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Abstract As a major source of air pollution, acidic pollutants such as SO2 and NO2 cause toxic effects on many of the environmental systems. In this study, the effects of acidic pollutants on two strains of photosynthetic microalgae were determined. Although, Chlorella sp. has been studied to remove air pollutants, Synechococcus sp. has not been reported yet. Microalgal cultures were cultivated into the 250 ml Erlenmeyer flasks containing 100 ml of BG 11 medium with triacontanol and sodium bicarbonate, as growth stimulators, and incubated at 25 ± 2 °C under continuous illumination (48 umol m-2 s-1 (2400 lx)). Cultures were aerated with SO2 or NO2 at 10', 15' and 20' exposing time on the 10 days of incubation period. Experiments showed that optimum pH was 7.5 for Synechococcus sp. and 8.5 for Chlorella sp. under effect of both SO2 and NO2 gases at 10' exposing time. Chlorella sp. showed much more tolerance to SO2 and NO2 than Synechococcus sp. Both of the microalgal cultures were better able to tolerate NO2 than SO2. The results indicated that the microalgal cultures used in this study may be applied for environmental control mechanisms with high tolerances of acidic pollutants under effect of growth stimulators.
Keywords: Environmental pollution, air quality, acidic pollutants, microalgae, growth stimulators
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. Traffic, flue gas depending on industry and the burning of fossil fuels is the key of the buildup carbon dioxide (CO2) in the environment, and they 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), trace amounts of heavy metals and other particles also have undesirable harmful effects on natural ecosystem. Therefore; air pollution is a global problem and both governments and scientist study to reduce the effects of this problem all over the world.
The technology for reducing of CO2 and other pollutants from environment has been based on biological assimilation as an effective way. For example, plants can reduce the atmospheric CO2 concentration by absorbing . The most common renewable energy source has a microalgal origin [4, 5]. It is also well known that microalgae grow faster than terrestrial plants and the efficiency of energy product is much higher . Microalgae have the remedy 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.
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 air pollution can be reduced by microalgal biomass.
On the other hand high amounts of air pollutants cause toxic effects on microalgae [9, 11, 12, 30]. To enhance the microalgal biomass and maintaining to photosynthesis some of the growth stimulators can be added to the growth media. The first of them is Triacontanol (TRIA) and the second is sodium bicarbonate (NaHCO3). Triacontanol is a natural plant growth hormone, a long 3 chain 30-carbon primary alcohol (C30H61OH) and stimulate photosynthesis and growth [13, 14]. Sodium bicarbonate, a type of inorganic carbon, can be fixed as a CO2 source by microalgae as Wang et al.  reviewed.
In the present work TRIA and NaHCO3 added to the culture media to overcome the inhibition of microalgal biomass by SO2 and NO2 acidic pollutants. Additionally, two strains of microscopic microalgae were tested their ability to tolerate air 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 identified to has an important role in the global carbon cycle  and to our knowledge has not been studied to test its ability to reduce of air pollutants. This is also the first report about the microalgal cultures with TRIA application under effect of air pollutants in the literature. The scope of the current search was to determine the interactive effects of highly toxic SO2, NO2 and growth stimulators on different microalgal cultures and to test 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 BG 11 media . The number of initial inoculated microorganism was PPPPPP for Synechococcus sp. and uuuuu for Chlorella sp. The concentration of TRIA was 1 mg l-1 and NaHCO3 was 34 mg l-1 for Synechococcus sp. and was 1 mg l-1 TRIA and 43 mg l-1 NaHCO3 for Chlorella sp. into 100 ml BG 11 media in a 250 ml Erlenmeyer flask (referans). Once inoculated, unshaken flasks were incubated at 25 ± 2 °C under continuous illumination at 48 umol m-2 s-1 (2400 lx) light intensity provided by cool white fluorescent lamps.
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 ml Erlenmeyer flasks, magnetic heat stirrer (Velp Scientifica ARE) 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 into the Na2SO3 or NaNO2 chemical solutions. As a result of HCl addition, SO2 and NO2 gases are being produced into the 500 ml flask 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 under the 500 ml flask to be formation of the reaction. The 250 ml flask 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 into 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 effects of initial pH of the culture media on microalgal SO2 and NO2 tolerance were firstly investigated. The initial pH of the microalgal cultures that inoculated in media was adjusted to 6.5, 7.5, 8.5 and 9.5 by 0.1 M H2SO4 and 0.1 M NaOH. The cultures incubated for 15 days and once aerated with SO2 or NO2 with 10' exposing time on 10 days of incubation period. Samples were taken before the microalgal cultures aerated with SO2 and NO2 on 10 days, and after the aeration on 11, 13 and 15 days for each experiment. All the experiments were performed in triplicates.
