The major environmental toxicity of acidic pollutant in the fossil fuel gas substances has long been well known. Macro and microalgae are biological sources with a large range of biotechnological uses, for e.g., bioremediation, bio-fuel, air pollutant absorber, and many more. This study addresses the use of Chlamydomonas sp., an effective biomaterial in their tolerance against 2 and 5% of the SO2 & NO2. Growth kinetics were improved by the addition of sodium bicarbonate to the culture conical media. SO2 and NO2 were provided to culture media by the use of sodium meta-bisulfite and nitrous acid. The control combination of SO2 and NO2 provides: 2% SO2, 5% SO2, 2% NO2, 5% NO2, (2% SO2 + 2% NO2), (5% SO2 + 5% NO2) at the seventh day of incubation. The optimum pH ranged between 7.1 and 8.6 when exposed to gas. Results suggested that the growth kinetics of Chlamydomonas sp. is greater in SO2 and low in the 5% exposure of NO2. The maximum absorbing concentrations of SO2 and NO2 were 921.625 μg/ml and 906.25 μg/ml respectively for Chlamydomonas sp. This work highlights the potential of algae in tolerance to NO2 & SO2 from the polluted air.

  • The study addresses the use of Chlamydomonas sp., an effective biomaterial in their tolerance against various combinations of 2 and 5% of SO2 & NO2.

  • Their growth kinetics were improved by addition of sodium bicarbonate to the culture conical media.

  • Results highlighted that the growth kinetics of Chlamydomonas sp. is greater in SO2.

  • This work highlights the potential of algae in tolerance to NO2 & SO2 from the polluted air.

Earth's natural resources are being destroyed for human civilization and technological development. The world's growing population is facing the challenging issue of air and land pollution that threatens lives and natural habitats (Khajouei et al. 2017). Air pollution is defined as any atmospheric condition in which substances are present above desirable levels, producing harmful effects on human beings and the life span of ecosystems. The primary origins of air pollution are natural (smoke and ash from forest fires, volcanic activities, storms, etc.) or artificial (combustion of fuels, world wars, industries, transportation, etc.). The main source of air pollution is the combustion of fossil fuel, emitting carbon dioxide, sulfur dioxide and nitrogen oxides: the chief sources of global warming (Chisti 2007). The pollutant substances can affect the biological and physiological processes of living beings and ultimately affect agricultural yield (Musselman et al. 1994). Humidity and temperature are sharply demarcated and directly impact on climate change. The increase in greenhouse gases leads to an increase in the temperature (Dacie et al. 2019). The oxides of carbon (CO2), nitrogen (NOx), and sulfur (SOx) were mostly responsible (60–63%) for the radiation between various atmospheric layers from 1979 to 2004 (Hofmann et al. 2006). Many new technologies have been developed to reduce the unpleasant outcomes of global warming. On 11 December 1997 in Kyoto, Japan, the Kyoto Protocol for global CO2 emissions was announced (Singh & Singh 2014).

According to the 2015 Global Burden of Disease Study, particulate matter with diameter of 2.5 microns contributed to 4.2 million deaths globally, 52% in China and India. In 2015, 254,000 deaths worldwide were due to ground-level ozone. Pollutants such as sulfur dioxide (SO2) and nitrogen dioxide (NO2) have more significant health hazards. These pollutants are emitted by the burning of fossil fuels, for example in vehicles, among others. In 2019, India emitted 21% of global anthropogenic SO2 emissions or 5,953 kilotons a year, and Russia emitted 3,362 kt/year. China at 2,156 kt per annum occupies third position. Biomass used as a renewable resource is well known to be biodegradable in terms of CO2, NO2 and SO2 over its life cycle (Kumar et al. 2019). Algal bio-fixation is one of the most successful methods to eliminate the hazardous gases from the atmosphere (Kaithwas et al. 2012). Algae during their life span are capable of reducing environmental pollution (Yu et al. 2017). It is estimated that the global market for products extracted from microalgae will hit USD 1,143 million by 2024 (Bold 1978). The algae are responsible for 50% of the photosynthetic processes on the Earth. A carbon source (CO2 or organic carbon such as sugars), nitrogen, phosphorus, salts, and vitamins are essential for algal growth (Costa et al. 2015). Photosynthesis is the process in green plants, microalgae and certain types of microscopic organisms by which carbon dioxide and water combine in the form of chlorophyll, utilizing light as an energy source, and as a result producing abundant oxygen (Sawyer et al. 2003). Macro- and microalgae are biological sources with a large range of biotechnological uses. This study addresses the use of Chlamydomonas sp. and Ulothrix sp. Both are effective biomaterials in their tolerance against 2 and 5% concentrations of SO2 and NO2. Their growth kinetics are improved by the addition of sodium bicarbonate (NaHCO3) to the culture conical media.

