Treatment of the Allura Red food colorant contaminated water by a novel cyanobacterium Desertifilum tharense

Cyanobacteria are photosynthetic prokaryotes, also called blue-green algae, and at least 2,654 newly isolated species have been reported all over the world, extensively living in the water systems (Kenne & Merwe 2013). Members of the Cyanophyceae class have chlorophyll, which can convert sunlight energy into chemical energy (Barsanti & Gualtieri 2014). Recently, there has been an increased interest in studies on the usability of aquatic microorganisms such as cyanobacteria as biosorbent in environmentally friendly biological wastewater treatment technologies (Tastan et al. 2017; Kumar et al. 2019).

Allura Red (AR) is an extensively used colorant/dye in different industries such as food, medical, and cosmetics (Alkahtani et al. 2017), as well as most of the food coloring applications such as coloring bakery products, meat, and fish products, soft drinks, and jams (Al-Degs 2009).

Recent studies indicated that pollutants in wastewater released as a result of industrial activities harm aquatic organisms in water environments (Töre et al. 2021). Since the wastewater of these applications contains a high amount of colorants, which can be hazardous for aquatic organisms and eventually for humans, it is unavoidable to treat the colorant contaminated effluents (Nguyen et al. 2020; Majhi et al. 2021). Compared with chemical and physical treatment technologies, biological treatment technologies are preferred because of their eco-friendly nature (Mnif et al. 2016; Tastan & Tekinay 2016).

The main purpose of the study is to identify newly isolated cyanobacteria using 16S rRNA sequencing analysis, and then, to investigate the food colorant biosorption performance of newly isolated and identified cyanobacteria including isotherm, kinetic and thermodynamic studies. As a last objective, we intended to characterize the biosorbent before and after dye biosorption by Fourier transform infrared (FTIR) and scanning electron microscopy (SEM) analyses. Unlike other biosorption studies with the cyanobacterial originated biosorbent, the effect of the dye biosorption process on chlorophyll content was examined in the present study. The cyanobacterium called Desertifilum tharense, which was newly isolated from water samples in Turkey, was reported as the successful food colorant biosorption.

The synthetic food colorant called Allura Red (AR) and all other chemicals were obtained from Merck (Germany). The stock solution was prepared by dissolving 200 mg of AR dye in 100 mL deionized water.

A novel cyanobacterium, which is used as a biosorbent to remove food colorant in water samples, was isolated from Denizli-Buharkent geothermal province located in Central Anatolia Region (Turkey).

In order to isolate cyanobacterial species from the water samples, BG11 agar-medium in the petri dishes was used (Rippka 1988). The samples were spread on the dishes and incubated under cool-white fluorescent, 25 μmol m₋₂ s₋₁ (1750 lx) light intensity at 25±2 °C temperature. The medial pH was adjusted to 8 using either the solutions of sulfuric acid or sodium hydroxide (0.01 M and 1 M, respectively). The incubated microbial cells of the colonies were isolated with micromanipulation from the Petri dishes. At the aseptic conditions, the purification of cyanobacterial cells was performed by proceeding with the repeated inoculations on BG11 medium agar. After obtaining a pure culture, the cyanobacterial cells were inoculated into the liquid medium.

The molecular identification method was applied to determine the new isolate. The genomic DNA was extracted from the cells harvested from an exponentially growing culture using GenJet Genomic DNA Purification Kit, according to the manufacturer's protocol. 16S rRNA is the elucidative region for isolate identification. The forward primer (106F: 5′-CGGACGGGTGAGTAACGCGTGA-3′) and the reverse primer (781R(a): 5′-GACTACTGGGGTATCTAATCCCATT-3′) were used to amplify the 16S rRNA region (Burja et al. 2001). The sequencing was performed in Applied Biosystems Genetic Analyzer 3130. The PCR reactions were carried out in a final volume of 50 μL containing 100 ng of purified DNA, 2.0 μL of MgCl₂ (2.0 mM), 1.5 U of Taq polymerase, and 0.3 μM of each forward and reverse primer. Amplifications were programmed on the thermocycler (Applied Biosystems Gene AMP PCR System 9700), at the following conditions: denaturation at 95 °C for 5 minutes, then 35 cycles of 1 minute at 94 °C, 1 minute at 60 °C, and 1 minute at 72 °C.

After all these processes the obtained sequences were compared to the reference sequences at National Center for Biotechnology Information (NCBI) by using an online program called The Basic Local Alignment Search Tool (BLAST 2020).

