are used to adjust the pH. These disadvantages
include (1) difficulty of pH control, (2) poor diffusion capability of
H+, and (3) harmful effects of sulfates, chlorides, or acetate ions derived from the corresponding acids. In this study, carbon dioxide
(CO2) was used to acidify the water while avoiding the disadvantages mentioned above.
Most of the research that has been conducted to evaluate the
influence of CO2 on microalgae growth to date has focused on
the effect of different CO2 aerated concentrations, rather than
changes in pH resulting from CO2 aeration. Several studies have reported that the growth rate of some algal strains was enhanced
when the cultures were aerated with elevated levels of CO2 such
as 2% CO2 (Riebesell et al., 1993; Schippers and Lürling, 2004;
Zou, 2005; Ge et al., 2011). In contrast, most microalgae have been
found to be susceptible to high concentrations of CO2 (e.g., P10%
CO2) due to damage to the PS II process at low pH and low CO2 concentrating mechanism (CCM) efficiency, as well as to photoinhibition (Ikuko and Qiang, 1998; Miyachi et al., 2003; Chiu and Kao,
2009). However, Miyachi et al. (2003) also found that some green
algae grew rapidly under a CO2 concentration higher than 40% with
an uncertain mechanism. In the context of these studies, CO2 was
seen as a reactant in the photosynthesis process of algae (e.g., carbon source). However, little information regarding the role of CO2
as an acidification reagent with respect to changes in the pH of surface water has been reported.
In this study, the effect of pH values ranging from 5.5 to 6.5, adjusted by the addition of various amounts of CO2 on two species of
blue-green algae during different growth stages, were investigated.
The purpose of this study was to evaluate the effects of the CO2
acidification method on the control and management of summer
blooms caused by blue-green algae. To the best of our knowledge,
this is the first study to use carbon dioxide to regulate the pH of the
culture medium to inhibit the growth of blue-green algae. In addition, CO2 is recognized as the main gas causing global warming;
however, our study can be used to identify methods of decreasing
the CO2 concentration in the atmosphere and thus reducing the
problem of global warming to a certain extent. Accordingly, the results presented herein will help manage summer blooms of surface
water and decrease greenhouse gas emissions.
2. Methods
2.1. Microalgae species and cultures
Two blue-green algal species, M. aeruginosa and Anabaena spiroides, were selected for this study because they are the dominant
species during the summer bloom. In addition, the toxicity of the
production of M. aeruginosa microcystin was considered. The species were obtained from the Innovation Base of Lake Eco-environment, Chinese Research Academy of Environmental Sciences
(CRAES) and maintained in M11 medium with the following composition (per liter): 100 mg NaNO3, 10 mg K2HPO4, 75 mg
MgSO47H2O, 40 mg CaCl22H2O, 20 mg Na2CO3, 6 mg ferric citrate
and 1 mg Na2EDTA2H2O, with the pH adjusted to 8.0. Stock cultures (approximately 105 cells mL1) of both algae were maintained in 500 mL flasks enclosed with sealing membrane. When
the cell number of the cultures was approximately 108–109 cells
mL1 (about 7 days of culture), the stock algae were transferred
to experimental reactors with 15 L M11 working medium at an initial cell concentration of around 105 cells mL1. All cultures,
including the stock and working cultures, were cultivated in an
5743
illumination incubator under the conditions of 26 °C, 2000 l,
and light/dark (L/D) 14 h/10 h cycles.
2.2. Experimental equipment
The lab-scale equipment used in this study consisted of an illumination incubator (GP-01, Hengfeng Medical Equipment, China),
four PMMA rectangle reactors (40 cm 25 cm 30 cm) with 15 L
cultures, a gas cylinder maintaining CO2 (99.99% purity), and three
flow meters used to control gas flow at a maximum rate of
100 mL min1. Each acidified reactor was sparged with CO2 from
the upper part of the reactor.
2.3. Experimental design
The experiment was designed taking into account two factors,
determination of the aeration conditions and the effect of acidification with CO2 on the two species of blue-green algae. In the first
part of the experiment, the optimal aeration rate and CO2 aeration
frequency were examined. In the second part of the experiment,
the growth of the two algal species at both the logarithmic growth
and stable breeding phases during the acidification period was
investigated to determine the control efficiency. Detailed information regarding all experiments is presented below.