Algae are biological sources with a large range of biotechnological uses, for example bioremediation, bio-fuel and air pollutant absorber. As we know CO2, SO2, and NO2 are the major air pollutants; hence we use photoautotrophic algae, which can absorb these air pollutants. Algal organisms produce 71% of environmental oxygen by photosynthesis. Algae are sunlight driven cell-processing plants, which convert CO2 to potential bio-fuels, nourishments, feeds and bioactive compounds. Thus, we incorporate a biochemical process to reduce pollutants present in air. Microalgae help in the reduction of CO2, generating oxygen in the process of photosynthesis. They are also capable of reducing SO2 and NO2. In this study, field samples of algae were taken. After isolation and identification of algae, the cultured algae were incorporated with sulfur dioxide and nitrogen dioxide. In this work we focus mainly on reduction of SO2 and NO2 through microalgae photosynthesis, for which we use sodium metabisulfite and nitrous acid for formation of SO2 and NO2, respectively. After that we take the solution and calculate absorbance by spectrophotometer; the plotted graph shows the growth kinetics of algae, and the concentrations of SO2 and NO2 in the broth are calculated. This work has practical application in the field of air pollution control. This also helps in the growth of algal biomass (Taştan et al. 2012a).

Chemicals used

Acidic pollutants SO2 and NO2 were prepared using sodium metabisulfite (Merck) and nitrous acid (Merck) dissolved in distilled water in concentrations of 100 μg/ml to 150 μg/ml; 10 ml of sample and standard solutions were taken. Sulfamic acid (1 ml) and rosaniline (2 ml), HCHO (Aldrich), H2O2 and HCl were prepared. Stock solution of NaHCO3 (Merck) was added to the cultured media for the growth of culture (Taştan et al. 2012b).

Media and growth conditions

After field sampling and identification of the algae (Figure 1) by the catalase test and under 100x compound microscope study of the sample, artificial culture media was prepared. The conical well was sterilized and moist conditions provided for the unicellular cyanobacterium Synechococcus sp. and microalgae (Figure 2) obtained by the full procedure described in Rippka (1988). In all of the experiments, the inoculations were made to ensure the initial biomass concentrations of both the microalgal cultures were about 0.05 g/L dry weight. Microorganisms were cultivated in 100 mL BG 11 media (Shekhar et al. 2021; Tripathi & Pandey 2021; Omar et al. 2022b). Erlenmeyer flasks containing a series of known concentrations of NaHCO3 in 250 mL stock solution at pH 7.1–8.0 were incubated at 25–27 °C under continuous illumination for 7 days.
Figure 1

Sample of algae used in the experiment.

Figure 1

Sample of algae used in the experiment.

Close modal
Figure 2

Structure of Chlamydomonas.

Figure 2

Structure of Chlamydomonas.

Close modal

BG 11 broth/media designed by M. M. Allen (Allen & Stanier 1968; Rippka 1988) allows growth of a wide range of cyanobacteria from soil and freshwater habitats. This broth is a medium for cultivation of green algae and shows the major changes in algal bloom (Omar et al. 2020; Tripathi & Pandey 2022). The major constituents of the media are shown in Table 1.