The pure culture of isolated Desertifilum tharense, a member of prokaryotic blue-green algae (cyanobacteria), was stored at the Culture Collection of Microalgae in Vocational School of Health Services, Gazi University, Ankara, Turkey. BG 11 culture medium (100 mL) was used for microalgal (cyanobacterial) incubation (Rippka 1988) in the Erlenmeyer flasks.

With the intention of large-scale microalgal (cyanobacterial) biomass production, sterile plastic reactors (with 5 L volume) containing BG11 were used. The application of dilute sodium hypochlorite and/or ethanol solutions was performed for sterilization. The motors with 8 W working capacity and 2–4 outputs were also administered for ventilation in the reactor system (Gül et al. 2019).

The surface zeta potentials (ZP) of biosorbents after and before biosorption were measured with the electrophoretic method by the usage of Malvern Zetasizer Nano ZS (Phoenix, EDAX, Mahwah, NJ, USA). The dispersant was deionized water with pH 8.5 (the pH value of experimental conditions). The biosorbent was characterized by analyses of FTIR and SEM. FTIR spectra of the biosorbents before and after biosorption of AR were measured in a Perkin Elmer 400 spectro photometer. SEM analysis was performed by using Leo 440 Computer Controlled Digital System.

In this equation: Q (mol kg⁻¹): adsorbed amount, C_e (mol L⁻¹): equilibrium concentration, X_m: the maximum adsorption capacity of the adsorbent, K_L: dispersion coefficient

In this equation: K_f: a measure of adsorption capacity, β: adsorbent surface heterogeneity

The value of E (kJ mol⁻¹) indicates whether the adsorption mechanism is physical or chemical. If the adsorption energy is 8<E<16 kJ mol⁻¹, the adsorption is chemically controlled, and E<8 kJ mol⁻¹ indicates that the adsorption proceeds physically.

In order to gain information about the cyanobacterial morphology, the living cyanobacterial biomass was viewed under a microscope (Zeiss-Primo Star). The morphological properties of the cyanobacterium were investigated as well.

In the previous studies, chlorophyll a, chlorophyll b, and chlorophyll (a + b) contents were frequently examined to evaluate the change in the physiological state of lichen (a symbiotic lifestyle of a fungus as mycobiont and an alga or a cyanobacterium as photobiont) under various environmental stress conditions (Boonpragob 2002). However, little is known about the effect of textile dye on chlorophyll concentration in photosynthetic organisms such as algae (Copaciu et al. 2013; Rane et al. 2014). D. tharense is a filamentous cyanobacterium (Oscillatoriales), with a trichome width size of 2–3.7 μm in diameter (Dadheech et al. 2012). The chlorophyll concentration of D. tharense was determined according to the method developed by Porra et al. (1989) at 646.6 nm for chlorophyll a. The chlorophyll concentrations were expressed in μg of chlorophyll per milliliter.

A newly isolated blue-green alga (cyanobacterium) was identified by molecular techniques and then the food colorant removal properties of this microorganism were investigated. The calculation of biosorption isotherms and kinetics, and also biosorbent characterization studies such as ZP, FTIR, and SEM before and after the biosorption process were carried out. Also, the changes in cyanobacterial physiology after the biosorption process were examined.

According to the information obtained from the BLAST program, the new cyanobacteria isolate was identified as Desertifilum tharense from 16S rRNA sequencing. The classification of this species is also submitted in Supplementary material 1 (Dadheech et al. 2012).

The morphology of the cyanobacterium was observed under the light microscope and the micrograph of the cyanobacterium is given in Figure 1. While the morphology of D. tharense was investigated, the cyanobacterial biomass had a multicellular filamentous structure similar to other species belonging to the order Oscillatoriales (Martins & Branco 2016). The morphological characteristics of the isolated cyanobacterium were observed as having thin thallus, densely entangled filaments, agglutinated trichomes, and cylindrical cells having homogeneous content (Figure 1). The cell wall lies between the plasma membrane and the mucilaginous sheet and the filaments were simple, which was previously explained for the appearance of other cyanobacterial cells by Barsanti & Gualtieri (2014). Similarly, Dadheech et al. (2012) isolated a cyanobacterium strain from Rajasthan, India, and identified this cyanobacterium as Desertifilum tharense. The cyanobacterium species newly isolated in this study showed a similar morphology identified by Dadheech et al. (2012). The algae/cyanobacteria had a characteristic green color due to the pigment Chlorophyll-a, which was responsible for photosynthesis (Sinetova et al. 2017).

