Study of acidic air pollutant (SO₂ and NO₂) tolerance of microalgae with sodium bicarbonate as growth stimulant

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 (NO_x), and sulfur (SO_x) 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).

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).

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.

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).

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.

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

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.

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.

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.

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).

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.