Biosorption of pollutants from chemically derived wastewater using Microcoleus sp.

Microcoleus sp. is a suitable model organism since it is widely available, easily identifiable, isolable, and culturable (Dvořák et al. 2012). They can be found within soil crusts, freshwater epipelon, and other subaerophytic environments (Garcia-Pichel et al. 2001; Boyer et al. 2002; Komarek & Anagnostidis 2005; Hašler et al. 2012). They are mat-forming, unbranched, uniform, and filamentous/colonial cyanobacteria. Despite being so accessible, the use of this species has not been in the limelight. Microcoleus sp. has been studied for oil degradation and soil crust restoration (Sorkhoh et al. 1995; Al-Hasan et al. 1998; Lan et al. 2012; Lababpour & Kaviani 2016). A study by Paniagua-Michel & Garcia (2003) considers the applicability of constructed microbial mats, including Microcoleus sp., in removing nutrients from shrimp culture effluent. However, no studies have been found with this specific species to determine its independent and unassisted removal efficiency of heavy metals and nutrients from effluent.

Industries, municipal wastes, and agriculture, aided by industrialisation, urbanisation, and technological development, contribute to the entrance of toxic pollutants into the environment. Two common contaminants in the environment are heavy metals and nutrients. Heavy metals in industrial effluent are hazardous to the environment. Heavy metals are metallic elements with a fairly high density, which makes them toxic even at low concentrations. The heavy metals in this study are Cr⁶⁺, Ni²⁺, and Zn²⁺ since they are the most hazardous pollutants due to their complex behaviour (DoE, Bangladesh). Correspondingly, eutrophication is caused by the presence of nutrients (nitrogen and phosphorus) in the form of nitrate, nitrite, ammonia/ammonium, or phosphate (Kosaric et al. 1974; Chan et al. 2014).

The health hazards associated with heavy metals are headache, dermatitis, lethargy, cancer, organ damage, damage of the nervous system, and even death; and those with the excessive presence of nutrients are gastric cancer caused by the conversion of nitrate to nitrosamines in the gastrointestinal tract and methaemoglobinaemia which further leads to respiratory problems, digestive problems, and even death of children (Kelter et al. 1997; Glass & Silverstein 1999; Foglar et al. 2005; Mayo & Hanai 2014). However, phosphate is only responsible for eutrophication which harms the aquatic fauna. Hence, disposing of them directly into water bodies harms all life forms. The freshwater content on Earth is 2.5%, of which only 31% is potable. Moreover, the spiralling water demand, water pollution, and the increasing shortage of freshwater resources necessitate wastewater reuse for worldwide sustainable development (Falkenmark & Widstrand 1992; Greenway 2005).

The maximum contaminated level (MCL) standards set by US EPA (Babel & Kurniawan 2003) indicate the acceptable concentrations of heavy metals along with the upper limits of the Total Nitrogen (TN) and Total Phosphorus (TP) content when being discharged into water bodies (US EPA 2012; see Table 1).

Conventional methods of heavy metal remediation include membrane filtration, coagulation and flocculation, oxidation, photocatalysis, and adsorption; those for nutrient removal are filtration, facultative ponds, advanced oxidation, adsorption, coagulation, activated sludge process, ion exchange, soil percolation, and constructed wetlands. Contrary to their effective pollutant removal efficiencies, these methods are not positively adopted by industries or treatment facilities since they require massive operation and maintenance expenditure, land area, and proficient operators. Consequently, the cost-effective alternative of the microalgae-based wastewater treatment process is gaining popularity.

Most algae can physically adsorb heavy metal ions to their bodies at the cell surface, both dead and alive (Aksu 1998; Satya et al. 2020). The proteins, lipids, and carbohydrates create a net negative charge on the cell surface, allowing them to act as binding groups for the positively charged heavy metals (Crist et al. 1981; Khummongkol et al. 1982; Gardea-Torresdey et al. 1990; Aksu 1998). Correspondingly, the study conducted by Sandau et al. (1996) reinforces the theory of biosorption of heavy metals by microalgae using a combined mechanism of extracellular and intracellular accumulation (Crist et al. 1988; Kuyucak & Volesky 1990; Sandau et al. 1996). Additionally, due to the photosynthetic property of microalgae, they uptake nutrients from wastewater for their metabolism and cellular growth. Microalgae form organic nitrogen from inorganic nitrogen (peptides, proteins, enzymes, chlorophyll, adenosine diphosphate, adenosine triphosphate), while inorganic phosphate generates adenosine triphosphate from adenosine diphosphate (Martinez et al. 1999; Gualtieri & Barsanti 2014). Blue-green microalgae can efficiently uptake pollutants in the range of 56–100% (Sibi 2016; Daneshvar et al. 2019; Moreira et al. 2019; Arias et al. 2020; Pham et al. 2020; Rueda et al. 2020; Touliabah et al. 2022). These data reinforce that phycoremediation is an excellent choice for an eco-friendly alternative.

