Phycoremediation of As(III) and Cr(VI) by Desmodesmus subspicatus: Impact on growth and biomolecules (carbohydrate, protein, chlorophyll and lipid)  – A dual mode investigation

doi:10.21203/rs.3.rs-3927238/v1

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Phycoremediation of As(III) and Cr(VI) by Desmodesmus subspicatus: Impact on growth and biomolecules (carbohydrate, protein, chlorophyll and lipid) – A dual mode investigation

https://doi.org/10.21203/rs.3.rs-3927238/v1

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published 20 Jul, 2024

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Microalgae are under research focus for the simultaneous production of biomolecules (e.g., biofuels) and bioremediation of toxic materials from wastewater. The current study explores the capability of indigenously isolated microalgae (Desmodesmus subspicatus) for phycoremediation As(III) and Cr(VI) along with the production of biomolecules by alternating its extracellular and intracellular compositions. Desmodesmus subspicatus survived up to the toxicity level of 10 mg/L for As(III) and 0.8 mg//L for Cr(VI). A decline in carbohydrate accumulation (⁓70%) at 10 mg/L of As(III) concentration was obtained. An increased content of protein (⁓28%) and lipid (by ⁓32%) within the cells of Desmodesmus subspicatus was observed when grown in 0.5 and 0.2 mg/L As(III) concentrations respectively. A descending trend in carbohydrate accumulation was noted with increasing Cr(VI) concentration and the least (~44%) was recorded at 0.8 mg/L Cr(VI). Desmodesmus subspicatus showed an excellent maximum removal efficiency for Cr(VI) and As(III) as 77% and 90% respectively.

Phycoremediation

Carbohydrate

Protein

Lipid

Chlorophyll

Arsenic(III)

Chromium(VI)

Bioaccumulation properties of arsenic and chromium have become a threat worldwide because of their toxic and hazardous nature (Tripathi &Poluri 2021). As per the standards given by the United States Environmental Protection Agency (USEPA), arsenic and chromium have been listed in group ‘A’ carcinogenic metal for humans (Biswas &Nag 2021, Elahi et al. 2020). The maximum limit of As(III) and Cr(VI) in potable water is 0.01 and 0.5 mg/L respectively, as per the guidelines of the World Health Organization (WHO) (Poonia et al. 2021). Inorganic arsenic compounds [As(III), As(V)] are more toxic to humans compared to organic arsenic compounds like dimethyl arsenic acid (DMA) and monomethyl arsenic acid (MMA) (Jomova et al. 2011). Arsenite [As(III)] shows more toxic effects on the biotic environment as compared to arsenate [(As(V)] (Hindmarsh et al. 1986). Along with groundwater contamination, other sources of arsenic contamination are wood preservatives, thermal power plants, electronic materials, alloy manufacturing industries, antifouling paints, leather preservatives, pharmaceutical, glass industries and agricultural industries (Chung et al. 2014). Skin disease, bronchitis, malfunctioning of the nervous system and digestive issues are the health problems created by arsenic poisoning (Daneshvar et al. 2019).

Inorganic chromium compounds usually exist in two valency states (hexavalent chromium and trivalent chromium). Chromium in hexavalant form [Cr(VI)] is almost 100 folds more carcinogenic, when compared to the trivalent chromium (Cr(III)). Cr(VI) has high solubility in water and capable of mutating the DNAs and RNAs in all living organisms. Significant sources of Cr(VI) are industrial wastewater and domestic sewage discharged from leather tanning, mining, electroplating, paints & pigment, textile, steel, metallurgical and alloy industries, etc. (Nag et al. 2020, Nag &Biswas 2021b).