2.3.1. Determination of the optimal CO2 aeration rate
Three acidified reactors were aerated with CO2 gas at different
flow rates (10, 30 and 50 mL min1). During the aeration process,
the pH of each reactor was determined at an interval of 5 min to
investigate the change in pH according to the gas rate.
2.3.2. Determination of the optimal CO2 aeration frequency
After the pH of the reactors was regulated at the setting points
(5.5, 6.0 and 6.5, respectively), the pH of each reactor was examined continuously to investigate the extent of the CO2 release
and determine the suitable aeration frequency. Both pure culture
medium and algae-maintained medium were examined.
2.3.3. Acidification during the logarithmic growth phase
The experiment involved two groups: one for M. aeruginosa
and another for A. spiroides. Each group consisted of three acidified reactors with various pH values (pH 5.5, 6.0 and 6.5, respectively) adjusted by different CO2 flow fluxes and one control
reactor without aeration. Since the 2nd day of inoculation, CO2
was sparged at the determined frequency to maintain the pH corresponding to each acidified reactor. The reactor pH and DO were
monitored, and sample solutions were collected daily to evaluate
the content of chlorophyll a throughout the acidification period.
In the M. aeruginosa experiment, samples were also collected
and analyzed on the 1st and 7th days after the acidification
period to investigate the tolerance of M. aeruginosa to acidic
conditions.
2.3.4. Acidification during the stable breeding phase
This experiment was only conducted for M. aeruginosa. The
same general set of processes as those used to evaluate the effects
at the logarithmic growth phase were conducted; however, the
aeration treatments were not carried out until after 14 days of culture, when the growth of the algal species had entered the stable
breeding phase.
2.4. Analytical methods
The measurement of pH and DO was conducted at regular,
predetermined intervals. The reactor pH and DO were directly
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X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
determined using a pH meter (HM-21P, Toadkk Corp., Japan) and a
DO meter (Orion 3 Star DO portable instrument, Thermo, USA).
The number of both algae was determined by direct counting
using a microscope (XSZ-4G, Chongqing Optical & Electrical Instrument Co., Ltd., China).
The growth of algae was estimated based on the chlorophyll a
content, which was extracted with 90% acetone and measured
using a spectrophotometer (752 N, Shanghai Precision & Scientific
Instrument Co., Ltd., China), according to the Water and Waste
Water Monitoring and Analysis Standard Methods (Editorial Committee of Ministry Environmental Protection of China, 2002),
rather than measuring the cell density to avoid possible errors
resulting from weak or dead cells.
2.5. Measurement of growth rate
l ¼ lnðEt =E0 Þ=Dt
where Et and E0 are the final and initial chlorophyll a concentrations, respectively, and Dt is the cultivation time in days (Ono and
Cuello, 2007).
3. Results and discussion
3.1. Optimal aeration rate of CO2
To determine the aeration rate for adjusting the pH, the change
in pH with time was investigated at three aeration rates (10, 30,
and 50 mL min1). The results showed that the pH of the three
reactors decreased continuously with time to an almost constant
value, i.e., there was a final pH for a definite rate of aeration
(Fig. 1). After 95 min aeration, the final pH was 5.91, 5.56 and
5.41 at 10, 30, and 50 mL min1 aeration rate, respectively. Additionally, a higher aeration rate was associated with a more rapid
decrease in pH and a lower final pH. These results suggested that
the ability of the pH to reach a low level is more dependent on a
high aeration rate than a long aeration time with a low aeration
rate. Consequently, to induce the pH of the three acidified reactors
to decrease from about 8 to 6.5, 6.0, and 5.5 within a short time
(<15 min), the aeration rates were set at 50, 80 and 100 mL min1,
respectively.
The capability of CO2 to generate hydrogen ions with H2O was
the reason for the decrease in pH when aerating CO2 into water
(Gao et al., 1991). The chemical reactions of CO2 and H2O in aquatic
environments were as follows:
9.0
V=10mL/min
V=30mL/min
V=50mL/min
pH
7.0
6.0
5.0
4.0
0
20
H2 CO3 $ HCO3 þ Hþ ;
HCO3
$
CO2
3
þ
þH ;
K H ¼ 3:38 102 mol atm1 L1 ð25 CÞ ð1Þ
ð2Þ
pK 1 ¼ 6:3
ð3Þ
pK 2 ¼ 10:3
ð4Þ
The final pH corresponding to the equilibrium state of the carbonate
system described above depends on the partial pressure of CO2 and
the proportion of carbonate and bicarbonate ions (Gorard, 2005).