Microalgae remove heavy metals and nutrients through the process of biosorption. The advantages offered by this method, out of many, include reduced energy costs and required chemicals, utilisation of the harvested biomass in food, feed, fertilisers and chemical production, reduction of carbon footprint, and zero toxic sludge generation (Schumacher & Sekoulov 2002; Brinza et al. 2007; Pittman et al. 2011; Singh & Olsen 2011; Arbib et al. 2014; Imanthi et al. 2023).

This study aims to fill in the knowledge gap and evaluate the adsorption capacity of Microcoleus sp. to see if it may be a state-of-the-art asset in the bioremediation field. Therefore, the efficiency of Microcoleus sp. as a low-cost biosorption medium is tested and analysed to apprehend the effect of different microalgal doses and hydraulic retention times on the uptake of the pollutants.

The microalgae strain was collected in January 2021 from a pond near Shahjalal University of Science and Technology (Bangladesh, Sylhet, 24.9172° N, 91.8319° E). It was collected in a 1 L plastic bottle from which 100 mL was added to a synthetic BG-11 medium (Stanier et al. 1971). The BG-11 medium composition was as follows: K₂HPO₄·3H₂O 0.04 g/L, MgSO₄·7H₂O 0.075 g/L, CaCl₂·2H₂O 0.036 g/L, Citric acid 0.006 g/L, Ferric ammonium citrate 0.006 g/L, EDTA (disodium magnesium salt) 0.001 g/L, Na₂CO₃ 0.02 g/L, NaNO₃ 1.5 g/L, trace metal mix 1 mL/L; the trace metal mix consisted of H₃BO₃ 2.86 g/L, MnCl₂·4H₂O 1.81 g/L, ZnSO₄·7H₂O 0.22 g/L, CuSO₄·5H₂O 0.079 g/L, NaMoO₄·2H₂O 0.39 g/L, and CoCl₂·6H₂O 0.05 g/L. The environmental factors that impact the culture are temperature, pH, oxygen, light intensity, and CO₂. The surrounding temperature directly influences photosynthesis and the biochemical processes. Most algal species thrive at a temperature of 20–30 °C, a luminous intensity of 5,000–7,000 lx, and a pH of 6–8 (Béchet et al. 2017; Nouri et al. 2021). Photosynthesis is directly affected by extreme temperatures, pH, and light intensities. As a result, inhibition of microalgal growth occurs (Atkinson et al. 2003; Juneja et al. 2013; Krzemińska et al. 2014). Hence, the batch cultures were conducted in 1 L Erlenmeyer flasks for 7 days at 26 °C, pH of 7, light intensities of 5,000 lumens at a 16:8 photocycle (simulating natural daylight for synchronised light and dark phases), and continuous aeration using aquarium pumps for atmospheric CO₂ supply.

The algal strain was isolated by serial dilution 30 times. The dilution gave us fresh and pure microalgal biomass for mass culture. The mass culture was cultivated for 30 days under the same culture conditions. A large 60 cm × 20 cm tank with a capacity of 10 L was used for this purpose: a larger surface area allowed the mat forming Microcoleus sp. to expand easily. The growth rate of the microalgae in the mass culture tank had to be visually observed by its expansion and microscopic observation for healthy cells since the cells were not in a suspended state for spectrophotometric measurement. The microalgal strain was identified based on the morphological features of the species.

Chemically derived wastewater was used in this experiment. Usually, heavy metal ion concentrations in industrial effluents vary widely. However, a challenge in industrial effluent treatment is that low concentrations of heavy metals become difficult to remove and very few processes can successfully bring the levels below the MCL. Hence, to ascertain whether post-treatment is required, the Cr⁶⁺, Ni²⁺, and Zn²⁺ concentrations have all been kept at a low concentration of 20 mg/L to simulate industrial effluent. The stock solutions containing heavy metals and nutrients were prepared using K₂Cr₂O₇ for Cr⁶⁺ ion, NiCl₂·6H₂O for Ni²⁺ ion, ZnCl₂ for Zn²⁺ ion, K₂HPO₄·3H₂O for PO₄³⁻ ion, NaNO₃ for NO₃⁻ ion, and (NH₄)₂SO₄ for NH₄⁺ ion. For the representation of industrial wastewater, 20 mL of each metal solution was mixed to obtain 1 L of synthetic wastewater with a concentration of 20 mg/L each. To simulate the difficult condition of municipal wastewater, the PO₄³⁻ concentration was set at 170 mg/L, NO₃⁻ was set at 17 mg/L, and that of NH₄⁺ was set at 100 mg/L (de-Bashan 2002; Olguín 2003; Zhou et al. 2012; Chaitee et al. 2022) by taking 170, 17, and 100 mL, respectively, from each of the stock solutions and adding them together to get 1 L in total finally.

