Two local Chlamydomonas strains were obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB) with the record ID of FACHB-2221 and FACHB-2220 and in the current experiment denoted as CHL-2221 and CHL-2220, respectively. The stock cultures were grown in the fresh medium of Tris–acetate–phosphate (TAP) and grown at ambient temperature (25 ± 1.5°C) under light illumination of 50 µmol photons m⁻² s⁻¹ prior to the treatment. The 20% inoculum size culture was prepared for the experiment by inoculating 100 ml of stock culture with an optical density (OD) of 0.2 at 750 ʎ (OD₇₅₀ = 0.2) into 400 ml fresh TAP medium in a 1-L conical flask. The inoculated cultures were grown at 25 ± 1.5°C and illuminated in a 12:12-h light–dark cycle with white fluorescent lamps with 50 µmol photons m⁻² s⁻¹ light intensity. An air pump fixed in a container was used to provide the aeration. To examine the influence of tobacco smoke on microalgae, every 1 h, one cigarette was burnt and kept in the container to provide the smoke to microalgae cultures, while for the control, cultures only aerated with atmospheric air. The schematic of the experimental rig used in the study is presented in Supplementary Figure S1. The maximum specific growth rate was calculated throughout the exponential growth phase based on the natural logarithm of optical density (OD₇₅₀) against time (day) following Eq. 1 (Barati et al., 2018): μ max ( day - 1 ) = ( Ln   D e - Ln   D i ) / ( t e - t i ) , (1) where D_e is OD₇₅₀ at t_e, D_i is OD₇₅₀ at t_i, and t_e and t_i are times in the exponential phase.

Total carbohydrate concentration was measured based on the widely accepted protocol offered by Borowitzka and Moheimani (2013) (Borowitzka and Moheimani, 2013) with minor modifications. In brief, 50 ml of each replicate was centrifuged, and the pallet was ground in HCl (2M) with a hand homogenizer. The ground samples were sonicated and incubated for 1 h at 80°C in an ultrasonicator. Next, the samples were centrifuged, and aliquots of samples were taken into glass tubes, followed by phenol, water, and gentle addition of H₂SO₄ (95.5% v/v). Then, the samples were incubated for 30 min, and the OD was determined at 485 nm (OD₄₈₅). Finally, the concentration of carbohydrates was calculated from the standard curve by considering glucose as a reference sugar. Lipids were recovered by means of methanol/chloroform/water (2:1:0.8 v/v) and calculated via the gravimetric technique by Bligh and Dyer (Bligh and Dyer, 1959). The solution in the glass vial was air-dried with nitrogen gas, and then the dried extract was retained for 24 h in a desiccator before determining the weight. The difference in weights of glass vials was considered as the lipid weight. Proteins were recovered with NaOH 0.5 N at 80°C for 30 min, and the protein concentration was calculated via the dye-binding technique by Bradford using the standard curve produced by bovine serum albumin as standard (Borowitzka and Moheimani, 2013). Additionally, carotenoid and chlorophyll extractions were done by filtration of 20 ml culture on the glass-fiber filter (Whatman GF/C, 0.45 μM), and subsequent homogenization in 10 ml of absolute methanol. After overnight incubation, the samples were centrifuged for 5 min at 1,409 × g to attain the clear supernatant prior to spectrophotometric quantification. Chlorophyll concentrations (mg/L) were calculated by analyzing the cell extracts’ absorbance at 645 and 665 nm using a UV–vis spectrophotometer following Eq. 2 (Lichtenthaler, 1987): Chlorophyll  ( C a + C b ) = ( 24 . 93 ×A 652 ) + ( 1 . 44  ×A 665 ) . (2)

Likewise, carotenoids (mg/L) were quantified from the same solution by determining the absorbance at 470 nm following Eq. 3 (Lichtenthaler, 1987): Total carotenoid content = ( 1000  A 470 - 1 . 63  Ca  - 104 . 96  C b ) / 214 . (3)

Lipid transesterification was performed by mixing 1 ml of 1% H₂SO₄–MeOH with the lipid extract and incubating for 60 min at 90°C by a test tube heater. Subsequently, the methylated fatty acids (FAMEs) were recovered by hexane, and then hexane was evaporated using the stream of nitrogen gas. Then, the samples were preserved at −20°C. The recovered FAMEs were analyzed via gas chromatography–mass spectrometry (GC/MS, Thermo Fisher Scientific, United States) equipped with a non-polar DB 35 column where helium gas was used as the carrier. From each sample, 1 µl of FAME was injected to the GC/MS controlled under systematic temperature settings of the initial oven temperature set at 40°C for 5 min, then elevated at a rate of 4°C min⁻¹ to 260°C, and held for 1 min.

