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Air pollution has been a major problem throughout history [1,2]. The sources of air pollution are both natural and human based. The naturals are fire, wind, volcanic eruptions and evaporation of organic components. The burning of fossil fuels is the key of the buildup carbon dioxide (CO2) in environment, traffic and flue gas depending on industrial source are the major sources of air pollution of the results of human activity. Air pollutants such as carbon monoxides (COx), sulfur dioxide (SO2), nitrogen oxides (NOx) and other particles or trace amounts of heavy metals also have harmful effects on natural ecosystem. As a result; air pollution is a global problem and both governments and scientist study to reduce effects of this problem.
The technology for reducing CO2 and other pollutants from environment has been based on biological assimilation. Plants can reduce the atmospheric CO2 concentration by absorbing . The most common renewable energy resource has a microalgal origin [4,5]. Microalgae have the answer to many of the global problems facing us today and they use CO2, the major Global Warming Gas, with sunlight as their inexhaustible energy source, produce O2 and convert it many of the useful products and energy . The truth is that, without microalgae environment would be poorer and energy crisis would be a more and more important problem. This was a major reason why there is interest in microalgae in different areas. It is also well known that microalgae grow faster than terrestrial plants and the efficiency of energy product is much higher .
Depending on the increasing amounts of environmental pollution, several studies have been published on the linkages between microalgae and toxic components [8-12, 24-26, 30, 35] and all these studies indicated that a considerable attention in environmental pollution can be reduced by microalgal biomass.
On the other hand high amounts of SO2 and NO2 cause toxic effects on microalgal growth. The present paper is proposed to overcome the inhibition of microalgal growth by SO2 and NO2 with the help of the growth stimulators. To enhance the microalgal biomass and maintaining to photosynthesis some of the growth stimulators added to the growth media. The first of them is Triacontanol (TRIA) and the second is sodium bicarbonate (NaHCO3). Firstly, TRIA is a natural plant growth hormone, a long 3 chain 30-carbon primary alcohol (C30H61OH) and stimulate photosynthesis and growth [13,14]. On the other hand sodium bicarbonate (NaHCO3), a type of inorganic carbon, can be fixed as a CO2 source by microalgae as Wang et al.  reviewed.In the present study two strains of microscopic microalgae were tested their ability to tolerate environmental pollutants. The selection of Chlorella sp., was based on previous works to tolerate to high concentrations of CO2 [24, 26] and SOx, NOx [9, 30]. Synechococcus sp. was selected because of it is known to play an important role in the global carbon cycle  and to our knowledge has not been studied to test its ability to reduce of environmental pollutants.
The scope of the current search was to determine the interactive effects of highly toxic environmental pollutants and growth stimulators on different microalgal cultures and to compare the tolerance of them.
2. Materials and Methods
Stock solution of TRIA (96 %, w/v; Aldrich) was prepared by dissolving of 0.5 g of the chemical in chloroform (Merck). Sodium bicarbonate solution (Merck) was prepared by dissolving 17.2 g l-1 of the chemical in distilled water. Appropriate volumes of the solutions were added to the media.
The SO2 and NO2 gases were prepared by dissolving of Na2SO3 (Merck) and NaNO2 (Merck) in distilled water to a final concentration of 1 mol l-1 and by dropping into these solution 37 % HCl (Merck) for each flask sample.
2.2. Microorganisms and culture conditions
A strain of unicellular cyanobacterium, Synechococcus sp. and unicellular green alga, Chlorella sp. were used in the study, as provided by Ankara University, Faculty of Science Biotechnology Laboratory's from the current culture collection.
The microorganisms were cultivated in BG11 medium  using shake flask method. The medium pH was adjusted to 7.5 by 0.1 M H2SO4 and 0.1 M NaOH, before autoclaving. A microorganism suspension of 20 ml was contacted with 80 ml of culture media at 1 mg l-1 TRIA and 34 mg l-1 NaHCO3 for Synechococcus sp. and at 1 mg l-1 TRIA and 43 mg l-1 NaHCO3 for Chlorella sp. in an Erlenmeyer flask. Once inoculated, unshaken flasks were incubated under continuous illumination at 2400 lx light intensity provided by cool white fluorescent lamps at 25 ± 2 °C on 10 days.