Table 1

Composition of BG 11 broth/media for isolation and growth

Constituents of the mediaConcentration
Sodium nitrate (NaNO31.50 g/L 
K2HPO4 0.0314 g/L 
Magnesium sulfate (MgSO40.036 g/L 
Calcium chloride dihydrate 0.0367 g/L 
Sodium carbonate 0.0200 g/L 
Disodium magnesium EDTA 0.001 g/L 
Citric acid 0.0056 g/L 
Ferric ammonium citrate 0.006 g/L 
HEPES buffer 2.3 g 
NaNO3 1.5 g 
P-IV metal solution 1 ml 
K2HPO4·7H2O (6 g/L) 5 ml 
MgSO4·7H2O (6 g/L) 5 ml 
Na2CO3 (4 g/L) 5 ml 
CaCl2·2H2O (2.5 g/L) 10 ml 
Na2SiO3·9H2O (4.64 g/L) 10 ml 
Constituents of the mediaConcentration
Sodium nitrate (NaNO31.50 g/L 
K2HPO4 0.0314 g/L 
Magnesium sulfate (MgSO40.036 g/L 
Calcium chloride dihydrate 0.0367 g/L 
Sodium carbonate 0.0200 g/L 
Disodium magnesium EDTA 0.001 g/L 
Citric acid 0.0056 g/L 
Ferric ammonium citrate 0.006 g/L 
HEPES buffer 2.3 g 
NaNO3 1.5 g 
P-IV metal solution 1 ml 
K2HPO4·7H2O (6 g/L) 5 ml 
MgSO4·7H2O (6 g/L) 5 ml 
Na2CO3 (4 g/L) 5 ml 
CaCl2·2H2O (2.5 g/L) 10 ml 
Na2SiO3·9H2O (4.64 g/L) 10 ml 

After dissolving all the necessary components in the conical flask the media was autoclaved at 121 °C at 103 kPa for 15 minutes. The media was poured into Petri dishes. Moreover, it was inoculated with the algal blooms present in the environment.

The catalase test was performed as follows: Use a loop or sterile wooden stick to transfer a small amount of colony growth onto the surface of a clean, dry glass slide. Spot a drop of 3% H2O2 on the glass slide. Development of oxygen bubbles can be observed (Omar et al. 2022a).

Gas preparation system

A system was maintained to add the acidic pollutants SO2 and NO2 in the laboratory to the culture media of the microorganisms. The system includes a 100-ml Erlenmeyer flask, magnetic heat stirrer, heat resistant hose, and another 100-ml conical flask with microalgal culture solution, cork and passing tube. The energy for the reaction was provided by the heat stirrer and sodium metabisulfite and nitrous acid were added to the 100-ml Erlenmeyer flask using a syringe technique that penetrates 0.5 cm into the culture solution (Zieliński et al. 2023). The acidic pollutants SO2 and NO2 are provided to the culture.

The chemical reaction involved in liberation of SO2 is:
formula
formula
The chemical reaction of nitrous acid with water in liberation of NO2 is:
formula

After the above process, absorbance was calculated by spectrophotometer for the analysis of SO2 and NO2 reduction in the sample by microalgae.

Sulfur dioxide (SO2) content preparation

To calculate the amount of SO2, standards of sodium metabisulfite were used and dilutions from 100 μg/ml to 1,500 μg/ml were made; 10 ml of sample and standard solutions were taken. Sulfamic acid (1 ml) and rosaniline (2 ml) were prepared. HCHO and HCl were added. After making up to 25 ml, the sample was kept for 30 minutes at room temperature. Absorbance was measured at 560 nm. Further, the graph was plotted on an Excel spreadsheet to extrapolate the values.

Nitrogen dioxide (NO2) content preparation

A standard solution of nitrous acid and then subsequent standard solutions of 0.20–2 μg/ml were prepared. A sample of quantity 10 ml was taken. Sulfanilamide (10 ml), H2O2 (1 ml) and NEDA (1.4 ml) were added. After making up to 25 ml, the sample was kept for 10 minutes at room temperature. Absorbance was measured at 560 nm.

Effect of NaHCO3, pH, and exposure time

To determine the effect of NaHCO3 concentration on the growth, the microalgae were cultivated in media containing 2–5% of 0.2 g/L. Samples were taken on days 3, 4, 5, 6 and 7 of the incubation period. The experiment was performed three times. System pH ranged between 7.1 and 8.5 and the exposure time was increased in the triplicate experiment.