Economic constraints of widely used conventional methods for wastewater treatment such as electrochemical treatment, ion exchange methods, evaporation, precipitation and reverse osmosis (RO) techniques have driven researchers to develop more cost-effective, biodegradable and sustainable processes for the remediation of heavy metals. In this direction, modifying the adsorbents or using biological systems or integrated processes are the ways, which will reduce the running cost of a treatment plant, along with producing useful by-products. Bio-based green adsorbents as well as microorganisms (like Chlorella sp., Chlorela vulgaris, Scenedesmus quadricauda, Scenedesmus abundans, Maugeotia genuflexa, Ulothrix cylindricum) (dead or alive) were used as one of the trending sustainable and eco-friendly bioremediation methods to overcome these drawbacks (Bahar et al. 2013, Daneshvar et al. 2019, Ganguly et al. 2023, Nag &Biswas 2021a, Sarı et al. 2011, Sibi 2016, Tuzen et al. 2009). Agricultural, forest and green domestic wastes are being used as adsorbents for heavy metal removal (Das et al. 2022, Nag et al. 2016). Recently, an increased number of studies have been conducted in which microorganisms like fungi, bacteria, yeast, cyanobacteria and microalgae were used to adsorb heavy metals from effluents (Cui et al. 2021, Joshi et al. 2011, Saha &Nag 2022, Soares &Soares 2012). The mechanism of remediation of toxic substances using microalgae as the biosorbent has been termed as phycoremediation (Moondra et al. 2021). Phycoremediation is a low-cost, sustainable and green process as well as produces considerable amounts of biomass, lipid and energy (Tamil Selvan et al. 2020). Present work has been executed with the aim of valorization of Desmodesmus subspicatus, for remediation of heavy metals [As(III) and Cr(VI)] along with the accumulation of lipids, protein, chlorophyll and carbohydrates. Further, the growth kinetics of Desmodesmus subspicatus when grown in As(III) and Cr(VI) containing media was investigated along with its removal percentage.

Microalgal strain and chemicals

Desmodesmus subspicatus, a isolated microalgae collected from the Deopani river in Arunachal Pradesh, India was used (Sarkar et al. 2023). The BG-11media was used for the growth of the microalgal species. The isolated microalgae was preserved at 19^o C, 14 − 10 night-day ratio and 29 µmol/m² of light intensity. All the chemicals were purchased from Merck, India and media used in the experiments was purchased from Hi-media laboratories, India. Chromium (VI) and Arsenic (III) solutions were prepared by adding the known amount of potassium di-chromate (K₂Cr₂O₇) and sodium arsenite (NaAsO₂) salt in distilled water.

Experimental outline

250 ml of BG-11 media was prepared for seed culture and the media was inoculated with 2% stock culture. The seed culture was allowed to grow for 15 days at 28^o C, 14 − 10 night-day ratio and 29 µmol/m² of light intensity. This seed culture was used as the inoculum for all the experiments. The experiments were executed in two sets for both As(III) and Cr (VI). In this first set of experiments, Desmodesmus subspicatus were cultivated at varying As(III) and Cr(VI) concentrations. 50 ml BG 11 culture media containing various concentrations of As(III) and Cr(VI) were inoculated with 4% seed culture and cultivated for 15 days in triplicate. The concentrations of arsenic and chromium were varied within 0.5-15mg/L and 0.2-1 mg/L respectively in BG-11 culture media. At the end of the 15th day, the biomass was obtained after centrifugation (1000 rpm, 10 mins) of samples. The growth, carbohydrate, protein, total chlorophyll and lipid content were obtained by evaluating the biomass collected through a centrifuge. The collected supernatant was used to evaluate the remaining concentrations of arsenic and chromium.

In the second experimental set, the kinetics and adsorption efficiency of Desmodesmus subspicatus grown at 10 mg/L of As(III) and 0.6 mg/L of Cr(VI) were investigated. Here, Desmodesmus subspicatus were cultivated in 250 ml culture media (containing arsenic and chromium) in triplicate for 30 days. Subsequently, samples were collected at three-day intervals for the determination of growth of Desmodesmus subspicatus and concentration of arsenic and chromium.