2.3. Gas preparation system
In laboratory experiments a design basis was devised for producing of SO2 and NO2 gases. The model was also used to transfer these acidic pollutants to the culture media. The diagram of the experimental system is comprehensively given in Fig 1. System is composed of 500 and 250 ml Erlenmeyer flasks, magnetic heat stirrer and heat resistant hose. The 500 ml flask contains a gum elastic dowel with two holes that named acid-inlet (9.5 cm) and gas-exit (4.5 cm) and HCl trickles into the acid-inlet hole with a help of 5.0 ml clysters to Na2SO3 or NaNO2 chemical solutions, as above mentioned. As a result of HCl addition, SO2 and NO2 gases is being produced in the 500 ml flasks with the help of stirrer magnet and these gases are transferred with 23.5 cm length heat resistant hose in 250 ml flask that contains microalgal culture solution. The heat is provided by the magnetic heat stirrer (Velp Scientifica ARE Heating magnetic stirrer) to formation of the reaction where under the 500 ml flask. The 250 ml flask that contains of 100 ml of culture solution includes a single-hole gum elastic dowel with a 19.0 cm length metal needle. The gases that are produced in 500 ml flask are transferred to the 250 ml flask by the help of the metal needle that enters up to 1.0 cm into the culture solution.
Figure 1. The diagram of experimental set-up
1: Magnetic heat stirrer
2: Erlenmeyer flask (500 ml)
3: Gum elastic dowel with acid inlet and gas exit holes
5: Heat resistant hose
6: Gum elastic dowel with cotton
7: Erlenmeyer flask (250 ml)
2.4. Effect of different parameters on microalgal gas tolerance
2.4.1. Effect of pH
The effect of medial pH value on acidic gas tolerance of microalgal cultures was determined in the samples that once aerated with 10' exposing time of SO2 and NO2 at the end of the 10 days of incubation period. The experiments were performed at pH 6.5, 7.5, 8.5 and 9.5 for each microalgal culture and the samples were taken at the end of the 11, 13 and 15 days.
2.4.2. Effect of gas exposing time
To determine the gas exposing time to effect on microalgal inhibition, cultures were once aerated with 10', 15' and 20' exposing time of each SO2 and NO2 at optimum pH values for both microalgal cultures after the 10 days of incubation period. Samples were taken at the end of the 11, 13 and 15 days for each experiment.
2.5. Analytical methods
The chlorophyll a, chlorophyll b and total chlorophyll concentrations were determined for each strain by recording optical absorption at 646.6 and 663.6 nm at the end of 11, 13 and 15 days with the method that developed by Porra et al. . The chlorophyll concentrations expressed in µg of chlorophyll per milliliter.
The OD600nm was measured with a Shimadzu UV 2001 model spectrophotometer at the same frequency. Cell dry weight was obtained by the measurement of the pellets that centrifuged at 5000 rpm for 10 min and after dried at 80 °C overnight. Centrifugation and drying were performed using a Hettich EBA 12 model centrifuge and Nüve FN 400 model sterilization, respectively. The colony number was determined by the counting of the colonies by microscopic examination. All the measurements were performed in triplicate.
2.6. Statistical analysis
The experiments were set in a completely randomized design up with three replicates. The data were subjected to analysis of variance using significant differences among treatments means were compared by descriptive statistics (±S.E.).
3.1. Effect of initial pH on microalgal gas tolerance
The pH is one of the most important environmental factors greatly influencing not only growth of microorganisms but also solution chemistry. The optimum pH of the culturing media is quite important to enhancing the tolerances of the microalgae to SOx and NOx . To determine the pH effect experiments were performed with 10' exposing time of acidic pollutants (Fig.2). As shown in the Fig.2a, b, c and d pH effect were determined at four different pH values for both microalgal cultures. Fig.2a and b shows the pH effect on chlorophyll a content of Synechococcus sp. The maximum chlorophyll a content was 0.93 µg ml-1 at pH 7.5 for SO2 and was 1.50 µg ml-1 at pH 7.5 for NO2 at the end of 15 days. The maximum chlorophyll a contents were 0.28, 0.35 and 0.19 µg ml-1 at pH 6.5, 8.5 and 9.5, for SO2, respectively. Synechococcus sp. had much more tolerance to NO2 than SO2. The chlorophyll a contents were 0.62, 0.91 and 0.99 µg ml-1 at pH 6.5, 8.5 and 9.5, respectively.
Figure 2. The effect of initial pH values on chlorophyll content of Synechococcus sp. and Chlorella sp. at 1 mol l-1 SO2 or 1 mol l-1 NO2 concentrations after 15 days (TRIA concentration, 1 mg l-1; NaHCO3 concentration, 34 mg l-1; exposing time 10'; illumination, 2400 lx). a) Synechococcus sp. - SO2, b) Synechococcus sp - NO2, c) Chlorella sp. - SO2, d) Chlorella sp. - NO2.
