Effects of abiotic stress induced by a standard mix of atmospheric pollutants in Chlorella mirabilis (Chlorophyceae)

This study evaluates the level of oxidative stress induced by a mix of standard atmospheric pollutants in the green microalga Chlorella mirabilis. Genus Chlorella has been found as one of the most widespread among the phototrophic organisms occurring also in urban context, where it forms biofilms on monumental and building surfaces (1, 2). Aerial and sub-aerial algae are considered suitable bio-indicators to study the effects of pollutants because their species-specific sensitivity may be greater than in higher plants along with faster physiological responses. Furthermore, they are easy to handle and to maintain in controlled conditions (3). For these reasons, Chlorella mirabilis was used in the present study as testing organism to evaluate the response to atmospheric pollutants. With the aim to apply a lot of pollutants without increase the variability of the system, we chose a standard mix of atmospheric pollutants, i.e. ASTM D-7897-2015 (American Society for Testing and Materials International). As result of studies set up on urban surfaces depositions (4,5), used to stress building materials and evaluate their “accelerate ageing” (6), it consists in four kinds of atmospheric particles: soot (carbon), dust, salts, and particulate organic matter, which together represent a dark solution called “soiling mix” (7). Chlorella mirabilis was firstly grown in a specific culture medium (Bold’s Basal Medium, BBM) in aseptic conditions at 23 °C, under 14 h photoperiod. To evaluate the effects of pollutants, algal cultures were carried out at the same time in a new medium (BBM-SM) obtained by mixing “soiling mix” with BBM (1:5 v/v). Stress level in algae growing inside BBM-SM was assessed through the quantitative determination of photosynthetic pigments (Chlorophyll a, b and carotenoids) and malondialdehyde (MDA), this last considered as marker of lipid peroxidation. The extraction of photosynthetic pigments and oxidative metabolites was performed according with the methods of Lichenthaler (8) and Heath and Packer (9), respectively. Cell density was monitored during the experimental tests and the algal growth curve was described in both cultures conditions, after 15 day of algal growth. Results of repeated tests on algae cultures stressed by pollutants underline an increase over 60% of MDA compared with control cultures, highlighting the occurrence of oxidative stress. At the same time the concentration of chlorophyll a and b and carotenoids decreases over 60%, 20% and 70%, respectively. Observation under light microscope shows shape and colour changes in algal cells during their growth in BBM-SM. Nevertheless, C. mirabilis proved to be able to survive in critical environmental conditions characterized by high concentration of pollutants and dark particulate. These results may represent an important first step to use this species in studies on cell response to different stress factors and moreover may have interesting effects on many fields of applied plant biology.