Measurement of growth

The biomass growth of the samples was measured by centrifugation of 2 ml of biomass sample at 10000 rpm using a centrifuge for 10 minutes (Eppendorf, USA, Model 5424). Afterward, the supernatant was separated from the biomass cells and collected for metal ion estimation. Thereafter, the cells were mixed with 2 ml of distilled water and a vortex was used for evenly mixing the sample solution. Subsequently, at an absorbance of 680 nm, biomass growth of all the samples was measured using a UV-visible spectrophotometer (Evolution 201, Thermo Fisher Scientific, USA) as detailed in some recent studies (Sarkar et al. 2022b).

Measurement of biomolecules (Carbohydrate, soluble protein, chlorophyll and lipid)

All the biomolecules were estimated using the procedures following the protocols in previous studies (Ansari et al. 2017, Ghosh et al. 2017a, Ghosh et al. 2017b, Herbert et al. 1971, Lichtenthaler 1987, Lowry et al. 1951, Sarkar et al. 2023, Sarkar et al. 2022a, Sarkar et al. 2022b). Carbohydrate accumulation, within the cells of Desmodesmus subspicatus was estimated by utilizing the phenol-sulfuric acid method (Sarkar et al. 2022a). Protein accumulation was estimated as per Lowry’s method (Lowry et al. 1951). Moreover, the total chlorophyll content of Desmodesmus subspicatus was examined using the procedure detailed in Lichtenthaler et al. (Lichtenthaler 1987). Lipid estimation was conducted by utilizing the Sulfo-phosphor-vanillin (SPV) method (Sarkar et al. 2023).

Arsenic quantification

The supernatant gathered after the microalgal cultivation and biomass separation (as briefed in section 2.3), was used for As(III) estimation. 15.2 ml of sample after suitable dilution was taken in a test tube and concentrated HCl (0.2 ml) was added. The As(III) concentrations to obtain the standard curve were prepared using sodium arsenite (NaAsO₂) salt. The resultant sample mixture was used to measure the total arsenic (which contains only As(III))concentration using Atomic Absorption Spectrophotometer (AAS, Model-iCE 3000 Series, Thermo Scientific, USA) in vapour phase mode. Subsequently, the removal percentage, metal uptake (mg/g-biomass) was determined using the equations given as follows:

Removal % = [(C₀-C_i)/ C₀] * 100% (4)

As(III) uptake (mg/g-biomass) = (C₀-C_i)/w (5)

Where, C₀, C_i are initial and final concentrations of arsenic in mg/L, w denotes the dry weight of biomass (g) obtained after cultivation time.

Chromium quantification

The collected supernatant obtained after centrifugation of the microbial culture was used for estimating the chromium concentration that remained. Carbazide-acetone solution has been used as an indicator for measuring the absorbencies of the samples at 540 nm in a spectrophotometer (Basak et al. 2018, Nag et al. 2016). The Cr(VI) concentrations to obtain the standard curve were prepared using potassium di-chromate (K₂Cr₂O₇) salt. Thereafter, final concentrations, removal percentage of Desmodesmus subspicatus and chromium uptake (mg/g-biomass) were determined utilizing the equations (4 and 5).

Variation of growth with initial As(III) and Cr(VI) concentrations

Experimental results depicted the biomass formation after 15 days of microalgal growth of (Desmodesmus subspicatus) in the presence of As(III) (0.5 to 15 mg/L) (Fig. 1a) and Cr(VI) (0.2 to 1 mg/L) (Fig. 1b) in the growth media. Growth of the biomass declined consistently with ascending As(III) concentrations and the least growth was recorded at 1 mg/L (40% decrease compared to control). Afterwards, the growth increased with ascending metal concentration up to 10 mg/L and growth ceased at 15 mg/L initial As(III) concentration. The growth of microalgal species was generally affected by the generation of reactive oxygen systems (e.g. hydrogen peroxide) which was triggered in the presence of As(III) (Arora et al. 2017). The ‘U’ shaped growth response of Desmodesmus subspicatus with increasing As(III) concentrations could be the result of the increasing response of enzymes (antioxidant) like superoxide dismutase (SOD), malondialdehyde and catalase (CAT) (Rai et al. 2013). SOD is a necessary component for the anti-oxidative defense process, capable of changing O^− 2 to H₂O₂ at a very fast rate. APX is highly attracted to hydrogen peroxide, scavenging trace amounts of H₂O₂ in more specified zones within cells. CAT is a major ROS-scavenging enzyme, that promotes the disproportion reaction of H₂O₂ in H₂O and O₂ (Rai et al. 2013).