The data given in Fig.2c and d shows the pH effect of acidic pollutants on Chlorella sp. The culture had much more tolerance to both of the acidic pollutants than Synechococcus sp. The chlorophyll (a+b) contents were 2.07, 2.72, 3.06 and 1.88 µg ml-1 at pH 6.5, 7.5, 8.5 and 9.5 for SO2, respectively. The culture also showed much more tolerance to NO2 and chlorophyll (a+b) content increased from pH 6.5 to 8.5 and then decreased to pH 9.5. The maximum chlorophyll (a+b) content was 3.3 µg ml-1 at pH 8.5.
3.2. Effect of gas exposing time on microalgal gas tolerance
Exposing time, has a substantial impact on the determination of target experiments, was determined at optimum pH values for both microalgal cultures. Exposing time may vary from minutes to hours according to the biomass and target analyses. In the light of the above information, a time range from 10' to 20' was tested for SO2 and NO2 gases. The exposing time effect on chlorophyll content, dry weight and colony number were comprehensively given above.
The acidic pollutants would cause toxic effects on the microalgal growth or photosynthesis. To address the photosynthetic inhibition or tolerance we also researched chlorophyll contents. To find the maximum gas exposing time that the cultures able to tolerate and were inhibited experiments were set up to 15' and 20'. The results were recorded on 10 (without gas) 11, 13 and 15 days (with 10',15' and 20' gas exposing time intervals). It was recorded that when exposing time increased, chlorophyll values of both microalgal cultures decreased. Fig.3 and 4 presents all of the exposing time effects. As shown in Fig.3a, exposing time of 10' did not inhibit the increasing of microalgal chlorophyll content until the day 13. The chlorophyll content was 0.8 µg ml-1 on day 10 and increased to 1.03 µg ml-1 on day 13. The culture began to be inhibited at the end of 15 days and chlorophyll content decreased to 0.93 µg ml-1.
There was only a little increase in 11 days at 15' (0.49 µg ml-1) and 20' (0.39 µg ml-1) SO2 gas exposing time according to the data and after 11 days Synechococcus sp was rapidly and considerably inhibited. The chlorophyll a content was 0.16 µg ml-1 at 15' and 0.13 µg ml-1 at 20' at the end of 15 days.
Fig.3b shows the effect of NO2 exposing time on Synechococcus sp. As seen in the figure NO2 did not inhibit the microalgal culture at 10' on the contrary had a positive effect. The reaching to chlorophyll values in a short time could be explained that photosynthesis is much less sensitive to the presence of nitrogen oxides than other pollutants . The chlorophyll a content had a rapid increase at 10' on 10 days (0.82 µg ml-1) and had a stable effect at 15'. For example chlorophyll a content was 0.48 µg ml-1 on 10 days and was 0.48, 0.45 and 0.43 µg ml-1 at 15' on 11, 13 and 15 days, respectively. There was a similar result between the 20' and 15' results until the day 13, but at the end of the 15 days chlorophyll a contents pronounced declined to 0.19 µg ml-1and photosynthetic inhibition of Synechococcus sp. was occurred.
Figure 3. The effect of SO2 and NO2 exposure time on chlorophyll a content of Synechococcus sp. after 15 days (TRIA concentration, 1 mg l-1; NaHCO3 concentration, 34 mg l-1; illumination, 2400 lx). a) SO2 effect, b) NO2 effect
The experiments showed that 20' exposing time of both SO2 and NO2 could significantly inhibit the growth of Synechococcus sp. cells. On the other hand we saw better results for Chlorella sp. than Synechococcus sp. when culture aerated with SO2 during 10' culture did not inhibit (Fig.4a). The photosynthetic inhibition effect of SO2 was more remarkable than NO2. Chlorophyll (a+b) content was 1.66 µg ml-1 at 0' on 10 days and increased to 3.06 µg ml-1 at 10' on 15 days. Culture was able to tolerated SO2 when was applied until 10' and than chlorophyll (a+b) content began to fall after 11 days and was 1.51 µg ml-1 on day 15 at 15' SO2 exposing time. Chlorophyll (a+b) content decreased significantly at 20' SO2 exposing time from 1.56 µg ml-1 on day 10 to 1.08 µg ml-1 on day 15.