To examine the biomass quality as a feedstock for biodiesel production, the physicochemical characteristics of FAMEs were calculated according to widely accepted formulas. The cetane number (CN), percentage of unsaturation degree (DU, %), long-chain saturation factor (LCSF, wt%), iodine value (IV, g I₂/100 g oil), cold filter plugging point (CFPP,°C), and saponification value (SV, mg KOH g⁻¹) were calculated according to Francisco et al. (2010) and Ashour et al. (2019), as presented in Eqs. 4–9: DU ( % ) = ∑ [ ( 2 ×PUFAs ) + MUFAs ] , (4) where PUFAs and MUFAs signify the proportion of polyunsaturated and monounsaturated fatty acids. SV ( mg KOH g - 1 ) = ∑ [ ( N%× 560 ) / M ] , (5) IV ( gI 2 / 100  g oil ) = ∑ [ ( N%×D× 254 ) / M ] ,   (6) CN = 46 . 3 + ( 5458 / SV ) − ( 0 . 225  × IV ) , (7) where N% signifies the ratio of each individual fatty acid, M signifies the molecular weight of each fatty acid, and D denotes the quantity of carbon–carbon double bonds. LCSF = ( C 16 : 0 × 0 . 1 ) + ( C 18 : 0 × 0 . 5 ) + ( C 20 : 0 × 1 ) + ( C 22 : 0 × 1 . 5 ) + ( C 24 : 0 × 2 )   (8) CFPP ( °C ) = ( LCSF× 3 . 1417 ) - 16 . 477 , (9) where C24:0, C22:0, C20:0, C18:0, and C16:0 signify the weight ratio of each fatty acid. In addition, the higher heating value (HHV) is estimated via Equation 10 (Demirbas, 2016): HHV ( MJ / KG ) = 49 . 43 - 0 . 041 ( SV ) - 0 . 015 ( IV ) . (10)

The influence of tobacco smoke on the specific growth rate, lipids, proteins, fatty acids, and carbohydrates was evaluated using one-way ANOVA. All calculations were done in SPSS 16.0 (SPSS Inc, United States), and the variances were assumed significant when p < 0.05.

Growth was analyzed by measuring the OD of the cultures cultivated under ambient atmospheric air and tobacco smoke for the duration of 7 days. The growth patterns were varied for the two studied microalgal strains upon exposure to tobacco smoke (Figure 1). It can be seen that by the increase in the exposure duration, the differences were intensified in CHL-2221, and by the end of the experiment, the cell concentration of smoke-treated CHL-2221 compared to the control was considerably lower. In CHL-2220, lower growth was observed till day 3; however, it was able to tolerate the tobacco smoke and recover its proliferation ability by the end of the experiment, which is possibly owing to the presence and activation of tolerance mechanisms under tobacco smoke, although their activation required time. The biomass productivity and specific growth rate (μ_max) of the investigated strains are shown in Figure 1. In ambient aeration, μ_max in CHL-2221 and CHL-2220 was, 0.445 days⁻¹ and 0.506 days⁻¹, individually, displaying a considerably faster growth in CHL-2220. The μ_max was not influenced in CHL-2220 (p > 0.05) by exposure to tobacco smoke, and it was almost 0.500 days⁻¹, whereas in CHL-2221, μ_max reduced meaningfully (p < 0.05) from 0.445 days⁻¹ to 0.376 days⁻¹. The difference in growth response of studied strains is due to their capability to tolerate various contaminations in tobacco smoke. For example, heavy metals can cause this growth inhibition. It was shown that inorganic chemicals As, Co, Cd, Cu, Cr, Hg, Ni, Mn, Pb, Se, Sb, Sn, Zn, and V were added to the microalgal growth medium of Nannochloropsis salina, which resulted in significant suppression of growth (Hess et al., 2017). Additionally, the presence of heavy metals in tobacco smoke can be considered a key growth inhibitor. Quinoline, acrylonitrile, and dimethylhydrazine are the three main components of tobacco smoke, and they are probably the main factors affecting microalgae growth. Quinoline is known as a common N-containing six-membered ring compound that is present in algal bio-oil and in organic chemical wastewater [31]. It is reported that it is biodegradable and organisms can use it as a carbon source [32]. Acrylonitrile is a resin derivative and is widely used in various industrial manufacturing fields [33]; its biodegradation by bacteria is reported [34]. The toxicity effect of 1,1-dimethylhydrazine can be mitigated by antioxidant molecules in microalgae [35]. The availability of mechanisms for the degradation of these compounds in bacteria suggests that our studied microalgal strains may comprise those mechanisms. Therefore, future research is required to confirm it.