The effect of pH was examined via the variety of pH values from 1.0 to 12.0 and as seen in Figure 2. The decolorization rate was not significantly changed from pH 4.0 to 8.5. The pHs at the point of zero charges (pHpzc) of blue-green algal strains were reported in the range of 4.6–8.5 in the literature (Martinez et al. 2008; Mishra & Mukherji 2012; Dotto et al. 2013). Similarly, the colorant biosorption rate was sharply decreased after pH 8.5 due to the pHpzc of the cyanobacterium strain (Figure 2(a)). The optimal pH was selected as 8.5 in the present study.

The effect of cyanobacterial biosorbent dosage on food colorant biosorption was tested with a variety of biosorbent closes in the range of 10–200 mg. The increase of biosorbent dosage caused the augmentation of the dye biosorption rate. Recently, Gül et al. (2019) showed that acid red textile dye biosorption of another algal species called Phormidium animale was increased by the increment of dye concentration. Similarly, Bayazit et al. (2020) reported that Remazol Black B textile dye removal by P. animale biosorbent was raised with the increasing biosorbent dosage. These increasing patterns were explained by the raising of available biosorption sites on the surface of the biosorbent (Saeed et al. 2010). The maximum AR biosorption was found 97% with 200 mg biosorbent dosage in a 10 ml working solution (Figure 2(b)).

In order to obtain more detailed information about the biosorption of AR by cyanobacterial biosorbent, the biosorption isotherm models were derived from the biosorption experiments. The graphs of biosorption isotherm models are given in Supplementary material 2.

The calculations of R² values from Langmuir and Freundlich biosorption isotherm models showed that biosorption of food colorant AR was fitted with the Langmuir isotherm model (Table 1). The maximum biosorption capacity was found to be 0.345 mol kg⁻¹, and the biosorption energy was found to be 5.73 kJ mol⁻¹, which indicated that the biosorption process was physical.

The experimental data was used to understand the fitted kinetic model for biosorption (Supplementary material 3). In the experimental series, it was observed that the biosorption process was stabilized after 420 minutes. According to the results of kinetic calculations, the biosorption process was better adapted to the pseudo-second-order kinetic model due to the correlation coefficients (Table 2).

At the same time, when the experimentally calculated Q_t values and the theoretically calculated Q_e values were compared, the results found from the pseudo-second-order kinetic model were found to be closer to each other. These results indicated that the biosorption process followed the pseudo-second-order kinetics.

The effect of temperature on the food colorant by cyanobacterial biosorbent was determined at 5 °C, 25 °C, and 40 °C. The increasing temperature caused decreasing biosorption rate (Supplementary material 4), which was reported as a characteristic phenomenon of physical adsorption (Aksu & Tezer 2005). And this situation also supported the results of D-R calculations derived in this study. The calculations of biosorption thermodynamics showed that the biosorption of food colorant by cyanobacterial biosorbent was endothermic, entropy increased, and spontaneous (Table 3).

To have more detailed information about the colorant and biosorbent interactions, ZP and SEM analyses were performed. The surface zeta potentials (E_zeta) of the cyanobacterial biosorbents before and after biosorption were measured as 0.226 and 0.140 mV at pH 8.5 (the pH of experimental conditions), respectively. After the AR biosorption process, the positively charged value of the biosorbent was decreased due to the negative charge of the anionic dye. The change in the E_zeta indicated the sorption of dye molecules onto the surface of the cyanobacterial biosorbent. In Figure 2(a), the colorant removal decreased sharply after pH 8.5 due to the changes in surface charge of the cyanobacterial biosorbent. Similarly, the pH values at the isoelectric point of Anabaena varialis and Prochlotrix hollandica, which were also filamentous cyanobacteria, were measured as 8.4 and 8.1, respectively (Davis et al. 1980; Babu et al. 1999). It is known that pH is the most important factor affecting the zeta potential of the cell surface (Martinez et al. 2008). It is assumed that while the pH increased from 8.5 to 12, the zeta potential was changed due to the isoelectric point of zero, which resulted in a decrease in decolorization.

The SEM micrographs of dried cyanobacterial biosorbent before and after the biosorption process are given in Figure 3(a) and 3(b), respectively. Recently, Hussein et al. (2018) reported that the surface morphology of a filamentous blue-green alga (Cyanobacterium) called Nostoc carneum was changed depending on the sorption of Methyl Orange dye after the biosorption process. Similarly, in this study, the surface morphology of the cyanobacterial biosorbent of D. tharense seems different after and before biosorption. The surface of cyanobacterial biomass was irregular and porous before biosorption (Figure 3(a)). After biosorption, the biomass particles were densely packed due to the presence of dye molecules that made a monolayer on the surface. A similar situation was shown by Kumar et al. 2019, who studied the biosorption of Orange G dye by the dried powdered algal biomass called Chlorella vulgaris. Also, Kiran et al. (2016) explained that the algal surfaces have a high affinity to the pollutants to form monolayers, which caused aggregations of the algal biomass. The SEM results of this study fitted with the recently published articles in the literature.