Analytical method

The optic density was measured using a double beam spectrophotometer at 560 nm. The chlorophyll concentration was determined at 646.6 nm and 663.6 nm for chlorophyll a and chlorophyll b, respectively. The result was shown in 1 g of chlorophyll per mL (Porra et al. 1989).

Effect of 5 and 2% concentration of sulfur dioxide

According to the experiment 5% (Table 2) and 2% (Table 3) solution of sodium metabisulfite was prepared in 100 ml of water. The absorption of the SO2 was calculated during the 7-day period of incubation (Table 4). The absorption of SO2 sharply increased on the second day of incubation. The maximum absorption was on the fifth day of SO2 exposure at 0.541 and the concentration of the sulfur dioxide was maximum at 831.2 μg/ml. The growth kinetics graph of algae with 5% dosage of SO2 (Figure 3) shows a linear growth trend. The absorbance (Table 5) and concentration at 2% solution showed moderate 618.75 μg/ml bio-fixation of SO2. The linear graph (Figure 4) of the experimental study indicated that the bio-fixation of pollutant increases as the time exposure increases. The growth kinetics graph of algae with 2% dosage of SO2 (Figure 5) also shows a linear growth trend.
Table 2

Absorbance and concentration of 5% SO2 on different days

DaySO2 absorbance at 560 nmConcentration of SO2
0.36 606.25 μg/ml 
0.47 743.75 μg/ml 
0.54 831.25 μg/ml 
DaySO2 absorbance at 560 nmConcentration of SO2
0.36 606.25 μg/ml 
0.47 743.75 μg/ml 
0.54 831.25 μg/ml 
Table 3

Absorbance and concentration of 2% SO2 on different days

DaySO2 absorbance at 560 nmConcentration of SO2
0.264 486.25 μg/ml 
0.370 618.75 μg/ml 
0.421 682.50 μg/ml 
DaySO2 absorbance at 560 nmConcentration of SO2
0.264 486.25 μg/ml 
0.370 618.75 μg/ml 
0.421 682.50 μg/ml 
Table 4

Absorbance obtained for 5% SO2 on different days

DayAbsorbance in spectrophotometer
0.390 
0.389 
0.410 
0.445 
0.456 
0.512 
0.513 
DayAbsorbance in spectrophotometer
0.390 
0.389 
0.410 
0.445 
0.456 
0.512 
0.513 
Table 5

Absorbance of algae 1 (2% SO2)

DayAbsorbance in spectrophotometer
0.35 
0.37 
0.39 
0.42 
0.49 
0.53 
0.62 
DayAbsorbance in spectrophotometer
0.35 
0.37 
0.39 
0.42 
0.49 
0.53 
0.62 
Figure 3

Growth kinetic curve for 5% dosage of SO2.

Figure 3

Growth kinetic curve for 5% dosage of SO2.

Close modal
Figure 4

Extrapolation curve of SO2.

Figure 4

Extrapolation curve of SO2.

Close modal
Figure 5

Growth kinetics curve for 2% dosage of SO2.

Figure 5

Growth kinetics curve for 2% dosage of SO2.

Close modal

Effect of 5 and 2% concentration of nitrogen dioxide

Solutions of 2 and 5% were made by dissolving nitrous acid in 100 ml of distilled water. The absorbance and concentration of 2% NO2 (Table 6) is maximum at day 1. Absorbance shows increasing trends with time (Table 7) and growth kinetics of algae are linear (Figure 6). The 5% solution of NO2 showed the maximum absorption (0.489) and concentration (795.375 μg/ml) on the first day of exposure at room temperature (Table 8). Absorbance recorded by spectrophotometer (Table 9) is increasing with respect to time (Figure 7). These data indicated that the nitrogen oxides are consumed faster than the sulfur dioxides.
Table 6

Absorbance and concentration of 2% NO2 on different days

DayAbsorbanceConcentration
0.36 634.123 μg/ml 
0.25 496.625 μg/ml 
3 0.19 421.625 μg/ml 
DayAbsorbanceConcentration
0.36 634.123 μg/ml 
0.25 496.625 μg/ml 
3 0.19 421.625 μg/ml 
Table 7

