Biomass characterisation and phylogenetic analysis of microalgae isolated from estuaries: Role in phycoremediation of tannery effluent

Algal Research 14 (2016) 92–99

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Biomass characterisation and phylogenetic analysis of microalgae isolated from estuaries: Role in phycoremediation of tannery effluent Balaji Sundaramoorthy, Kalaivani Thiagarajan 1, Shalini Mohan, Sankari Mohan, Priya Rajendra Rao, Siva Ramamoorthy, Rajasekaran Chandrasekaran ⁎,1 School of Bio Sciences and Technology, VIT University, Vellore 632014, India

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Article history: Received 5 August 2015 Received in revised form 7 December 2015 Accepted 30 December 2015 Available online xxxx Keywords: FTIR Microalgae Phylogenetic analysis Phycoremediation Tannery effluents

a b s t r a c t Chrome tanning activity has contributed intensively towards environmental pollution in the form of effluents. To study the specific contribution of microalgae towards phycoremediation, four heavy-metal tolerant microalgal species were isolated from the estuaries receiving tannery effluents, and their biomass was examined by their interaction. The sequences of the four microalgal species, namely Anabaena (VITMA1), Oscillatoria acuminate (VITMA2), Phormidium irriguum (VITMA3) and Spirogyra maxima (VITMA4), were clustered after isolating their ribosomal DNA. Phylogenetic analysis revealed that VITMA1 showed a distant relationship with Anabaena, having only 63% sequence similarities, and other species such as VITMA2, VITMA3 and VITMA4 showed 82%, 95% and 92% sequence similarities, respectively. Microalgal species were grown in BG11 media along with chromiumcontaminated tannery effluents for analysing their growth, biomass and protein contents. The binding site characterisation was assessed by Fourier transform infrared spectroscopy, nuclear magnetic resonance and X-ray diffraction spectral studies. The results obtained from these studies advocate that the interactions are due to the presence of alkynes and aromatic functional groups. Scanning electron microscopy analysis showed the presence of intact cells with chromium accumulation. The biosorption activity was found to be 90% for O. acuminate (VITMA2), followed by 80% for P. irriguum (VITMA3), 65% for Anabaena (VITMA1) and 55% for S. maxima (VITMA4), respectively, and proves their impressive potential for phycoremediation activity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Heavy metals are toxic substances, that are non-biodegradable, and non-essential and are adsorbed into the living organisms, which constitutes a global environment problem [1]. Chromium is one of the widely used heavy metals that exists in predominant oxidation states such as the hexavalent and trivalent states. Hexavalent chromium exerts a detrimental effect and poses hazardous health issues in humans [2–4]. Chromium pollution is caused by tanning of leather in tanning industries and includes other industrial activities. In India, particularly Vellore district of Tamil Nadu is well known for heavy-metal pollution because of tanneries and its associated industries. Ranipet is an industrial area that is about 120 km from Chennai and is one of the major tanned leather exporting places in India. The total number of industries in and around Ranipet is about 240, including tanneries and boiler auxiliary's plants. Surface water in this area is polluted due to the release of contaminated water from these industries. The concentration of heavy metals in surface water is above the normal limits (10 μg/L), due to ⁎ Corresponding author at: Plant Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India. E-mail address: [email protected] (R. Chandrasekaran). 1 Equally contributed.

http://dx.doi.org/10.1016/j.algal.2015.12.016 2211-9264/© 2016 Elsevier B.V. All rights reserved.

which the people in this area are affected and suffer with asthma, cancer and skin diseases [5]. Chromium remediation is usually achieved employing various physical and chemical processes, including coagulation, ion exchange, precipitation and adsorption [6]. These conventional treatment methods are often expensive and lead to chemical toxicity. To overcome this problem, numerous attempts have been made to develop a biological method using microorganisms for the remediation of heavy metals [7,8]. Microalgae adapt to stress by attaining homeostasis between their physiology and heavy metals present in the environment and can be used for remediation of heavy metals [9]. Microalgae have been used as adsorbents by various researchers to remove the heavy metals from industrial effluents [10]. Microalgae are successfully employed as biosorbents because they are cost effective, economic, eco-friendly and abundant and have metal-removal capability and a high generation potential [11–14]. Therefore, the significance of the current study is to explore the ability of microalgal isolates to grow in media containing tannery effluents with chromium. This is the first study to report the growth and chromium adsorption capability of microalgae isolated from estuaries receiving tannery effluents. A phylogenetic tree was constructed for species identification and the morphological changes in microalgal

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isolates with or without effluent treatment were studied through scanning electron microscopy. Characterisation studies like Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) analysis were carried out to study the interactions between the functional groups of microalgae with chromium present in the effluent. 2. Materials and methods 2.1. Isolation and identification of microalgae Microalgal samples were collected from estuaries of the Palar River near Vaniyambadi, Vellore district. The collected microalgal species were identified by Prof. V. Krishnamurthy (late), Director, Krishnamurthy Institute of Algology, Chennai, Tamil Nadu, India, with reference to the monograph of T.V. Desikachary [15].

