The Effect of Chromium on Photosynthesis and Lipid Accumulation in Two Chlorophyte Microalgae

Heavy metals have adverse effects on microalgae metabolism and growth. Photosynthesis and lipid profile are extremely sensitive to heavy metal toxicity. The impact of hexavalent chromium – Cr(VI) on photosynthesis and lipid accumulation in Mucidosphaerium pulchellum and Micractinium pusillum exposed to different concentrations (0 – 500 μg L-1) was investigated for 11 days. A significant (p < 0.05) increase in lipid content was observed with increasing Cr(VI) concentration. However, growth was suppressed at higher concentrations exceeding 100 μg L-1. Addition of Cr(VI) in the cell culture medium showed a negative effect on quantum yield (Fv/Fm) and a photosynthetic inhibition of > 65% was noted in both species at 500 μg L-1. However, the lipid gravimetric analysis presented inner cell lipid content up to 36% and 30% of dry weight biomass for M. pulchellum and M. pusillum, respectively. The fatty acids profiles of both microalgae species showed higher levels of hexadecenoic acid as well as ω3, ω6, and ω7 fatty acids. The effect of Cr(IV) on photosynthesis and lipid accumulation in both microalgae species was concentration and exposure time dependent. This shows that an appropriate concentration of Cr(VI) in culture medium could be beneficial for higher lipid accumulation in freshwater eukaryotic microalgae species.


Introduction
The use of microalgae in the pharmaceutical, medical and food industry is increasing due to their no reported side effects on human health. Thus, it is necessary to investigate microalgae growth parameters and potential growth inhibitors [1]. Heavy metals, antibiotics and herbicides are toxic to microalgae even at low concentrations however, to a certain extent, microalgae could [13], however, non-biodegradable heavy metals resist bioremediation and show long environmental persistence times [14,15], which is posing a serious threat for the biotic life. Heavy metals are abundantly available in nature. Unlike other heavy metals such as zinc (Zn) and copper (Cu), chromium (Cr) is not required for plant respiration or growth. However, due to rapid industrialization, an abundant quantity of Cr is being dumped into the water, causing unfavorable effects on animals and plants [12]. Furthermore, Cr toxicity is form-dependent, with hexavalent chromium -Cr(VI), being the significantly more toxic to humans, than the trivalent chromium -Cr(III) [16]. Trace amounts of Cr(III) are needed daily for adult humans [17]. On the other hand, Cr(VI) has carcinogenic and mutagenic effects on human [17]. Severe acute effects including gastrointestinal disorders, hemorrhagic diathesis, and convulsions could occur when ingesting 1 -5 g of chromate [17,18].
The exposure of microalgae to heavy metals could cause inhibited growth, suppressed cell division, reduced photosynthesis, and restrained enzymatic activity [19][20][21]. Advancement in chlorophyll fluorescence technology has made easier to analyze the photosynthesis processes, which has enabled researchers in finding new factors affecting photosynthesis. Even though the impact of heavy metals on microalgae have gained considerable attention and have been extensively reported, the effects of Cr(VI) toxicity on chlorophyte microalgae species Mucidosphaerium pulchellum (formerly Dictyosphaerium pulchellum) and Micractinium pusillum have not been yet reported.
In the present study, the toxic effects of heavy metal, hexavalent chromium, on growth and modulated fluorescence of freshwater eukaryotic non-model microalgae species M. pulchellum and M. pusillum were analyzed and compared. Furthermore, the effect of chromium on lipid accumulation and lipid composition was also investigated.

Microalgae Culture and Treatment
The eukaryotic freshwater microalgae species Mucidosphaerium pulchellum and Micractinium pusillum (phylum Chlorophyta, class Trebouxiophyceae) were obtained from the Korea Marine Microalgae Culture Center (KMMCC) Busan, Republic of Korea. Stocks were maintained on modified-AF6 agar slants [22]. The microalgae species were cultured in 250 mL flask bioreactors containing modified-AF6 medium [23], without citrate and ETDA, at a constant light intensity of 50 ± 2 µmol photons m -2 s -1 , 25 ± 2ºC temperature, and 50% humidity. The effects of potassium dichromate (K2Cr2O7, Yakuri Pure Chemicals, Osaka, Japan), a hexavalent form of chromium -Cr(VI), on photosynthesis and lipid accumulation at different concentrations (0 to 500 µg L -1 ) were investigated. All experiments were repeated at least three times.
3 of 13 5 mL sample was collected from each culture flask after a thorough hand mixing and cell densities were determined using hemocytometer (Marienfeld Superior, Germany) under a light microscope at a magnification of 400×. Furthermore, optical density at 750 nm (OD750) was recorded using UV/VIS spectrophotometer (WPA Biowave II, Biochrom, UK) on every alternating day [5].