Absorbance obtained at 2% dosage of NO2 on different days

DayAbsorbance in spectrophotometer
0.41 
0.46 
0.54 
0.62 
0.69 
0.74 
7 0.79 
DayAbsorbance in spectrophotometer
0.41 
0.46 
0.54 
0.62 
0.69 
0.74 
7 0.79 
Table 8

Concentration of 5% NO2 at different days

DayAbsorbanceConcentration
0.489 795.375 μg/ml 
0.316 579.125 μg/ml 
0.254 501.625 μg/ml 
DayAbsorbanceConcentration
0.489 795.375 μg/ml 
0.316 579.125 μg/ml 
0.254 501.625 μg/ml 
Table 9

Absorbance of algae 1 (5% NO2)

DayAbsorbance in spectrophotometer
0.64 
0.69 
0.75 
0.79 
0.82 
0.89 
0.94 
DayAbsorbance in spectrophotometer
0.64 
0.69 
0.75 
0.79 
0.82 
0.89 
0.94 
Figure 6

Growth kinetics curve for 2% dosage of NO2.

Figure 6

Growth kinetics curve for 2% dosage of NO2.

Close modal
Figure 7

Growth kinetics curve for 5% dosage of NO2.

Figure 7

Growth kinetics curve for 5% dosage of NO2.

Close modal

Effect of combination of SO2 and NO2 on Chlamydomonas

This combination study of NO2 and SO2 on Chlamydomonas sp. estimated that the consumption of SO2 is slow (Figure 8) but the amount of SO2 consumed for growth was greater than the concentration of NO2 (Table 10). The extrapolation curve for NO2 is shown in Figure 9. The concentrations of NO2 and SO2 increased to 671.625 μg/ml and 718.75 μg/ml respectively (Table 11). The combined 2% dosage of SO2 and NO2 on algae shows a linear trend in its growth (Figure 10).
Table 10

Absorbance and concentration of 2% NO2 and 2% SO2

DayAbsorbance for SO2Conc. of SO2Absorbance for NO2Conc. of NO2
0.45 718.75 μg/ml 0.59 671.625 μg/ml 
0.32 556.25 μg/ml 0.45 532.750 μg/ml 
0.26 481.25 μg/ml 0.39 452.875 μg/ml 
DayAbsorbance for SO2Conc. of SO2Absorbance for NO2Conc. of NO2
0.45 718.75 μg/ml 0.59 671.625 μg/ml 
0.32 556.25 μg/ml 0.45 532.750 μg/ml 
0.26 481.25 μg/ml 0.39 452.875 μg/ml 
Table 11

Absorbance and concentration of 5% NO2 and 5% SO2

DayAbsorbance for SO2Conc. of SO2Absorbance for NO2Conc. of NO2
0.61 918.75 μg/ml 0.58 768.50 μg/ml 
0.52 806.25 μg/ml 0.43 754.29 μg/ml 
0.48 756.25 μg/ml 0.32 678.26 μg/ml 
DayAbsorbance for SO2Conc. of SO2Absorbance for NO2Conc. of NO2
0.61 918.75 μg/ml 0.58 768.50 μg/ml 
0.52 806.25 μg/ml 0.43 754.29 μg/ml 
0.48 756.25 μg/ml 0.32 678.26 μg/ml 
Figure 8

Growth kinetics curve for 5% dosage of SO2 and NO2.

Figure 8

Growth kinetics curve for 5% dosage of SO2 and NO2.

Close modal
Figure 9

Extrapolation curve of NO2.

Figure 9

Extrapolation curve of NO2.

Close modal
Figure 10

Growth kinetics curve for 2% dosage of SO2 and NO2.

Figure 10

Growth kinetics curve for 2% dosage of SO2 and NO2.

Close modal

The experiment successfully used Chlamydomonas sp. cultures exposed to sulfur dioxide (SO2) and nitrogen dioxide (NO2) to increase their tolerance of acidic pollutants.

This study demonstrates not only the inhibitory effect of the hazardous gaseous air pollutants but also the ability of these organisms to survive and stimulate photosynthesis and growth rate. NO2 does not show much negative effect on the algae but it is a helpful growth factor. Overall, the algae show significant growth and tolerance levels against the acidic pollutants.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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