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2.5. Microalgal growth and biomass Growth rate of Anabaena (VITMA1), Oscillatoria (VITMA2), Phormidium (VITMA3) and Spirogyra (VITMA4) were monitored turbidimetrically at 560 nm, and their doubling time along with specific growth rate was calculated. The formula used for calculating doubling time (td) was 2.303 ∗ (log day 2 − log day 1) / (day 2 − day 1) and specific growth rate (μ) was 0.693/td = μ [24,25]. Biomass content of all the microalgal species was estimated using the formula (weight [g/L] = OD primary ∗ 0.238 [empirical data, dependent on cell strain, etc.]) [26]. 2.6. Total protein content Protein estimation was carried out following Lowry's method with modification [27]. Samples were collected on different days, and the amount of protein present in the samples was expressed in μg/mL. 2.7. Scanning electron microscopy

2.2. Collection of tannery effluent Tannery effluent was collected from the Ranipet industrial area of Vellore district. The presence of chromium was analysed by atomic absorption spectroscopy.

Dried biomass powders of all microalgal species and the corresponding metal ion–loaded powders were coated with ultra-thin film of gold by an ion sputter (JFC-1100), exposed under electron microscope (JEOL, JX-8100) at a working height of 15 mm with voltage ranging between 10 and 25 kV.

2.3. DNA extraction, polymerase chain reaction amplification and sequencing

2.8. Interaction studies of biomass with heavy metals

For DNA isolation, aliquots of the four different microalgal cultures were taken after reaching the exponential growth phase. DNA was extracted by Cetyl trimethyl ammonium bromide method [16] and the concentration of DNA was measured at 260 nm using a Gene Quant 1300 spectrophotometer (GE Health Care, Uppsala, Sweden). To amplify the D1–D2 (large sub unit) coding region of the ribosomal DNA, amplification reactions were performed on a T-gradient thermocycler (Eppendorf, Hamburg, Germany) using the universal eukaryotic primers 5-AGCGGAGGAAAAGAAACTA-3′ as the forward primer and 5TACTAGAAGGTTCGATTAGTC-3′ as the reverse primer [17], according to the polymerase chain reaction (PCR) protocol described by Sonnenberg et al. [18]. The reaction mixtures were analysed using agarose gel electrophoresis to confirm the presence of PCR products. PCR products were purified using the Gel PCR Clean-Up System (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were performed using ABI 373096 capillary, Big Dye Terminator Cycle Sequencing Ready Reaction Kit and the sequencing fragments were analysed on Applied Biosystems 3730xI DNA analyser.

2.8.1. FTIR study The dried microalgal biomass of four different species before and after adsorption was characterised by FTIR (Perkin Elmer, Massachusetts, USA) using potassium bromide pellets [28]. 2.8.2. NMR study Dried microalgal biomass of four different species were analysed for the identification of sorption (binding) sites using 1H NMR (Bruker, Massachusetts, USA). The NMR spectrum was obtained using deuterated cadmium chloride solution as a solvent. Microalgae with or without effluent treatment were analysed separately to observe the metal complexation on the microalgal sorption sites. 2.8.3. XRD study The X-ray diffractogram of dried microalgal biomass powder sample was obtained (Bruker) and the intensities were recorded as a function of 2 scan speed of 1.2°/min and XRD patterns were recorded from 10° to 70°. 2.9. Analysis of heavy-metal ions

2.4. Sequence alignment and phylogenetic analysis To identify the isolated microalgal species, namely VITMA1, VITMA2, VITMA3 and VITMA4, using phylogenetic study through evolutionary relationship, the homologous sequences were searched and retrieved from GenBank using the BLASTP tool [19]. The data set was constructed with great attention by considering only complete coding sequences and eliminating the sequences containing fewer than 200 nucleotides. In order to construct a phylogenetic tree, the isolated microalgal sequences were aligned using Clustal W [20] with default settings. Furthermore, to increase the quality of phylogenetic analysis, the aligned sequences were trimmed using a trimAI tool [21] by deleting the spurious sequences or poorly aligned regions. The most appropriate nucleotide substitution model was selected by Akaike Information Criterion applied in Modeltest v3.7 [22]. The phylogenetic reconstruction was performed by Neighbour Joining (NJ) method with 1000 bootstrap values through MEGA v5.05 [23]. NJ analysis was implemented using the p-distance model with gaps, and the missing data were analysed by pairwise deletion.