Accordingly, the finding that different CO2 ventilation rates lead
to different final pH values was attributed to the balance between
the pressure of the CO2 ventilation and the partial pressure of CO2
in the solution. Consequently, the ventilation rate was the decisive
factor for the final pH value of the solution.
3.2. Optimal aeration frequency of CO2
The specific growth rate (d1) was calculated as follows:
8.0
CO2 ðgÞ $ CO2 ðaqÞ;
CO2 þ H2 O $ H2 CO3
40
60
80
100
t (min)
Fig. 1. Change of pH in the three reactors corresponding to the three aeration rates
(V = 10 mL min1, 30 mL min1, 50 mL min1) with time.
CO2 was used as the medium in our experiment to decrease the
pH of the water from alkaline to acidic. However, the CO2 volume
in the algae solution might decrease continuously after stopping
aeration via two routes, CO2 release from the solution to the air
and CO2 absorption by algae, both of which result in a change from
acidic to alkaline conditions. Therefore, it is essential to study the
extent of CO2 released in culture medium (no algae contained)
and the absorption by algae to determine the CO2 aeration frequency and maintain the pH at the desired level.
3.2.1. Extent of CO2 released in pure culture
As shown in Fig. 2a, the pH of the three reactors (pH 5.5, 6.0, 6.5)
all increased slightly with time. Specifically, increases in the pH of
0.09, 0.13 and 0.16 were observed within 6 h, while increases of
0.32, 0.30 and 0.34 were observed within 12 h, respectively. The
pressure of CO2 outside the solution returned to normal levels after
aeration was stopped, destroying the balance between the CO2 pressure outside the solution and that in the solution (the CO2 partial
pressure of the solution was higher than that of the outside). These
findings indicate that the increase in pH after stopping aeration
was due to the release of CO2 from the solution to the atmosphere.
These results suggest that the aeration frequency does not need to
be set at a high value (>2) to maintain the pH at the desired level
when only considering the effect of CO2 release on the pH in 1 day.
3.2.2. Comparison of the inhibitory effects on the growth of
M. aeruginosa corresponding to different aeration frequencies
As shown in Fig. 2c, a significant difference was observed in the
growth of M. aeruginosa under conditions corresponding to various
aeration frequencies with the final pH 6.0 of each aeration during
11 days culture. Specifically, M. aeruginosa cells grew logarithmically from the second day of culture when aeration was conducted
twice a day. Conversely, M. aeruginosa was found to be completely
inhibited when aeration was conducted four times a day. The chlorophyll a at the end of culture were 0.048, 1.451 and 1.875 mg L1
for two times a day, four times a day and Control respectively.
Thus, this significant difference in the growth of M. aeruginosa in
response to the two aeration frequencies was due to the different
change in pH during the acidification period (Fig. 2b). The suitable
pH range for M. aeruginosa growth was found to be 6.5–11.5. The
pH of the reactor subjected to aeration twice a day was maintained
at about 7.01 prior to the first aeration throughout the entire acidification period (Fig. 2b), which facilitated the growth of M. aeruginosa. The pH stabilized at around 6.2 in the reactor that was
subjected to aeration four times a day (Fig. 2b). In addition, the
photosynthesis of M. aeruginosa contributed greatly to the higher
increase in pH when aeration was conducted four times due to
CO2 absorption. It is worth noting that the growth of algae and
the pH of the culture medium would interact with each other
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X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
2.50
10.0
7.0
6.0
5.0
pH=5.5
pH=6.0
pH=6.5
Control
a
2.00
-1
Chlorophyll a (mg L )
8.0
pH
a
pH=5.5
pH=6.0
pH=6.5
9.0
1.50
1.00
0.50
4.0
0
2
4
6
8
10
12
0.00
t (h)
0
2
4
2 times a day
9
4 times a day
b
-1
pH
8
10
12
2.50
Chlorophyll a (mg L )
10
8
7
6
5
4
2.00
pH=5.5
pH=6.0
pH=6.5
Control
b
1.50
1.00
0.50
0
2
4
6
8
10
12
t (d)
0.00
0
2.50
8
12
16
20
24
Fig. 3. Change in chlorophyll a of Microsysitis aeruginosas (a) and Anabaena spiroides
(b) in the three acidified reactors (pH 5.5, 6.0, 6.5).