2.4.2. Effect of gas exposing time
To determine the effect of gas exposing time on microalgal SO2 and NO2 tolerance, cultures were inoculated in media with selected optimum pH values and incubated for 15 days. Each Erlenmeyer flasks once aerated with SO2 or NO2 at 10', 15' and 20' exposing time on 10 days of incubation period. Samples were taken before the aeration (on 10 days) and after the aeration (on 11, 13 and 15 days) for each experiment. All the experiments were performed in triplicates.
2.5. Analytical methods
In the experiments the number of microorganisms, chlorophyll content, optic density and dry weight were determined for each strain. Thoma haemocytometer (TH-50 and TH-100; Hecht-Assistent, Sondheim, Germany) 0.02 mm. in depth were used for initial inoculation concentration. Samples of 10 µl were gently loaded after the dilution into each side without any air bubbles got and after than counted under microscope (ffffffffff). In this way, total counts of all microalgae, viable and nonviable, were made by means of a Thoma haemocytometer, while counts of viable microalgae were determined by colony counts on agar plates.
The number of alive microalgal colonies was determined by the diluting and spreading the samples onto agar plates. Colonies were incubated at 25 ± 2 °C under continuous illumination 48 umol m-2 s-1 (2400 lx) for 15 days and then counted. The number of colonies expressed as colony forming units (CFU).
The chlorophyll a, b and total chlorophyll concentrations were determined for each strain by recording optical absorption for chlorophyll a at 646.6 and chlorophyll b at 663.6 nm at the end of 10, 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.
All of the experiments described below to follow the changes in the samples throughout the incubation period were performed in triplicate.
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. As shown in the Fig.2 the effect of pH of the culture media on chlorophyll contents was more remarkable on Synechococcus sp. than Chlorella sp. Fig.2a and b shows the pH effect on chlorophyll a content of Synechococcus sp.
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. Synechococcus sp. showed much more tolerance to both SO2 and NO2 at pH 7.5. 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. This optimum pH value was selected for further experiments. <Here Fig.2>
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 (48 umol m-2 s-1 (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 effect of pH on chlorophyll (a+b) content of 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 for SO2 at pH 6.5, 7.5, 8.5 and 9.5, respectively. The culture also showed much more tolerance to NO2. The chlorophyll (a+b) content increased from XXX at pH 6.5 to YYY at 8.5 and then decreased to XXX at pH 9.5. The maximum chlorophyll (a+b) content was 3.3 µg ml-1 at pH 8.5. The optimum pH was selected as 8.5 for Chlorella sp.
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. The exposing time effect on chlorophyll content, dry weight and colony number were comprehensively given below.
To find the maximum gas exposing time that the cultures able to tolerate experiments were set up to 10', 15' and 20'. It was recorded that when exposing time increased, chlorophyll values of both microalgal cultures decreased (Fig.3 and 4). As shown in Fig.3a, exposing time of 10' had not had a negative effect of the chlorophyll content of Synechococcus sp. 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 the 15 days and chlorophyll content decreased to 0.93 µg ml-1.
The effect of SO2 at 15' and 20' exposing time was more remarkable. There was only a little increase on the 11 day (0.49 µg ml-1) at 15' and (0.39 µg ml-1) at 20' exposing time of SO2 according to the data. Synechococcus sp was rapidly and considerably inhibited after 11 days of incubation period. The chlorophyll a content was 0.16 µg ml-1 at 15' and 0.13 µg ml-1 at 20' exposing time at the end of 15 days.
Fig.3b shows the effect of different exposing time of NO2 on Synechococcus sp. As seen in the figure NO2 did not inhibit the microalgal culture at 10' exposing time and on the contrary had a positive effect. The chlorophyll a content had a rapid increase from 0.82 µg ml-1at 10' exposing time on 10 days to XXX on 13 day. The exposing time of 15' had not had a significant effect on chlorophyll content. 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 on 11, 13 and 15 days, respectively. There was a similar result at the 20' exposing time 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, (48 umol m-2 s-1 (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. (Fig.4a). The photosynthetic inhibition effect of SO2 was more remarkable than NO2. Chlorophyll (a+b) content was 1.66 µg ml-1 on 10 days and increased to 3.06 µg ml-1 at 10' exposing time on 15 days. Culture was able to tolerate SO2 when was applied until 10' exposing time and than chlorophyll (a+b) content began to fall at 15' exposing time. The chlorophyll (a+b) content was (:::::::J on day 11 and decreased to 1.51 µg ml-1 on day 15. Chlorophyll (a+b) content decreased significantly at 20' SO2 exposing time from 1.56 µg ml-1 to 1.08 µg ml-1 from day 10 to 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, (48 umol m-2 s-1 (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' exposing time. Chlorophyll (a+b) content was 2.02 µg ml-1 10' exposing time on day 10 and increased to 3.14 µg ml-1 and 3.30 µg ml-1 on day 13 and 15, respectively. But, it decreased from 2.34 µg ml-1 to 2.23 µg ml-1 at 15' exposing time from day 10 to 15. There was significant negative effect on the culture at 20' exposing time of NO2, chlorophyll (a+b) content decreased from (…..) on day 10 to 1.13 µg ml-1 on day 15. Table 1 is a summary of all the data that was determined at the end of 15 days.