It could be noted from Fig. 1b, that the biomass formation (after 15 days) of Desmodesmus subspicatus was almost unaffected in the presence of Cr(VI) at low (0.2 mg/L) concentration compared to the control (biomass grown at BG-11 media). At low concentrations (0.2 mg/L of Cr(VI)), the metal removal occurred primarily by the process of biosorption without much influencing the intracellular metabolic activities within the cells of Desmodesmus subspicatus (Danouche et al. 2020). However, with growing values of initial Cr(VI) concentrations, its toxicity accelerated the oxidative stress within the cells, which influenced the intracellular metabolism by generating a reactive oxygen system (H₂O₂, O₂^-) (Danouche et al. 2020). Therefore, it resulted in a descending trend of microalgal growth with growing Cr(VI) concentrations. Moreover, the growth came to an end at a Cr(VI) concentration of 1 mg/L and declined by almost 90.7% compared to the control (p < 0.0005). Moreover, enzymes like chromium reductase can inhibit the oxidative stress by reducing harmful Cr(VI) compounds to less toxic Cr(III) compounds that were secreted by the microalgal species (Kamaludeen et al. 2003).

Variation in carbohydrate accumulation with initial concentration of As(III) and Cr(VI)

The consequences of initial concentrations of As(III) and Cr(VI) imparted on the carbohydrate accumulation within microalgal cells were investigated by measuring their carbohydrate content at different initial arsenic and chromium concentrations (Fig. 2) after microalgal growth (15 days). It could be observed, that the carbohydrate content within the microalgal cells declined, when initial As(III) concentration increased from 0 to 0.5 mg/L. Carbohydrate production of the microalgal cells was affected by the oxidative stress within the cells which might resulted in the synthesis of reactive oxygen system (ROS) like superoxide anion (O₂⁻ ), hydrogen peroxide (H₂O₂) and hydroxyl radical (HO) (Mahana et al. 2021). Increased arsenic concentration increased the oxidative stress of the cells, depleting the carbohydrate content by almost 50% at 0.5 mg/L of As(III) concentration. Microalgae possesses ROS-depleting mechanisms that include the generation of antioxidant enzymes like SOD, malondialdehyde, CAT, APX, GR and GPOX (Danouche et al. 2020). Studies have shown a bell-shaped response (arsenic concentration 0.5–10 mg/L, Fig. 2a) in the production of malondialdehyde, ascorbate peroxide and superoxide dismutase (SOD) when microalgal cells are exposed to heavy metals (Rai et al. 2013). The initial decline in carbohydrate accumulation (at 0.5 mg/L) might be due to the oxidative stress generated by As(III) toxicity. Furthermore, at a higher initial As(III) concentration (1 mg/L), arsenic toxicity might have stimulated increased synthesis of anti-oxidant enzymes thus producing more carbohydrates. Additionally, a decline in carbohydrate content at 5 and 10 mg/L initial As(III) concentration could be the result of increased ROS synthesis. Higher carbohydrate production (15 mg/L initial As(III) concentration) by microalgal cells could be a defensive strategy of Desmodesmus subspicatus against the arsenic(III) stress.