4 times a day
-1
4
t (d)
c
2 times a day
2.00
Chlorophyll a (mg L)
6
t (d)
11
Control
1.50
3.3. Effects of acidification treatments on the logarithmic growth phase
6.5) were both significantly lower than those of the control. Moreover, the growth rates of M. aeruginosa under the three acidic conditions showed a positive relationship with pH (Table 1). These
findings indicate that M. aeruginosa was inhibited significantly during the acidification period, which was similar to the results of a
study conducted by Chiu and Kao (2009) who found that the
growth of Nannochloropsis oculatain NCTU-3 aerated with 5%, 10%,
and 15% CO2 was completely inhibited. Chungching et al. (2006)
also obtained similar results in a study of the effects of pH on
the growth of Synechococcus lividus. The excess CO2 and H+ resulting from aerating CO2 into the algae solution might be responsible
for the inhibition of M. aeruginosa that was observed in the present
study. For example, excess CO2 could lower CCM efficiency because
of the weak carbonic anhydrase activity (Cheng et al., 2005). Conversely, it is assumed that excess hydrogen ion is directly harmful
to algal cells. The interior of algal cells would be acidified under
acidic conditions, which may damage the PSII of the photosynthetic system (Pronina et al., 1993). Specifically, a decrease in the
pH in the cytoplasm and possibly in the chloroplasts would stop
the Calvin–Benson cycle, thereby decreasing the population of
the open-state PSII reaction centers (Ikuko and Qiang, 1998).
Accordingly, one reason for the inhibition of M. aeruginosa might
be the decrease in the pH of its cytoplasm to different levels under
various pH conditions (pH 5.5, 6.0 and 6.5).
3.3.1. Growth of M. aeruginosa under three acidic conditions (pH 5.5,
6.0, 6.5)
The chlorophyll a concentration (Fig. 3a) and the growth rate
(Table 1) of M. aeruginosa under all three conditions (pH 5.5, 6.0,
3.3.2. Growth of A. spiroides under the three acidic conditions (pH 5.5,
6.0, 6.5)
The chlorophyll a concentration (Fig. 3b) and growth rates
(Table 1) of A. spiroides under the three acidic conditions (pH 5.5,
1.00
0.50
0.00
0
2
4
6
8
10
12
t (d)
Fig. 2. Change of pH in the three acidified reactors (pH 5.5, 6.0 and 6.5) with pure
medium after stopping aeration (a), and changes in pH (b), and chlorophyll a (c) in
two acidified reactors with Microsysitis aeruginosas under different aeration
frequencies (two times a day and four times a day).
during the acidification process. Hence, to ensure excellent inhibition of blue-green algae, the aeration frequency should be
increased properly to maintain the pH value at the desired level
as constantly as possible. Similarly, Xu and Liu (2009) found that
the pH of the aquatic environment was influenced greatly by algal
growth, and that the pH must be kept constant when controlling
the growth of algae by adjusting the pH, otherwise the inhibition
effects would not be remarkable.
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X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
Table 1
Growth rates of two species of blue-green algae under three acidic conditions (pH 5.5,
6.0 and 6.5) and control.
pH
5.5
6.0
6.5
Control
l (d1)
Table 2
Comparison of the chlorophyll a of Anabaena spiroides in the upper part and the entire
solution of the three acidified reactors (pH 5.5, 6.0 and 6.5).