<Here Table 1>
Table 1. The effect of SO2 and NO2 exposure time on dry weight (X) (g l-1) and CFU colony forming unit (x10-4) 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, (48 umol m-2 s-1 (2400 lx)); SO2 and NO2 concentration, 1 mol l-1)
X (g l-1)
0.592 ± 0.078
2.83 ± 0.190
0.479 ± 0.040
1.66 ± 0.577
0.281 ± 0.091
0.50 ± 0.577
0.696 ± 0.038
4.50 ± 0.860
0.494 ± 0.020
2.00 ± 1.550
0.403 ± 0.043
2.00 ± 0.289
0.654 ± 0.069
4.50 ± 1.730
0.502 ± 0.061
2.00 ± 0.289
0.371 ± 0.070
1.00 ± 0.173
0.698 ± 0.055
7.33 ± 0.190
0.602 ± 0.017
4.00 ± 0.866
0.445 ± 0.000
2.66 ± 0.485
The dry weight decreased from 5.01 g l-1 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 x10-4 colony ml-1 at 10', 15' and 20', respectively. The maximum dry weight was 6.44 g l-1 and microalgae/ml by CFU was 4.5 x10-4 at 10' exposing time of NO2.
The dry weight results of Chlorella sp. that exposed to SO2 were similar with the results of Synechococcus sp. that exposed to NO2. Dry weight was 6.23 g l-1 at 10' and decreased to 3.70 g l-1 at 20' exposing time of SO2. 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 CFU at 10', 15' and 20' exposing time of NO2, respectively.
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.
There are two kinds of major important problems, environmental pollution and higher energy consumption. Microalgae have the answer to many of the global problems. They fix CO2 and produce energy and also tolerate other pollutants produce energy and help to clean the environment, so they may potentially solve all of our problems.
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 onto the culture media was an effective method to enhance the tolerances of microalgae to acidic pollutants.
The present 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.
Three main conclusions can be explained in this study; First, CO2 in NaHCO3 can be significantly enhancing 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].
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. A question is why these negative effects may be minimal at lower exposing time levels. It is also interesting to compare which species is better for this process. For comparison of applicability of microalgal cultures under effect of 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 show differences in aquaculture as it can be expected. Therefore research of the pollutant tolerant species will be need for further studies. 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 and the other can be explained as SO2 and NO2 firstly dissolves in aquaculture and at higher contact times react with microalgae. The reduction in NO2 occurred with in 10' and continued slowly depending on TRIA and NaHCO3 in aquaculture. The pH change is one of the substantial differences between the two types of microalgae which the effect of different initial pH levels.
It was also reported that the initial optimum pH level was 7.5 for Synechococcus sp. and was 8.5 for Chlorella sp. for 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.
It is not definite that selection of effective microalgae for reduces of SO2 and NO2 is a key to achievement.
These results indicated that 10' and 15' exposing time was not inhibitory to microalgal biomass under these conditions.
It is well known that acidic pollutants had a negative effect on microalgal biomass [9,11,12,30]. We also investigated how to SO2 and NO2 show their effect on microalgal biomass and determined dry weight and colony number for both microalgal cultures.
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.
The biomass productivity and total chlorophyll content of Chlorella sp. was higher than that of Synechococcus sp.
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 at the end of 15 days, respectively and decreased at about 2.50 when exposing time increased to 20'. In a previous report, pH markedly decreased from 7.3 to 2.3 for
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 Chlorella vulgaris . 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. 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.
A different approach to usage of Synechococcus sp. and Chlorella sp. cultures on the tolerance of acidic components with growth stimulators was investigated for the first time in the literature. The significant results from the study can be summarized as follows;
Both Chlorella and Synechococcus sp. were suitable microorganisms for control of SO2 and NO2,
TRIA and NaHCO3 helped to microalgal growth under effect of SO2 and NO2,
Chlorella sp. was much more tolerance to acidic pollutants than Synechococcus sp.,
NO2 did not cause negative effects and behaved as an enhancer on the growth of both microalgal cultures at lower exposing time intervals.
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 for environmental studies, with no major technology and cost are needed.