Interestingly, the trend of carbohydrate accumulation on the microalgal cells showed a declined carbohydrate content with an increasing initial concentration of Cr(VI). The highest carbohydrate accumulation was noted when Desmodesmus subspicatus was grown in BG-11. At the exposure of 0.2 Cr(VI) concentration, carbohydrate accumulation declined by almost 23%, when compared with the control. The highest decrease (43%) in carbohydrate accumulation by the Desmodesmus subspicatus was noted when grown at 0.8 mg/L Cr(VI) concentration. The oxidative stress produced by the cells due to the increasing toxicity of Cr(VI) metals could be the probable reason for declining carbohydrate accumulation. Carbohydrate is the primary source of energy required for various steps of bioremediation mechanism (membrane transport, sequestration). Carbohydrates could be incorporated simply by initiating biochemical reactions to create energy in the form of adenosine triphosphate (ATP) (De Coen et al. 2001).

Variation in protein accumulation with initial As(III) and Cr(VI) concentration

A maximum surge in protein accumulation (~ 27% compared to control) after 15 days of microalgal growth was observed at 0.5 mg/L Cr(VI) concentration (p < 0.005) (Fig. 3a). Thereafter, the protein content was almost stable with increasing As(III) concentration up to 5 mg/L As(III) concentration. This increase in protein content with increased As(III) might be the self-protecting mechanism of microalgal cells by forming protein-As(III) complexes, synthesizing thiol-rich proteins and phytochelatins to fight the stress generated by the toxicity of As(III) (Gómez-Jacinto et al. 2015, Priatni et al. 2018). At 10 and 15 mg/L As(III) concentration, a declining protein accumulation was observed (~ 50% compared to control) (p < 0.005). The decrease in protein accumulation might be the outcome of the oxidative stress, triggered by the toxicity of arsenic. This stress might resulted the generation of ROS, which has given rise to the depletion of cells, carbohydrates and proteins (Pawlik-Skowrońska et al. 2004).

Protein content of the Desmodesmus subspicatus (after 15 days of biomass growth) grown on different Cr(VI) concentrations showed a similar trend ((Fig. 3b). It was noted that with increasing Cr(VI) concentration, the protein content in the cells has shown a growing trend. Further, the change in protein accumulation (at 0.6, 0.8, 1 mg/ L Cr(VI)) was almost negligible. Maximum protein accumulation (~ 1.5 times) was observed at 0.8 mg/L Cr(VI) concentration compared with the control. Therefore, increasing the synthesis of proteins (such as phytochelatins, heat-stable proteins, heat-denaturatable proteins) by the cells of Desmodesmus subspicatus could be the possible action of the microalgal species to decrease the toxic effects of Cr(VI) by forming complexes (Aharchaou et al. 2017, Gómez-Jacinto et al. 2015, Priatni et al. 2018). Some proteins (phytochelatins) convert toxic metal ions into harmless metal complexes by forming protein-binded metal complexes, called chelation (Arora et al. 2017). Therefore, it seems that the cells started to produce more protein to encounter excessive toxicity.

Variation in chlorophyll accumulation with initial As(III) and Cr(VI) concentrations

The chlorophyll content of Desmodesmus subspicatus (after 15 days of growth) grown at varying metal concentrations (chromium and arsenic) was evaluated and represented in Fig. 4. From the figure, it could be noted that chlorophyll content increased up to As(III) concentration of 1 mg/L (p < 0.005). The enhanced chlorophyll accumulation within the microalgal cells at higher As(III) concentrations may be due to the chlorophyllase synthesis being inhibited by the microalgal cells which enhanced the chlorophyll synthesis and prevented chlorophyll degradation (Zhang et al. 2013). However, at higher As(III) concentrations (from 5–15 mg/L), the chlorophyll content declined by almost 41–66%. The heavy metal's toxicity affected the microalgal cells' photosynthesis activity. The ROS synthesized due to stress caused by toxic heavy metal probably resulted in the accumulation of H₂O₂ and degraded the chloroplast of the microalgal cells (Arora et al. 2017).