pH
Microcystis aeruginosa
Anabaena spiroides
0.0030
0.0325
0.1105
0.2397
0.1175
0.1334
0.1219
0.1462
6.0, 6.5) were both lower than those of the control. In addition, the
growth rate of A. spiroides differed slightly among the three conditions (Table 1), unlike M. aeruginosa. These results demonstrated
that A. spiroides was inhibited when the acidification treatment
was conducted from the beginning of the logarithmic growth
phase, but that the effect was not as remarkable as for M. aeruginosa. Taken together, these results indicated two points: (1) both
species of blue-green algae were affected by acidic conditions,
which is consistent with a previous finding that cells with cellulosic cell walls, such as most blue-green algae, may be easily influenced by an acidic environment (Wolfgang, 2000); (2) various
species of algae preferring the same suitable pH have different abilities to adapt to acidic conditions, which supports another previous
finding that the growth rate of different strains of unicellular algae
can be influenced to different extents by pH (Nalewajko et al.,
1997). Because M. aeruginosa usually exists in a unicellular state
rather than an assembled state, it can easily be destroyed under
unfavorable conditions. Conversely, the Anabaena species, which
has a linear shape, is likely to consist of conglomerates; therefore,
it would be more resistant to damage under unfavorable conditions. This point was supported by the results of Chiang et al. (in
press) revealing that Anabena sp. CH1 had excellent CO2 tolerance
even at 15% CO2 level. Moreover, various species of algae have different abilities to maintain the pH of the chloroplasts and cytoplasm in environments with low pH or extremely high CO2. Ge
et al. (2011) reported that Botryococcus braunii grew well without
obvious inhibition when the culture pH changed from 6.0 to 8.0.
Specifically, PS II was protected from photoinhibition by control
of the state transition in some algae cells, but this protection mechanism did not function in other algal cells that were not resistant to
high CO2 (Miyachi et al., 2003; Ikuko and Qiang, 1998). Therefore,
the capability of maintaining the pH of the cytoplasm and chloroplasts might contribute in part to the difference in growth between
M. aeruginosa and A. spiroides observed in our study. Luo and Shen
(2007) and Ye et al. (2007) obtained similar results in their studies
of algae inhibition.
The effects of pH on the growth of algae were investigated
through biomass or other factors relating to photosynthesis rather
than the morphology of algae growth during the acidification process. However, in the present study, the morphology of A. spiroides
was also examined. Although A. spiroides was found to undergo
logarithmic growth under the three acidic conditions evaluated
herein, a significant sedimentation phenomenon of A. spiroides
was observed during the acidification period. In addition, the sedimentation rates of A. spiroides cells corresponding to three acidic
conditions were found to be substantially different and inversely
proportional to the pH (Table 2). These results indicated that the
cells of A. spiroides were much healthier at pH 6.5 than under the
other two conditions (pH 6.0, 5.5). It has been reported that vacuoles could be generated in blue algae cells when they exist in environments with unsuitable pH (Wu et al., 2001). Consequently, we
inferred that the sedimentation of A. spiroides cells might have been
due in part to the generation of vacuoles in A. spiroides cells under
three acidic conditions (pH 5.5, 6.0, 6.5), thereby influencing the
suspension capability of the algal cells. Moreover, it is possible that
5.5
6.0
6.5
Chlorophyll a (mg L1)
Sedimentation rate (%)
Upper part (suspension)
Whole (blended)
0.066
0.444
0.588
0.755
1.385
1.305
91.26
67.94
54.94
the bubble responsible for the suspension of algal cells in aquatic
environments might be damaged during the acidification process,
which would contribute to sinking of the algae to the bottom.
However, further studies are necessary to determine if this is the
case.
3.3.3. Tolerance of M. aeruginosa to different acidic conditions
As mentioned above, M. aeruginosa was found to be greatly
inhibited under all three acidic conditions. However, it was not
clear if M. aeruginosa was revived when the pH value returned to
alkaline. To resolve this problem, the chlorophyll a of M. aeruginosa
was measured after stopping the aeration treatment. Aside from a
study conducted by Shu et al. (2008) to evaluate the effects of
ultrasound at low power on the removal of M. aeruginosa, few studies have been conducted to evaluate the revival of algae after transfer from unsuitable conditions to suitable conditions. As shown in
Fig. 4a, the pH of the three reactors (pH 5.5, 6.0, 6.5) was 7.18, 6.81,
and 6.21, respectively, on the 1st day after stopping aeration. On
the 7th day after aeration, the pH of two reactors (pH 6.0, 5.5)
increased to about 8, whereas that of the remaining reactor (pH
6.5) increased to about 10. Correspondingly, the chlorophyll
a(Fig. 4b) and DO (Fig. 4c) levels at pH 6.5 were both much higher
than those at pH 6.0 and 5.5. The reason for the large differences in
pH, DO, and chlorophyll a between the reactor at pH 6.5 and the
two other reactors was the revival of M. aeruginosa, which grew
at pH 6.5, but died under the other two conditions. In our previous
study, we inferred that the reason for the large inhibition of the
growth of M. aeruginosa at pH 6.5 was the decrease in the cytoplasm and chloroplast pH and the damage to PSII. If this assumption was accurate, M. aeruginosa cells must have a complex
system for repairing the damage. If not, the M. aeruginosa was
greatly inhibited, despite its ability to protect its cells from damage
at pH values as low as 6.5. To date, the mechanism of the capability
of M. aeruginosa has not been identified. In contrast, M. aeruginosa
cannot thrive at pH values below 6.0.