Measurement of Toxicity, Modulated Fluorescence and Photosynthetic Inhibition
The sensitivity of M. pulchellum and M. pusillum in media supplemented with different concentrations of Cr(VI) was evaluated by toxy-PAM dual channel yield analyzer (Heinz Walz GmbH, Effeltrich, Germany) [5]. This toxicity analyzer is extremely sensitive to chlorophyll fluorescence and uses saturation pulse method to determine the effective fluorescence yield of photosystem II (PSII) [24,25]. To induce an equilibrium state for the photosynthetic electron transport, microalgae samples were dark adopted by placing in complete dark for 30 min before analysis and fluorescence intensity was measured using low intensity modulated light to avoid the reduction of the PSII primary electron acceptor (QA) [24].
Fluorescence intensity of microalgae cells excited by toxy-PAM blue light was measured at 650 nm. The minimal fluorescence level (F0; fluorescence measured shortly before the application of a saturation pulse), and the maximal fluorescence level (Fm; fluorescence measured during a saturation pulse) were recorded and the effective overall quantum yield (Y) of PSII was calculated using the following equation.

Lipid Extraction
The tested microalgae species were grown in modified-AF6 medium supplemented with different concentrations of Cr(VI) till pre-stationary phase and total lipids were extracted following the Bligh and Dyer method with slight modifications [26]. Briefly, microalgae cells were harvested by centrifugation at 4ºC and 5000×g for 20 min. Pellets were rinsed with distilled water and freeze-dried at -85ºC. Dried pellets were weighed, dissolved in 10 mL of methanol: chloroform and increased at a rate of 4°C to 240°C. Helium (He) was used as a carrier gas at a flow rate of 1 mL/min. Injection temperature and volume were set at 230°C and 1 µL, respectively. Fatty acids were identified by comparison with retention times of 37-component FAME mix standards (Sigma-Aldrich) and were expressed as mg/L. The corresponding fatty acids were further cross checked with the instrument database containing the NIST Ⓡ library [28].

Statistical Analysis
The statistical significance of the results was calculated using Analysis of Variance via SPSS ver. 27 (SPSS, Chicago, IL, USA). The significance among the samples was assessed using Duncan's multiple-range test and the results were considered statistically significant at p < 0.05.

Influence of chromium on microalgal growth
The culture medium, modified-AF6 medium, was supplemented with different concentrations of Cr(VI) ranging from 0 to 500 μg L -1 and their effects on growth parameters was investi-  Table   1). The 50, 100 and 250 μg L -1 chromium treatments showed reduction in growth after day nine, while 500 μg L -1 chromium treatment showed reduction in growth rate and cell number after day 7.  (Figures 3, 4).

Effect of chromium on lipid accumulation
The significant (p < 0.05) increases in lipid content were observed with the increasing Cr(VI) concentration in the culture medium. A lipid content of up to 36% was observed in M. pulchellum cultures at the maximum tested Cr(VI) concentration in this study (500 µg L -1 ), which was approximately 12 times higher than the control (0 µg L -1 ). Whereas a maximum lipid content of 30% was observed in M. pusillum cultures, which was approximately 3 times higher than the control.
A drastic increase in Cr(VI) concentration-dependent lipid content was observed in M. pulchellum however, the increase in concentration-dependent lipid content was not consistent in M. pusillum ( Figure 7). However, both tested microalgae species showed significantly (p < 0.05) increased lipid content, which shows that the addition of chromium could significantly enhance the lipid accumulation in the tested freshwater microalgae species.