The chlorophyll content of the cells after 15 days of growth at varying initial Cr(VI) concentrations did not vary significantly (Fig. 4b) signifying chlorophyll content was least influenced by the toxicity of Cr(VI). Peroxidation of chloroplast is the primary reason for the decline in chlorophyll content within the cells due to increased toxicity of Cr(VI). APX acts as a scavenger to H₂O₂ produced primarily in chloroplast and to maintain the cell's redox state (Rai et al. 2013). Also, the negligible change in chlorophyll content could result from higher protein production by the microalgal cells at higher concentrations of Cr(VI). Some proteins may act as a medium for metal binding (phycochelatin) and act as an antioxidant, reducing the toxicity of heavy metals (Arora et al. 2018).

Variation in lipid accumulation with initial As(III) and Cr(VI) concentrations.

The lipid content of Desmodesmus subspicatus grown in different As(III) concentrations containing BG-11 for 15 days was depicted in Fig. 5a. It has been observed that with ascending concentration of arsenic(III), the lipid content on the cells initially showed a declining trend with growing initial As(III) concentration. Minimum lipid content was recorded at 15 mg/L initial As(III) concentration and declined by ⁓64.2% (when compared with the control). Oxidative damage of lipid biomolecules caused by toxicity of As(III) could be the significant reason for declining lipid content at 10 and 15 mg/L initial As(III) concentration (Xiao et al. 2023).

Notably, lipid accumulation increased (⁓32% compared with the control) at an initial Cr(VI) concentration of 0.2 mg/L (p < 0.01) (Fig. 5b). Afterwards, lipid accumulation within the cells of Desmodesmus subspicatus declined with increasing Cr(VI) concentration. Minimum lipid accumulation was recorded at a Cr(VI) concentration of 1 mg/L and decreased by ⁓80% compared with the control. Lipids consist of fatty acids and their increased amount of generation within Desmodesmus subspicatus could be included as their self-protection strategy to overcome the cellular distress created by low Cr(VI) exposure (0.2 mg/L) (Bashir et al. 2021). Thereafter, the decrease in lipid accumulation might be the result of the peroxidation of lipid biomolecules caused by the oxidative stress generated because of the Cr(VI) toxicity (Xiao et al. 2023).

Removal efficiency of As(III) and Cr(VI) and metal uptake capacity of Desmodesmus subspicatus with initial metal concentrations

Removal efficiency and metal uptake of chromium and arsenic by Desmodesmus subspicatus after 15 days of microalgal growth was represented in Fig. 6. From Fig. 6a, it was observed that with the growing initial As(III) concentration, the removal efficiency of Desmodesmus subspicatus declined and was minimum (⁓32%) at an As(III) concentration of 15 mg/L. Additionally, the highest final As(III) concentration (~ 10 mg/L) was at 15 mg/L initial As(III) concentration. Moreover, the metal uptake by Desmodesmus subspicatus in remediating As(III) showed an increased trend with growing initial metal concentration. It can be observed that the (Fig. 6a) that the maximum arsenic uptake was 10.07 mg/g-biomass, at an exposure of 15 mg/L initial As(III) concentration. As(III) primarily exists in the form of H₃AsO₃ at a pH ranging between 8 and 8.8, which was the pH range of the present study (Arora et al. 2017). Intracellular metabolism of As(III), includes As(III) oxidation, complex formation with thiol compounds and sequestration into vacuoles, biomethylation of As(III) methylarsenicals (MA, DMA, and TMA) and excretion from cells are the probable detoxification mechanisms (Cullen et al. 1994, Rahman &Hassler 2014, Rahman et al. 2014). Therefore, at higher As(III) concentrations, the stress generated within the microalgal cells due to the toxicity of As(III) damaged various cell organelles (mitochondria, chloroplast) which in turn imbalanced the stability of microalgal cells. Due to declining growth, the surface biosorption of As(III) declined with increasing initial concentration.