Overall, the lipid concentration of the studied strains was reduced in response to tobacco smoke. CHL-2220 accumulated lipid by 15.16 %DW under control condition, while upon smoke exposure, it decreased to 13.82 %DW (p > 0.05) (Figure 2A). Likewise, a similar pattern was seen in CHL-2221, when its lipid level of 15.55 %DW under ambient conditions (p > 0.05) reduced considerably to 13.37 %DW, which indicates lipid biosynthesis inhibition upon smoke exposure in both strains. This is not in agreement with some studies, as microalgae usually accumulate lipid bodies under stress conditions (Wang et al., 2009). However, the reduction of the lipid content was similarly observed in microalgae consortia when the culture was exposed to unfiltered flue gas, and it was suggested that the suppression of fatty acid synthesis was due to the presence of impurities and acidifications (Aslam et al., 2018a). In addition, the increase in carbohydrates in microalgae often occurs during environmental stresses. In addition, microalgae are shown to accumulate starch as the leading energy and carbon storage molecule (Zhu et al., 2014). The influence of smoke on levels of carbohydrate varied among the studied strains (Figure 2B). In CHL-2221, the carbohydrate level amplified meaningfully (p < 0.05) when it was grown under tobacco smoke conditions, while in CHL-2220, it did not vary significantly. In CHL-2221, by considering its growth inhibition and carbohydrate level increases, it could be proposed that this strain stored carbohydrates as a stress response to tobacco smoke because carbohydrate metabolism regulation is vital in keeping the balance between proliferation and photosynthesis. Moreover, starch is the central storage compound in Chlamydomonas sp. (Puzanskiy et al., 2018), which is produced predominantly and more compared to lipids in moderate stress conditions, and also it is altered quicker subsequent to recovering to the ambient conditions (Siaut et al., 2011). Conversely, a different pattern was seen in the protein content. In CHL-2221 and CHL-2220, protein content increased from 35.48% and 30.91% to 36.55% and 34.56%, individually upon exposure to tobacco smoke (Figure 2C). A noteworthy shift in protein level was detected in CHL-2220, whereas it was not significant in CHL-2221. In fact, this trend was predictable as CHL-2220 presented tolerance to tobacco smoke exposure; thus, it might be due to the synthesis of proteins that confer tolerance. For instance, heat shock proteins (HSPs) are the commonly known proteins that are generally overexpressed in stress conditions; nevertheless, more advanced molecular analyses are essential to recognize the proteins that are differentially expressed (Barati et al., 2019). Alterations induced by different stressors are primarily noticeable in changes of photosynthetic pigments compared to the evidence of any variations in the cell growth (Haire et al., 2018). In CHL-2220, the carotenoid level was not influenced (p > 0.05), and it marginally fluctuated between 0.32 and 0.34 (Figure 2D), suggesting that this strain did not experience significant stress by the smoke exposure, while considerable changes (p > 0.05) were seen in CHL-2221 when the Car/Chl ratio was higher (0.43) upon treatment than under normal conditions (0.28). The elevated Car/Chl ratio proposes the occurrence of stress upon tobacco smoke. The increase in carotenoid levels is confirmed in Chlamydomonas sp. as a response to diverse stressors (Almarashi et al., 2020), including exposure to heavy metals stress (Nowicka et al., 2016). This proposes the conceivable influence of toxic chemicals present in tobacco smoke.