Discussion
Chromium exists in the environment as trivalent -Cr(III) and hexavalent form -Cr (VI), where Cr (VI) being the highly toxic, carcinogenic and mutagenic [29,30]. The discharge of chromium from anthropogenic sources such as household, industry, transport, mining, and agriculture increases its concentration several times above normal levels [31]. The aquatic ecosystems are seriously affected by Cr (VI) toxicity, which depends on its physiochemical, oxidation, and structural properties [32]. The Cr (VI) constituents are generally soluble and mobile in the environment [33]. It can easily pass cell membrane due to the structural similarity to inorganic anions which makes Cr(VI) as an alternative substrate in the sulfate transport system (34,35).
The cytotoxic effects of Cr (VI) on living organisms including plants, animals, and human are well reported and it is also a source of a variety of human cancers [32,36]. The freshwater eukaryotic non-model microalgae species, M. pulchellum and M. pusillum were chosen for this study due to their economic potential as they can grow at low light intensity as well as in CO2-deficient conditions [37]. They are diverse green phytoplankton species which occasionally inhabit freshwater lakes. Furthermore, the genetic transformation of M. pulchellum (formerly D. pulchellum) for higher erythropoietin protein accumulation was reported by our group [38], which shows the broad scope of this microalgae species. During this study, the toxicity of pulchellum as compared to M. pusillum, which shows higher sensitivity of M. pusillum to chromium even at low concentration. All tested Cr(VI) concentrations showed a significant decline in quantum yield and photosynthetic inhibition as compared to the control. A maximum photosynthetic inhibition of up to 67% was observed in M. pulchellum at 500 μg L −1 whereas, M. pusillum showed a maximum photosynthetic inhibition of up to 66%. This shows that Cr(VI) has a negative effect on normal growth of both microalgae species by interrupting photosynthesis.
Interestingly, both tested microalgae species showed quit similar trends for growth and fluorescence yields, this could be because both microalgae are freshwater eukaryotic microalgae species and belong to a same taxon -Chlorophyta and thus showed similar Cr(VI) uptake and a similar PSII structure. Contrary to the present study, a 50% inhibition in growth was observed by Hörcsik et al. [40] when analyzing Cr(VI) toxicity using chlorophyll composition of Auxenochlorella pyrenoidosa (Chlorella pyrenoidosa) for 72 h in media supplemented with 2 mg L -1 of Cr (VI) [40]. Similarly, in another study, a very minor amount of Cr(VI), 5 µmol L -1 , showed up to 40% inhibition in the maximal quantum yield of PSII of Chlorella vulgaris when treated for 96 h [41]. The variation in the present study could be due to different microalgae species and different experimental conditions. The lipid accumulation results were quite interesting, both tested microalgae showed increases in lipid content. A lipid content of up to 36% was observed in M. pulchellum at the maximum tested Cr(VI) concentration in this study (500 µg L -1 ), which was approximately 10 times higher than the control. Whereas a maximum lipid content of up to 30% was observed in M. pusillum. The fatty acids composition analysis showed higher levels of polyunsaturated fatty acids (hexadecenoic acid) and saturated fatty acids (ω3, ω6, and ω7). However, chromium exposure significantly affected the saturated fatty acids content. In this study, linoleic acid (C18:2; ω6) and α-linoleic (C18:3; ω3), were among the mostly affected fatty acids by Cr(VI). This agrees with the previously reported studies of Barsanti et al. [42], and Rochhetta et al. [43], which states that chloroplast structure related lipids such as linoleic acid and α-linoleic are mostly affected by chromium. Furthermore, Cr(VI) treated cultures showed no significant differences for the non-photosynthetic structure related fatty acids such as arachidonic acid (C20:4; ω6), which agrees with Rochhetta et al. [43]. This suggests that chloroplasts would be the main target organelle of Cr(VI) toxicity in M. pulchellum and M. pusillum.
Despite significant decreases in observed saturated fatty acids content at higher Cr(VI) concentration (especially ω3 and ω6), total lipid content showed a significant increase. This could be a microalgal defense mechanism to counteract oxidative damage [43]. Both microalgae species showed higher lipid accumulation at higher tested Cr(VI) concentration than the control, this shows that the addition of Cr(VI) could significantly enhance the lipid accumulation in M. pulchellum and M. pusillum.
The results of this study indicate that Cr(VI) can affect total lipids and fatty acids content, especially affecting the fatty acids related to photosynthetic activity. Changes in fatty acids composition in the treated cells could be due to their defense mechanism to reduce cellular damage caused by Cr(VI) and its route outlined above. Additionally, intracellular Cr(VI) reduction consumes intracellular antioxidants which could induce synthesis of simple and poly unsaturated fatty acids as a defense mechanism. However, further analytical and biochemical analyses are necessary to assist the findings. The present study could aid in aquaculture industry, in maintenance of microalgae stock cultures, and to estimate the possible side effects of using hexavalent chromium in microalgae cultures. It can further aid in the design and construction of biomarkers using eukaryotic freshwater microalgae species.
Data Availability Statement: The data generated or analyzed during this study are included in this article and the primary data could be provided by the corresponding author upon request.