3.4. Effects of acidification treatments during the stable breeding phase
As shown in Fig. 5a, the concentration of chlorophyll a of the
control and the three acidified conditions increased logarithmically
and had almost the same growth rate prior to aeration (2nd–14th
day). During the acidification period, the concentration of chlorophyll a at pH 6.5 was still similar to that of the control, whereas
it was lower under the other two conditions (pH 5.5, 6.0). Moreover, the concentration of DO (Fig. 5b) was lower under all three
acidic conditions than in the control, and was proportional to the
pH value. It was apparent from these results that the inhibition
effects of acidic conditions on the growth of M. aeruginosa were less
remarkable during the stable breeding phase than the logarithmic
growth phase. Similarly, Ye et al. (2007) reported that M. aeruginosa was more easily inhibited during the logarithmic growth phase
than the stationary phase in a study of the algae-inhibition performance of a hydraulic cavitation system combined with the electrolytic method. When algae cells transfer from logarithmic growth
phase to stable breeding phase, nitrogen limitation may result in
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X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
4.00
14
8
pH
3.50
pH=5.5
pH=6.0
3.00
pH=6.5
Control
a
-1
10
a
Chlorophylla (mg L )
12
The 1st day after stopping
acidification
The 7th day after stopping
acidification
6
4
2
2.50
2.00
1.50
1.00
0.50
0
5.5
6
0.00
6.5
0
pH
5
10
15
20
25
t (d)
25
-1
0.60
30
b
The 1st day after stopping
acidification
The 7th day after stopping
acidification
DO (mg L )
0.80
-1
Chlorophyll a (mg L )
1.00
0.40
pH=5.5
pH=6.0
pH=6.5
Control
b
20
15
10
0.20
5
0.00
5.5
6
0
14
6.5
-1
DO (mg L )
25
20
The 1st day after stopping
acidification
15
The 7th day after stopping
acidification
c
10
5
0
5.5
6
pH
16
18
20
22
24
t (d)
pH
6.5
Fig. 4. Chlorophyll a, pH and DO in three acidified reactors (pH 5.5, 6.0, 6.5) at the
1st and 7th days after stopping acidifying treatments for Microcystis aeruginosa.
the reduction in protein content and relative or absolute changes in
lipid and carbohydrate content, and light limitation will result in
increasing pigment content of most species and shifts in fatty acid
composition. And due to the distinct proportion and composition
of cell at different growth phases, cyanobacteria may show various
metabolic activities and sensitivity towards toxicant (Tay et al.,
2009). Additionally, membrane potential was proved to be decreased in stable breeding phase (Sahl, 1985), and the membrane
potential may influence the cell permeability. It is possible that
the permeability of M. aeruginosa cell at stable breeding phase
was low due to the decreased membrane potential, resulting in
the low concentration of H+ and CO2 entering into the cell.
Fig. 5. Chlorophyll a of the control and three acidified reactors (pH 5.5, 6.0, 6.5)
during the culture process (a) and DO of the control and three acidified reactors (pH
5.5, 6.0, 6.5) during the acidification period (b).
Therefore, the relative resistance of the stable breeding phase cells
to high concentration of H+ and CO2 might due, in part, to the metabolic activities distinct from logarithmic phase cells and a membrane potential lower than that found in logarithmic growing
phase cells.
Additionally, the biomass concentration and microcystin content in the inner cells were thought to be at the highest level when
M. aeruginosa was in the stable growth phase. During the stable
breeding phase, the microcystin content under these two conditions might have been higher than that of the control, even though
M. aeruginosa was inhibited to some extent at pH 5.5 and 6.0. This
is because the structure of some M. aeruginosa cells was destroyed
after acidification treatment (sedimentation phenomenon), which
would result in the release of microcystin from the cell interior.