The removal efficiency of Desmodesmus subspicatus for Cr(VI) removal was evaluated. It could be noted that the percentage removal of Desmodesmus subspicatus increased with growing initial Cr(VI) concentration. Moreover, the percentage removal of Cr(VI) was maximum (⁓95%) at a chromium concentration of 1 mg/L and least (⁓50%) at a chromium concentration of 0.2 mg/L. Additionally, the metal uptake by Desmodesmus subspicatus in remediating Cr(VI) increased with the growing initial metal concentration. From Fig. 6b, it can be observed that the maximum chromium uptake was ⁓0.94 mg/g-biomass, at an exposure of 1 mg/L initial Cr(VI) concentration. Functional groups that were majorly found accountable for chromium adsorption were alkyl chains, amine, amide, alcoholic groups, carboxylic groups, aldehydes complexes, halide compounds, sulfoxide and phosphate (Leong &Chang 2020). In a study, it was observed that with ascending values of initial Cr(VI) concentration (10.6 to 21.2 mg/L), the removal efficiency of Botryocossuss sp. NJD-1 increased, when grown in co-contaminated (organics and chromium) wastewater (Shen et al. 2019). Therefore, the increase in removal efficiency of Desmodesmus subspicatus can be justified by analyzing the increased protein content at higher Cr(VI) concentrations. Additionally, to mitigate the toxicity of Cr(VI), remediation was executed by the microalgal cells by forming Cr(VI) complexes. Desmodesmus subspicatus has shown almost 1.6 times better binding and metal accumulation capacity for Cr(VI) compared to As(III). Therefore, it could be noted that Desmodesmus subspicatus was more efficient in removing Cr(VI) as compared to As(III), when grown under similar environmental conditions.

Growth kinetics of Desmodesmus subspicatus and removal efficiency of As(III)

The kinetics and removal percentage of As(III) by Desmodesmus subspicatus, were executed for 30 days in a media containing an initial As(III) concentration of 10 mg/L (Fig. 7). At 10 mg/L initial As(III) concentration, the growth of Desmodesmus subspicatus was closest to that of control. It has been observed that the influence of As(III) on the growth of Desmodesmus subspicatus was much less since the growth curves of control and under the exposure of As(III) were almost the same (Fig. 7a). Moreover, biomass production and removal percentage of As(III) was increasing with time. Therefore, further studies could be done by increasing the cultivation time of Desmodesmus subspicatus. It could be predicted that a significant amount of As(III) diffusion into the cell cytoplasm was not happening through the microalgal cells. Alternatively, to nullify the toxic effects of As(III) and balance the structure of the cells, protective actions were executed by the microalgal cell by producing more proteins (SOD, CAT, APX, GR) at higher As(III) concentrations. These produced proteins act as antioxidant enzymes and helps in decreasing the carcinogenic effects of As(III) by forming complexes (protein-As(III)) and acting as a chelator.

Percentage removal and metal uptake of Desmodesmus subspicatus were evaluated and presented in Fig. 7b. It could be observed that with increasing cultivation time (days), the percentage removal of As(III) was increased steadily and reached 87% after 30 days of cultivation. Metal uptake was increasing with cultivation time and was at its peak on the 6th day of cultivation. Thereafter, it was declining steadily with days and least uptake was recorded at 30th day.

Growth kinetics of Desmodesmus subspicatus and As(III) removal

Similar to As(III), the growth kinetics of Desmodesmus subspicatus cultivated in 0.6 mg/L of initial Cr(VI) concentration was compared with the control (Fig. 8a). The growth of Desmodesmus subspicatus, grown in Cr(VI) environment was less as compared to control. The biomass growth of Desmodesmus subspicatus was influenced by the existence of chromium ions in the culture media. The growth rate of Desmodesmus subspicatus growing under control, decreased consistently with increasing days and the lowest growth rate was observed after 30 days. During the first three days, a decline in growth rate (~ 17.8% less compared to control) was observed.