Figure 4. The effect of SO2 and NO2 exposure time on chlorophyll (a+b) content of Chlorella sp. after 15 days (TRIA concentration, 1 mg l-1; NaHCO3 concentration, 43 mg l-1; illumination, 2400 lx). a) SO2 effect, b) NO2 effect
As present in the Fig.4b, NO2 had quite a lot of positive effect at 10' and partially at 15'. Chlorophyll (a+b) was 2.02 µg ml-1 on day 10 and increased to 3.14 µg ml-1 and 3.30 µg ml-1 on day 13 and 15, respectively. Chlorophyll (a+b) decreased from 2.34 µg ml-1 to 2.23 µg ml-1 from day 10 to 15 at 15' NO2 exposing time. There was significant negative effect on the culture at 20' NO2 exposing time, chlorophyll (a+b) decreased to 1.13 µg ml-1 on day 15. This might be explained that NO2 and SO2 firstly dissolves in aquaculture and at higher times react with microalgae. The reduction in NO2 occurred with in 10 minute and continued slowly depending on TRIA and NaHCO3 in aquaculture.
It is well known that acidic pollutants had a negative effect on microalgal biomass [9,11,12,30]. For that reason we wanted to investigate how to affect SO2 and NO2 on microalgal biomass and determined dry weight and colony number after the gas application for each SO2 and NO2 gases and both microalgal cultures. Table 1 is a summary of all the data that was determined at the end of 15 days. As discovered in the chlorophyll contents, both dry weight and colony number were high in cultures that were exposed to NO2.
<Here Table 1>
Table 1. The effect of SO2 and NO2 exposure time on dry weight (X) (g l-1) and colony number (x104 colony ml-1) of Synechococcus sp. and Chlorella sp. after 15 days (TRIA concentration, 1 mg l-1; NaHCO3 concentration, 34 mg l-1 for Synechococcus sp., 43 mg l-1 for Chlorella sp.; illumination, 2400 lx; SO2 and NO2 concentration, 1 mol l-1)
X (g l-1)
Colony number (x104 colony ml-1)
2.79 ± 0.40
2.83 ± 1.29
0.48 ± 0.54
1.66 ± 1.36
0.39 ± 0.11
1.00 ± 0.58
4.32 ± 0.55
4.50 ± 0.86
1.29 ± 0.27
3.60 ± 0.58
0.57 ± 0.36
1.66 ± 1.37
2.65 ± 0.47
9.00 ± 0.89
1.31 ± 0.10
3.00 ± 1.13
0.94 ± 0.18
2.00 ± 1.03
2.86 ± 0.17
10.5 ± 1.03
1.93 ± 0.23
8.60 ± 0.52
0.98 ± 0.37
2.66 ± 0.89
The dry weight decreased from 5.01 g l-1 to 3.73 g l-1 and 2.38 g l-1 for Synechococcus sp. when SO2 exposing time increased from 10' to 20'. In parallel with these results, colony number was 2.83, 1.66 and 1.00 x104 colony ml-1 at 10', 15' and 20', respectively. The maximum dry weight was 6.44 g l-1 and colony number was 4.5 x104 colony ml-1 at 10' NO2 exposing time.
The dry weight results of Chlorella sp. that exposed to SO2 were similar with the results of Synechococcus sp. that exposed to NO2. So that dry weight was 6.23 g l-1 at 10' and decreased to 3.70 g l-1 at 20' SO2 exposing time. The highest results in table were NO2 experiments. The maximum dry weight was 6.63 g l-1, 4.85 g l-1 and 3.76 g l-1 and the colony number was 10.5, 8.60 and 2.66 x104 colony ml-1 at 10', 15' and 20' NO2 exposing time, respectively. These results indicated that 10' and 15' exposing time was not inhibitory to microalgal biomass under these conditions.
There are two kinds of major important problems, environmental pollution and higher energy depletion problems. Microalgae have the answer to many of the global problems. The use of microalgae to fix CO2 and tolerate other pollutants produce energy and help to clean the environment, so microalgae may potentially solve all of our problems.
The study was designed to acquire a better understanding of some basic microalgal growth and inhibition responses under effect of acidic pollutants and the possible physiological enhance over biomass production in a stimulated culture. The effect of different parameters was studied by the help of the gas producer and transfer system. The advantages of the system are (1) can be easily established and used in each laboratory without any cost; (2) allows the produce and direct transfer of different gases through variety of chemicals; (3) the transfer of SO2, NO2 or other gases can be achieved by increasing gas exposing time and this time can be adjusted at the optimal range of different gases.
Acidic pollutants in flue gas, such as SO2 and NO2 might cause negative effects on the growth and physiology including inhibition of photosynthetic pigments, acidification of culture and inhibition of microalgal growth. Therefore, it may be concluded that adding growth stimulators to the culture media was an effective method to enhance the tolerances of microalgae to acidic pollutants. A question is why these negative effects may be minimal at lower exposing time levels. Our experiments pointed out that lower expose levels of acidic pollutants can be significantly tolerated by both of the microalgal culture by the help of growth enhancers. It is also interesting to compare which species is better for this process. For comparison of applicability of microalgal cultures under directly SO2 and NO2 gases, Chlorella sp. reveals a better tolerance to acidic pollutants than Synechococcus sp. It could be explained by the response of different genus and spp. show differences in aquaculture as it can be expected. Therefore research of the pollutant tolerant species will be need for further studies.