Laboratory-scale batch experiments were done with synthetic wastewater. The algal mats were harvested using a spatula and sieve cloth and then washed with distilled water. The excess water from the mats was removed using a vacuum filter. The biomass was then divided into the required doses. A magnetic stirrer was used to constantly stir the biomass within the reactor to maintain the mat evenly distributed. For each data collection (variable dose-constant HRT and variable HRT-constant dose), three 1 L solutions of synthetic wastewater were used. The hydraulic retention time (HRT) was fixed at 3 days for the first experiment set with variable algal dose, with doses ranging from 0.5 to 25 g/L. For the second experiment set with variable HRT, the dose was fixed at 20 g/L with HRT varying from 1 to 7 days. The environmental conditions remained the same as the culture period conditions, except the light was continuously provided during the entire treatment. The initial and final data were then collected spectrophotometrically using the HACH DR6000™ UV Spectrophotometer; the system programs for Chromium (Hexavalent), Nickel, Zinc, Ammonia (Nitrogen), Nitrate (Nitrogen), and Phosphorous (Total) were adopted. A dilution factor of 10 was used to detect all the contaminants since the spectrophotometer has specific detection ranges.

To express the feasibility of the treatment, biosorption modelling is done using the Langmuir and Freundlich isotherm models.

Moreover, analysis of variance (ANOVA) for a single factor is used to determine the statistical significance of the variable parameters on the output; all the tests are conducted for a significance level of α = 0.05. Finally, to judge the fitness of the models, the coefficient of determination (R²) and root-mean-square error (RMSE) is calculated. R² is a non-negative quantity, and its limits are 0 ≤ R² ≤ 1. If it is close to zero, it indicates that the prediction is not much improved by knowing the independent variable. While it moves away from 0 to 1, knowing the independent variable will be increasingly useful in predicting the dependent variable (Islam 2004). As the value of RMSE approaches zero, the modelled and the experimented data are defined to be increasingly closely fitted with each other; when it is zero, they are exactly fitted with each other.

Experiments were carried out in batches: three experimental setups for each variable. The average of the three readings was taken as C_e (mg/L). Additionally, the single-factor ANOVA test confirms the statistical significance of the varying algal doses and hydraulic retention times on the remediation efficiency.

The HRT was kept constant at 3 days, and the algal doses were taken as 0.5, 1, 3, 5, 7, 10, 15, 20, and 25 g/L. The removal efficiency of the biomass increased linearly with increasing dose in the case of Cr⁶⁺ and the maximum removal of 94% was found with 25 g/L of algal biomass. Similar results were found for Ni²⁺ and Zn²⁺, where 98% of Ni²⁺ and 97% of Zn²⁺ were removed at 15 and 25 g/L algal dose, respectively; also, 70% removal was found for PO₄³⁻ at 25 g/L. Unfortunately, this particular species of algae showed very poor to zero removal of NO₃⁻ and NH₄⁺. This can be explained by the study by Omoregie et al. (2004), where they found that Microcoleus sp. could not fixate nitrogen from the environment during the day.

The biosorption activity for all trends peaks on the 1st day (0.65 mg/g-day for Cr⁶⁺, 0.83 mg/g-day for Ni²⁺, 0.72 mg/g-day for Zn²⁺, and 4.22 mg/g-day for PO₄³⁻) (see Figure 2(b)), decreases steadily on the 2nd and 3rd day while a drastic reduction is observed from the 5th day onwards.

Langmuir isotherm model defines the monolayer adsorption feasibility and the Freundlich isotherm model defines surface heterogeneity. Since no significant data were found for TN, no further discussions will be made.

The novelty of this study is the initiatory use of Microcoleus sp. in the treatment of heavy metals and nutrients in wastewater. This specific species of blue-green microalgae can tolerate extreme temperature conditions and salinity. They can be found in arctic areas, deserts, and saline water bodies; therefore, using this species in those regions may bring revolutionary advancements. Microcoleus sp. has shown a promising removal efficiency of Cr⁶⁺, Ni²⁺, Zn²⁺, and PO₄³⁻. It could not remove the nitrogen content in the wastewater since the experiments were conducted with continuous lighting and they cannot perform nitrogen fixation during the day. Future studies may be carried out to validate the findings of this study.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.