Fourier transform infrared (FTIR) analysis is a kind of vibrational spectroscopy, and the FTIR spectra reflect the changes of functional groups on the surface of cyanobacterial biomass after the biosorption process. The changes on the peaks before and after biosorption indicated the functional groups which were responsible for the colorant sorption (Figure 4). The peaks observed at 2,917.24 and 2,919.02 cm⁻¹ (before and after) corresponded to the C–H stretching vibration. The peak that appeared at 1,625.95 cm⁻¹ shifted to 1,620.39 cm⁻¹ after colorant sorption, indicating the involvement of C-O stretching in the biosorption process (Bhattacharya et al. 2014). The peak which appeared in the region of 1,546.92 cm⁻¹ represented protein amide II band (mainly δ(N-H) bending) on the surface of algal biomass (Theivandran et al. 2015), which disappeared after biosorption. The new peak at 1,174.60 cm⁻¹ occurring after biosorption, which was found in the frequency ranges of 1,250–1,020 cm⁻¹, represented the C–N s stretching vibration presence of aliphatic amines (Mona et al. 2011b). Also similarly, the peak at 1,024.48 cm⁻¹ before biosorption shifting to 1,032.04 cm⁻¹ after biosorption revealed the C–N s stretching. These changes on the surface functional groups of algae or cyanobacteria showed that there were some interactions between the surface of algal biosorbent and the colorant molecules after the biosorption process. The results of FTIR showed that the functional groups like hydroxyl, amide, and carboxylate groups found on the surface of cyanobacterial cells were mainly responsible for the biosorption of food colorant.

The ZP, SEM, and FTIR analysis indicated the adsorption of the colorant molecules on the surface of the biosorbent, and the thermodynamic and D-R model results showed that the biosorption occurred physically.

The cyanobacterial biomasses were used as biosorbents for the removal of dye molecules (Mona et al. 2011a, 2011b; Gül et al. 2019; Bayazit et al. 2020). According to our best knowledge, there is no study to determine the relationship between dye molecules and the chlorophyll content of cyanobacterium. The chlorophyll-a concentration of dried D. tharense culture was 3.7 μg mL⁻¹ before AR colorant biosorption. This concentration decreased to 3.06 μg mL⁻¹ after AR colorant biosorption onto the cyanobacterium biomass. It is assumed that the decrease of chlorophyll-a content indicated the interaction of dye molecules with chlorophyll-a on the surface of the cyanobacterium, and therefore the sorption of dye molecules affects the amount of chlorophyll-a.

In the last studies, the usage of cyanobacterial biomasses as the low-cost and eco-friendly biosorbents to remove a variety of dye molecules from wastewaters was investigated. Some of these studies about cyanobacterial or algal dye biosorption were reviewed and the results are given in Table 4. As seen in Table 4, our newly isolated cyanobacterial biomass called D. tharense performed a very successful decolorization rate at the highest initial dye concentration of 1,500 mg L⁻¹.

Industrial wastewaters contain varying concentrations of dyestuffs. Despite the high decolorization efficiency of some strains, they may fail to remove dyes and some environmental problems may arise. The biosorbent originated from D. tharense removed 97% of AR dye from an aqueous solution containing a high concentration of the dye (1,500 mg L⁻¹). According to our present knowledge, there isn't any study focusing on the dye biosorption potential of D. tharense identified in this study. The reviewed literature about dye removal by cyanobacterial or algal biosorbents (Table 4) showed that the identified cyanobacteria D. tharense in the present study performed dye removal capacity at approximately 10 times higher dye concentration than other biosorbents obtained from cyanobacteria or algae. The results of isotherm, kinetic, thermodynamic calculations, and characterization studies (FTIR and SEM) supported that the food colorant molecules physically adsorbed on the surface of biosorbent (D. tharense originated). It is concluded that the identified novel cyanobacterium was a useful candidate to remove high concentrations of food colorants from wastewater according to this preliminary lab-scale study. Further pilot-scale studies may show the utilization of this biosorbent in wastewater treatment plants.

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