When compared with the stable phase, the microcystin content
in the culture medium might be at an extremely low level when
the acidification treatment was conducted from the beginning of
the logarithmic phase due to the complete growth inhibition of
M. aeruginosa. However, it is also possible that the gene controlling
the generation of microcystin would be damaged by high concentration of CO2 or H+ during the acidification process, which would
result in the less generation of microcystin under the acid conditions. Therefore, the change of microcystin during the acidification
process needs to be further investigated.
4. Conclusions
M. aeruginosa and A. spiroides were both inhibited throughout
the entire acidification period, when the experiment was
5748
X. Wang et al. / Bioresource Technology 102 (2011) 5742–5748
conducted from the beginning of the logarithmic phase. When
compared with the logarithmic phase, the effect of pH on the
growth of M. aeruginosa was less remarkable when acidification
treatment was conducted during the stable breeding phase. Therefore, the method of CO2 acidification is much more applicable for
the prevention of blue-green blooms in lakes than for the removal
of algae after algal blooms have already occurred. It was also found
that different blue-green algae species responded differently to the
same acidic condition.
Acknowledgement
This work was supported by the 863 project (2007AA06Z351),
key project of the Science and Technology Research of Educational
Ministry (No. 108027).
References
Agrawal, S.C., Singh, V., 2000. Akinete formation and germination in three bluegreen algae and one green alga in relation to light intensity, temperature, heat
shock and UV exposure. Folia Microbiol. 45, 439–446.
Axelsson, L., Mercado, J.M., Figueroa, F.L., 2000. Utilization of HCO
3 at high pH by
the brown macroalgae Laminaria saccharina. Eur. J. Phycol. 35, 53–59.
Cheng, L.H., Zhang, L., Chen, H.L., 2005. Advances on CO2 fixation by microalgae.
Chin. J. Biotechnol. 21 (2), 177–181.
Chiang, C.L., Lee, C.M., Chen, P.C., in press. Utilization of the cyanobacteria Anabaena
sp. CH1 in biological carbon dioxide mitigation processes. Bioresour. Technol.
Chiu, S.Y., Kao, C.Y., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis
oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838.
Chungching, L., Shaolun, L., Weilung, W., 2006. Effects of temperature and pH on
growth and photosynthesis of the thermophilic cyanobacterium Synechococcus
lividus as measured by pulse-amplitude modulated fluorometry. Phycol. Res. 54,
260–268.
Dnailov, R.A., Ekelund, N.G.A., 2001. Effects of pH on the growth rate, motility and
photosynthesis in Euglena gracilis. Folia Microbiol. 46 (6), 549–554.
Editorial Committee of Ministry Environmental protection of China, 2002. Methods
of Water and Waste Water Monitoring and Analysis, 4th ed. China Environment
and Science Publisher, Beijing, MA.
Ferrier, M.D., Butler Sr, B.R., Terlizzi, D.E., Lacouture, R.V., 2005. The effects of barley
straw (Hordeum vulgare) on the growth of freshwater algae. Bioresour. Technol.
96 (16), 1788–1795.
Gao, K., Aruga, Y., Asada, K., Ishihara, T., Akano, T., Kiyohara, M., 1991. Enhanced
growth of the red algae Porphyra yezoensis Ueda in high CO2 concentrations. J.
Appl. Phycol. 3, 356–362.
Ge, Y.M., Liu, J.Z., Tian, G.M., 2011. Growth characteristics of Botryococcus braunii
765 under high CO2 concentration in photobioreactor. Bioresour. Technol. 102,
130–134.
Gorard, J.E., 2005. Principles of Environmental Chemistry. Jones and Bartlett
Publishers, Sudbury, MA.
Hrudey, S., Burch, M., Drikas, M., Gregory, R., 1999. Remedial measures. In: In
Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to their
Public Health Consequences, Monitoring and Management. WHO/Spon, London,
pp. 275–312.
Ikuko, I., Qiang, H., 1998. Effect of extremely high-CO2 stress on energy distribution
between photosystem I and photosystem II in a ‘high-CO2’ tolerant green alga,
Chlorococcum littorale and the intolerant green alga Stichococcus bacillaris. J.