The fatty acid composition of CHL-2221 and CHL-2220 grown under tobacco smoke exposure and ambient conditions is displayed in Table 1. The ratio of saturated fatty acids (SFAs) in both strains significantly varied. The SFA level of CHL-2220 upon exposure to tobacco smoke declined significantly (p < 0.05) from 29.15 to 24.14%, whereas no substantial variations were detected in the SFA level of CHL-2221, and it remained at about 20%. Among SFAs, palmitic acid was the main constituent and displayed significant (p < 0.05) decreases in response to tobacco smoke exposure. Palmitic acid (C16) is the most frequently found constituent of the membrane cell wall and is also one of the main SFAs in microalgae (Osorio et al., 2019). Also, its substantial alterations in response to diverse stressors, including salinity stress conditions, have been previously reported (Yang et al., 2018), indicating its probable participation in acclimation under environmental stress conditions. Mutually, both studied strains presented reduced palmitic acid in response to tobacco smoke as it declined from 10.68 to 7.31% in CHL-2221 and from 20.18 to 11.22% in CHL-2220. Both CHL-2220 and CHL-2221 MUFAs’ content demonstrated a significant increase in response to the tobacco smoke by demonstrating an increase in MUFA from 11.59 to 15.97% and from 13.10 to 20.73%, respectively. Palmitelaidic acid (C16:1) and oleic acid (C18:1) were the dominant MUFAs in both strains. In CHL-2220, oleic acid (C18:1) content has no significant (p > 0.05) alterations, while its content in CHL-2221 significantly (p < 0.05) increased from 7.64 to 17.09% in response to tobacco smoke. Oleic acid is the major constituent of triacylglycerols (TAGs); therefore, the increase in its content can be considered as TAG accumulation (de Jaeger et al., 2017). Moreover, according to the previous study on green microalgae (Srivastava et al., 2017), the acclimation process in the current study could be considered the CHL-2221 response to tobacco smoke.

The main FAME components obtained from both tobacco smoke-treated strains and control strains in this experiment were the PUFAs. Its content has no significant alteration in control and tobacco smoke-treated CHL-2220 strains and was around 59%, while a significant decline was observed in CHL-2221 treated by the tobacco smoke (declined from 65.69 to 60.93%). Alpha-linolenic acid (C18:3) was the main PUFA in mutually both treated and control strains, which is a common dominant unsaturated fatty acid in many microalgae species (Moon et al., 2013). The CHL-2220 control strain and CHL-2221 control strain have the alpha-linolenic acid (C18:3) content of 29.97 and 29.72%, respectively. However, treating the CHL-2221 strain with tobacco smoke dramatically decreased the alpha-linolenic acid content (from 29.72 to 21%). On the other hand, the alteration of the alpha-linolenic acid content of CHL-2220 strains treated with tobacco smoke remained unchanged, while phosphorous (Qari and Oves, 2020) and nitrogen (Sun et al., 2018) stress in the Chlamydomonas strains have caused alpha-linolenic acid accumulation. Meanwhile, hexadecadienoic acid (C16:2) was another evident PUFAs in this study. It was not detected in both control strains under ambient conditions, while the exposure to tobacco smoke led to an increase of C16:2 in CHL-2221 and CHL-2220 to 3.81 and 3.88%, respectively. Dihomo-γ-linolenic acid (C20:3) has demonstrated the same pattern as hexadecadienoic acid (C16:2). Although C20:3 was not found in the studied strains grown in ambient conditions, exposure to tobacco smoke led to its accumulation by 3.42 and 3.94% in the CHL-2220 and CHL-2221, respectively. These results suggest that dihomo-γ-linolenic acid might have the acclimatization role in response to the tobacco smoke ambient. Moreover, the synthesis of omega-3 and omega-6 fatty acids has been provoked in both strains in response to tobacco smoke. This can be considered as a positive impact. Due to the essential role of fatty acids such as omega-3 and omega-6 in human well-being, additional analysis is necessary to identify the active compounds triggering their synthesis. However, molecular studies and toxicological screening should be considered prior to any decision to use them as a food supplement.