The removal percentage and metal uptake of Cr(VI) by Desmodesmus subspicatus were represented in Fig. 8b. It could be noted that around 51% of the removal of chromium happened within the first 3 days and reached around 70% within 6 days of microalgal cultivation. Thereafter, it reached to steady state and maximum removal (75%) was achieved at 30 days Moreover, the metal uptake was maximum (~ 8.5 mg/g-biomass) within 3–6 days of cultivation followed by a decreasing trend. This result may be due to the availability of functional groups, that got saturated after six days of cultivation. Afterward, the rate of bioremediation decreased with the number of days, resulting in a steady slow variation in removal percentage (73% in 15 days and 75.7% in 30 days with the cultivation time.

The study demonstrated the capability of Desmodesmus subspicatus for remediation of As(III) and Cr(VI) and change in the accumulation of biomolecules within the microalgae cells. Further, the removal efficiency of Desmodesmus subspicatus for effectively removing various concentrations of Cr(VI) and As(III) was investigated. The isolated Desmodesmus subspicatus survived the toxic exposure of up to 10 and 0.8 mg/L of As(III) and Cr(VI) concentration respectively. The toxicity stress caused by Cr(VI) and As(III) was adjusted by remodeling the biomolecule compositions within the cells (carbohydrate, protein, chlorophyll, lipid). Biomass production under chromium and arsenic exposure has decreased by almost 90% as compared to control. Protein content increased by almost 49% under the exposure of Cr(VI) and As(III). The chlorophyll accumulation increased by almost 39% under As(III) exposure. The lipid content of Desmodesmus subspicatus increased by almost 32% when grown under exposure of Cr(VI). After 30 days of growth, the microalgal strain detoxified about 87% and 76% of As(III) and Cr(VI) respectively. This approach exhibits dual benefits, which include the removal of harmful metals (trivalent arsenic and hexavalent chromium) from wastewater sources, along with a high yield of proteins and pigments. This study has provided insight into the possible increased accumulation of proteins and pigments even at higher As(III) and Cr(VI) concentrations and could be explored more using the microalgal strain (Desmodesmus subspicatus). Additionally, a good amount of As(III) and Cr(VI) was remediated. Therefore, this combined research holds a strong scope for large-scale cost-effective phycoremediation of wastewater along with the production of value-added substances like proteins and pigments.

Acknowledgments

AG thankfully acknowledges the Ministry of Human Resources and Development (MHRD), India for the scholarship of PhD work and support provided by National Institute of Technology Agartala, India

Authors Contribution

Anisha Ganguly and Kalyan Gayen contributed to the conception and design of this work. Material preparation, data collection and analysis were performed by Anisha Ganguly and Kalyan Gayen. The first draft of the manuscript was written by Anisha Ganguly. Soma Nag, Tridib Kumar Bhowmick and Kalyan Gayen reviewed the manuscript. All authors read and approved the final manuscript.

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Conflict of Interest

Authors declare that there is no competing financial and/or non-financial interests for this work.

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Phycoremediation of As(III) and Cr(VI) by Desmodesmus subspicatus: Impact on growth and biomolecules (carbohydrate, protein, chlorophyll and lipid) – A dual mode investigation

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Abstract

Figures

Introduction

Materials and Methods

Microalgal strain and chemicals

Experimental outline

Measurement of growth

Measurement of biomolecules (Carbohydrate, soluble protein, chlorophyll and lipid)

Arsenic quantification

Chromium quantification

Results and Discussion

Variation of growth with initial As(III) and Cr(VI) concentrations

Variation in carbohydrate accumulation with initial concentration of As(III) and Cr(VI)

Variation in protein accumulation with initial As(III) and Cr(VI) concentration

Variation in chlorophyll accumulation with initial As(III) and Cr(VI) concentrations

Conclusion

Declarations

References

Supplementary Files

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