Photochem. Photobiol. B: Biol. 44, 184–190.
Liang, H., Nan, J., 2009. Algae removal by ultrasonic irradiation–coagulation.
Desalination 239, 191–197.
Lora, E., Fleming, Carlos R., 2002. Blue green algal (cyanobacterial) toxins, surface
drinking water and liver cancer in Florida. Harmful Algae 1, 157–168.
Luo, W., Shen, J.Y., 2007. Responses of growth of two cyanobacteria species to
different pH environments. J. Shanghai Jiaotong Univ. (Agric. Sci.) 25 (6),
566–569.
Miyachi, S., Iwasaki, I., Shiraiwa, Y., Yoshihiro, S., 2003. Historical perspective on
microalgal and cyanobacterial acclimation to low- and extremely high-CO2
conditions. Photosynthesis Res. 77, 139–153.
Nalewajko, C., Colman, B., Olaveson, M., 1997. Effects of pH on growth,
photosynthesis, respiration, and copper tolerance of three Scenedesmus
strains. Environ. Exp. Bot. 37, 153–160.
Oliveira-Filho, E., Lopes, R., Paumgartten, F., 2004. Comparative studies on the
susceptibility of freshwater species to copper-based pesticides. Chemosphere
56, 369–374.
Ono, E., Cuello, J.L., 2007. Carbon dioxide mitigation using thermophilic
cyanobacteria. Biosyst. Eng. 96, 129–134.
Pronina, N. A., Kodama M., Miyachi S., 1993. Changes in intracellular pH values in
various microalgae induced by raising CO2 concentrations. In: XV Int. Botanical
Cong., Yokohama, Japan, p. 419.
Riebesell, U., Wolf-Gladrow, D.A., Smetacek, V.S., 1993. Carbon dioxide limitation of
marine phytoplankton growth rates. Nature 361, 249–251.
Sahl, H.G., 1985. Influence of the staphylococcin-like peptide pep 5 on membrane
potential of bacterial cells and cytoplasmic membrane vesicles. J. Bacteriol. 162,
833–836.
Schippers, P., Lürling, M., 2004. Increase of atmospheric CO2 promotes
phytoplankton productivity. Ecol. Lett. 7, 446–451.
Shu, T.G., Yuan, B.L., Wang, S.R., 2008. Studies on the removal of Mircocystis
aeruginosa by low-power ultrasoni. J. Huaqiao Univ. (Nat. Sci.) 29 (1), 72–75.
Spiros, K., Antje, S., Michiel, P., 2009. Sonic cracking of blue-green algae. Appl.
Acoust. 70, 1306–1312.
Tay, C.C., Salm, S., Lee, Y.H., 2009. The behavior of immobilized cyanobacteria
Anabaena torulosa as electrochemical toxicity biosensor. Asian J. Biol. Sci. 2 (1),
14–20.
Twist, H., Edeards, A.C., Codd, G.A., 1998. Algal growth responses to waters of
contrasting tributaries of the river Dee, North-East Scotland. Water Res. 32,
2471–2479.
Wicks, H., Thief, P.G., 1990. Environmental factors affecting the production of
peptide toxins in floating scums of the cyanobacterium Microcystis aeruginosa in
a hypertrophic african reservoir. Environ. Sci. Technol. 24, 1413–1418.
Wolfgang, Gross, 2000. Ecophysiology of algae living in highly acidic environments.
Hydrobiologia 433, 31–37.
Wu, Y.H., Zhao, Y.J., Guo, H.L., 2001. The formation of vacuole in blue-green algae
under unsuitable pH conditions. Prog. Nat. Sci. 11 (7), 761–765.
Xu, H., Liu, Z.P., 2009. Effect of pH on growth of several freshwater algae. Environ.
Sci. Technol. 32 (1), 28–30.
Ye, D.N., Jia, J.P., Xu, Y.F., Xu, D.H., 2007. Research on algae-inhibition performance
of hydraulic cavitation system combined with electrolytic method. Chin. J.
Environ. Eng. 1 (12), 67–71.
Zou, D.H., 2005. Effects of elevated atmospheric CO2 on growth, photosynthesis and
nitrogen metabolism in the economic brown seaweed, Hizikia fusiforme
(Sargassaceae, Phaeophyta). Aquaculture 250, 726–735.