In order to study the impact of tobacco smoke exposure on biodiesel features, the key biofuel parameters such as SV, IV, CN, CFFP, DU, HHV, and LCSF were investigated in the selected strains (Table 2). Also, we compared these parameters with other Chlamydomonas sp. grown under nitrogen-rich (N+) conditions that previously showed a promising result (Maneechote et al., 2021). The DU indicates the total mass ratio of MUFAs and PUFAs; therefore, the higher the DU, the higher the unsaturation of fatty acids. Although the treated and control samples of CHL-2221 showed no significant alteration in the DU value, the tobacco smoke-treated CHL-2220 displayed a slight increase in the DU value (from 129.91 wt% to 135.74 wt%). However, a significantly lower DU value of 58.20 was observed in other Chlamydomonas sp. grown under enriched N conditions. CN is another critical biofuel property that contributes to the ignition quality of the biofuels. The CN value strictly depends on the DU value. Surprisingly, by CHL-2220 exposure to tobacco smoke, the CN value decreased from 30.34 to 25.28, while CHL-2221 treatment with tobacco smoke increased the CN value from 26.07 to 29.73. However, a relatively higher CN value of 57.92 was observed in Chlamydomonas sp. grown under elevated nitrogen concentration. It shows the relatively lower quality of CN value of the studied strains in both ambient and treatment conditions of the current study than that of other Chlamydomonas sp.

Another key factor in investigating fatty acid unsaturation is the iodine value (IV), which measured the grams of iodine reacting with 100 g of the FAME sample (Ramos et al., 2009). The IV represents the double bonds available in the unsaturated fatty acid chain structure (EN 14214, 2008). The tobacco smoke exposure increased the IV of the CHL-2220 from 180.34 g I₂ 100 g⁻¹ to 201.73 g I₂ 100 g⁻¹, while declining the IV of CHL-2221 from 199.81 g I₂ 100 g⁻¹ to 184.53 I₂ 100 g⁻¹. By comparison, Chlamydomonas sp. was reported to have almost three times lower IV value (61.91 g I₂ 100 g⁻¹), which was in the acceptable range suggested by EN. As it has been determined by the European Standard organization [48], biodiesel with higher IV than 120 g I₂ 100 g⁻¹ will polymerize and form glycerides. This phenomenon at elevated temperatures leads to deposit buildup or increase in lubricating oil corrosivity (Shaltout et al., 2019). The milligrams of potassium hydroxide (KOH) required to saponify 1 g of FAMEs is described as the saponification value (SV). The SV has an opposite correlation with the chain length of fatty acids. The SV of tobacco smoke-treated CHL-2220 culture was not altered significantly (slightly increased from 221 mg KOH g⁻¹ to 223 mg KOH g⁻¹) in comparison to the control. Similarly, the tobacco smoke-treated CHL-2221 strain SV alteration was not significant compared to the control strain (declined from 220.73 mg KOH g⁻¹ mg KOH g⁻¹ to 218.79 mg KOH g⁻¹). The SV value of Chlamydomonas sp. was 213 mg KOH g⁻¹, which was comparatively lower. The high SV of both strains means a low yield of biodiesel, while extra methanol is required during the biodiesel production process and increases with the production of glycerol (Bart et al., 2010).