Three main conclusions can be explained; First, CO2 in NaHCO3 can significantly enhance biomass yield with a growth stimulator TRIA. Second, acidic components may cause significant damage to microalgal chlorophyll content and biomass and thus they need to be used to a safe time level and thirdly, the presence of NOx in microalgal culture did not inhibit in according with the findings of Doucha et al. and Hauck et al. [10,12].
Some of the important parameters such as temperature and pH also were detected during the experiments. A significant difference was not observed on the initial temperature of the culture media after the transferring of SO2 and NO2 gases at different time. The temperature was 25 ± 2 °C before the acidic pollutants, and decreased to max 25.75 ± 2 °C after the acidic components.
It was reported that the initial optimum pH level was 7.5 for Synechococcus sp. and was 8.5 for Chlorella sp. into the SO2 and NO2 experiments. Depending on nature of the biomass, this finding optimum pH values for different studies have been reported for cyanobacteria and Chlorella sp. For example, Lawry and Jensen  reported that Synechococcus sp. showed its maximum phosphate uptake at pH 7.5-8.5 range and Baebprasert et al.  explained that Synechocystis sp. showed its maximum hydrogenase activity at pH 7.5. On the other hand Suárez et al.  discovered the pH effect of the binding of metals in solution by Chlorella vulgaris with a range of pH 4.0, 6.0 and 8.0 and they found the higher percentage of binding at alkali pH 8.0.
The biomass productivity of Chlorella sp. was higher than that of Synechococcus sp., also the total chlorophyll content of Chlorella sp. was higher. The next remarkable subject is why Synechococcus and Chlorella sp. cells are inhibited under high transfer levels of acidic pollutants? One of the possibilities is an acidification of the culture media. The pH change is one of the substantial differences between the two types of microalgae which the effect of different initial pH levels. It has been well documented that when the acidic pollutants are transferred into the culture media, the pH of the media go down and this was a reason of the dissolution of acidic pollutants in culture media . In this study the pH of the growth medium was about 3.0 and 4.8 for Chlorella sp. and 2.59 and 2.72 for Synechococcus sp. at 15' exposing time of SO2 and NO2, respectively and went down at about 2.50 when exposing time increased to 20'. In a previous report, pH markedly decreased from 7.3 to 2.3 for Chlorella vulgaris and the decreases in the pH levels did not inhibited the growth of Cyanidium caldarium . It was also possible to control the pH range to be able to tolerate the SO2 and NO2 gases  and they reported that a significant decrease in pH from 5.8 to 3.0 was observed after 12 h. for Chlorella sp. and they found that Chlorella sp. was completely inhibited from the beginning if pH was not controlled therefore, they controlled pH to 7.00 by adding NaOH solution. In this study the pH of the Chlorella sp. was more alkali than Synechococcus sp. before the transfer of the acidic pollutants and more tolerated to these components. On the other hand the Chlorella cells did not completely inhibit. This was particularly encouraging because the presence of acidic pollutants in aquaculture could cause totally inhibition of growth.These findings suggested that a large amount of microalgal biomass may be used for further studies. It is clear that all of the above results have provided many of the advantages and properties to microalgal tolerance of acidic pollutants under effect of growth stimulators toward the future practical applications.
The usage of Synechococcus sp. and Chlorella sp. cultures on the tolerance of acidic components with growth stimulators was investigated for the first time. The significant results from the study can be summarized as follows;
TRIA and NaHCO3 helped to microalgal growth under effect of SO2 and NO2,
Chlorella sp. dominant microalgal culture was much more tolerance to acidic pollutants than Synechococcus sp.,
NO2 did not only cause negative effects but also behaved as an enhancer on the growth of both microalgal cultures.
A different approaches to tolerate acidic gases by the use of two different microalgal species was investigated. The study suggested that usage of this method is feasible and an instance for the further investigation into culture physiology under effect of acidic components and environmental studies, with no major technology and cost are needed.
The authors are grateful to the Scientific and Technological Research Council of Turkey (TÜB°TAK-B°DEB) for financial support and wish to express their gratitude to Prof. Dr. Orhan ATAKOL and Prof. Dr. Mustafa TaÅŸtekin (Ankara University, Faculty of Science, Department of Chemistry) for their valuable comments.