Another key factor that should be considered is cold filter plugging point (CFPP) which indicates the minimum temperature that the biodiesel can run an engine without any problem (Vidyashankar et al., 2015). The increase in the SFA concentration increases the CFPP value. In the current study, different CFPP responses were observed from the Chlamydomonas strains exposed to tobacco smoke. The CFPP of tobacco smoke-treated CHL-2220 dropped considerably from −3.41°C to −7.75°C, while this value has increased in the tobacco smoke-treated CHL-2221 strain from −6.05°C to −5.94°C. Therefore, it can be suggested that tobacco smoke has positive impacts on CHL-2220 and enhance the properties of biodiesel extracted from tobacco smoke-treated CHL-2220, while the Chlamydomonas sp. presented a poor CFPP value of 2.23°C, which limits the applicability of the biodiesel to warm climates. This is one of the drawbacks of biodiesel with a high CN value that usually exhibits poor CFFP, due to the high SFA level. LCSF is another key property that describes CN and IV. The higher LCSF represents more favorable physicochemical features of the generated biodiesel. In this study, tobacco smoke exposure led to the decline of the LCSF value in both strains. In CHL-2220, it caused a 33% decline, while the LCSF value of CHL-2221 was not significantly altered. Higher heating value (HHV) is the volume of heat energy produced through the combustion of 1 g of fuel and reports through units of energy per unit mass or volume of substance (Demirbas, 2016). It has been suggested that the fuel with higher HHV is more efficient for small-size engines (Mondal et al., 2019). The HHV values of both strains in the current study were 37, which were perfectly comparable with the suggested acceptable range defined by EN (Brithish Standard EN 14214, 2008). European standards (EN14214) and Australian standards have indicated the optimum CN value of 51 (Kemp and Kemp, 2003; Brithish Standard EN 14214, 2008), while the CN values of the studied strain did not meet it due to the high DU value. Therefore, it can be suggested that these tobacco smoke-treated strains can be used for biodiesel production if the DU could be decreased. Therefore, a proper balance of MUFAs, including oleic acid, and lesser PUFAs and SFAs, would serve this purpose. Blending microalga biomass with other feedstock is a suggested solution to overcome this matter and enhance the biodiesel quality (Abomohra et al., 2020).

This study contributes to developing more sustainable biological air purifiers. The main implication of microalgal air purifiers is that the CO₂ of indoor environments such as offices, shopping malls, train stations, and airports can be reduced and instead O₂ produced. This is of great significance as the indoor places are usually sealed to control temperature, while CO₂ produced by crowd is accumulated and decreases the air quality for people. In microalgal air purifiers, O₂ is produced as the culture grows, and CO₂ is transformed into the cell building block (e.g., lipids and carbohydrates). Therefore, a circular economy platform can be achieved by converting biomass to high-value products. For example, biodiesel can be generated from microalgae as a more environmentally friendly method to fuel production, or pigments can be recovered to be used as natural colorants. Several patents were published describing reactor design for air purification purposes in recent years, but the potential inhibitory of smoke was not considered. Thus, this study provides valuable information on the possibility of reactor failure due to the accumulation of toxins of tobacco smoke over time and consequently warns the researchers to consider the influence of air pollutants on microalgae.

Tobacco smoke is composed of various compounds that are released in the form of gas during aeration; studying the composition of inlet and outlet gas can provide important information on the absorbance capability of microalgae. However, this was not studied in this study due to the limitation of experimental tools such as gas sensors. Therefore, we only studied the effect of the entire tobacco smoke composition on the performance of microalgae. Many studies have already discussed the effect of heavy metals, SO_X, NO_X, and pharmaceutical pollutants on microalgae growth. Nevertheless, the effect of important chemicals such as acrylonitrile and benzene has not been studied so far, suggesting an interesting potential research gap for future studies. In the current study, both strains’ lipid extracts presented poor biodiesel characteristics, whereas their substantial PUFA ratio recommended its applicability for pharmaceuticals, nutrients, and animal feed. Therefore, it is recommended to investigate the toxicity of FAME extracts after smoke exposure. Moreover, the tolerance of microalgae can be enhanced to achieve higher microalgae performance using adaptive laboratory evolution (ALE). ALE is a process of selecting the most suitable mutations to particular stresses or conditions that naturally occur in the cell population (Arora et al., 2020). It was found that it was a successful approach to enhance the tolerance of microalgae to unfiltered flue gas (Aslam et al., 2017) and chemical contaminants such as phenol (Wang et al., 2016). Therefore, it can be considered as a method to enhance the tolerance of microalgae to air pollutants in future studies.