The concentration of heavy metals in control and effluent-treated microalgal solutions was determined using atomic absorption spectrometry analysis (Varian 250 model) by the American Public Health Association standard methods [28]. The biosorption efficiency was calculated by Sorption (%) = C0 − Ce / C0 × 100, where C0 and Ce are the initial and equilibrium concentrations of chromium (mg/L) [29]. 3. Results and discussion 3.1. Isolation and identification of microalgae Four freshwater microalgal samples were isolated from the estuaries receiving tannery effluents from the Palar River near Vaniyambadi in Vellore district. The collected microalgal isolates were indicated as VITMA1, VITMA2, VITMA3 and VITMA4 and cultured in BG11 media in culture flasks. Light microscopic images of the isolated species used in this study are shown in Fig. 1. Morphologic and microscopic analyses allowed preliminary identification of isolates VITMA1, VITMA2,

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Fig. 1. Microscopic images of microalgal species isolated from estuaries: (A) Anabaena, (B) Oscillatoria, (C) Phormidium and (D) Spirogyra.

VITMA3 and VITMA4 as genera Anabaena, Oscillatoria, Phormidium and Spirogyra, respectively, by Prof. V. Krishnamurthy (late), Director, Krishnamurthy Institute of Algology, Chennai, Tamil Nadu, India, with reference to the monograph of T.V. Desikachary [15]. The morphological heterogeneity of microalgae makes the microscopic identification difficult. Hence, DNA from these species were isolated and PCR amplified to confirm the morphology-based genera identification and also to identify the species of the given genera.

3.2. DNA amplification, sequencing and phylogenetic analysis PCR amplification of the genomic DNA of microalgal isolates using the universal forward and reverse primers revealed efficient amplification. A single band of amplified DNA with a product size of 1000 base pairs was recorded for all the four microalgal isolates (Fig. 2). Based on the sequencing results, the microalgae with high similarity were

subjected to phylogenetic analysis. BLAST search also facilitates the identification of close relatives of new isolates [30]. An NJ phylogenetic tree was constructed with 39 species of microalgae inferred from the nucleotide sequences using the MEGA v5.05 [23] to identify the microalgal species through evolutionary relationship. The phylogenetic tree showed four clusters and was unambiguously separated into distinct groups, namely Phormidium, Anabaena, Oscillatoria and Spirogyra. The phylogenetic tree suggests that each isolated sequence was clustered with each of the four distinctly separated microalgal species. For instance, the isolated sequence VITMA1 was clustered with Anabaena showing distant relationship with Anabaena species and having 63% sequence similarity. When compared to VITMA1, the other three isolated sequences from VITMA2, VITMA3 and VITMA4 were closely related to the other microalgal species Oscillatoria acuminate, Phormidium irriguum and Spirogyra maxima based on 82%, 95% and 92% sequence similarities, respectively (Fig. 3). Thus, the result suggests that VITMA1 had evolutionary relationship with Anabaena. Similarly, VITMA2, VITMA3 and VITMA4 had relationship with Oscillatoria, Phormidium and Spirogyra. Similar phylogenetic analysis has been reported recently for freshwater microalgae that produce lipids [17]. Phylogenetic analysis may afford the basis for understanding the functional diversity within conserved families [31]. 3.3. Specific growth rate, doubling time and biomass

Fig. 2. Agarose gel electrophoresis of 18S rDNA PCR products from isolated microalgal species.

Specific growth rate, doubling time and biomass are the important parameters to determine the detrimental effect of heavy metals on the growth of microalgae. The microalgal species were grown in B11 media with tannery effluent containing chromium. The average specific growth rate of microalgal species in control and with effluent treated for Anabaena (VITMA1) was 0.166 ± 0.08 μ and 0.105 ± 0.07 μ, for Oscillatoria (VITMA2) it was 0.185 ± 0.06 μ and 0.128 ± 0.05 μ, for Phormidium (VITMA3) it was 0.168 ± 0.05 μ and 0.111 ± 0.05 μ and for Spirogyra (VITMA4) it was 0.150 ± 0.08 μ and 0.101 ± 0.07 μ, respectively (Fig. 4A, B, C, D). The average doubling time of microalgal species in control and with effluent treated for Anabaena was 3.866 ± 1.23 td and 3.615 ± 1.24 td, for Oscillatoria it was 4.205 ± 1.17 td and 3.868 ± 1.10 td, for Phormidium it was 4.091 ± 1.18 td and 3.703 ± 1.09 td and for Spirogyra it was 3.600 ± 1.17 td and 3.565 ± 1.21 td, respectively (Fig. 4E, F, G, H). When compared to the controls, the reduction in growth was 93.50% for Anabaena, 91.98% for Oscillatoria, 90.51% for Phormidium and 97.36% for Spirogyra. The average biomass content of microalgal species in media with or without effluent for Anabaena was 5.245 ± 0.82 g/L and 2.508 ± 0.63 g/L, for Oscillatoria was 6.471 ± 1.02 g/L and 2.713 ± 0.84 g/L, for Phormidium was 5.771 ± 0.26 g/L and 2.663 ± 0.42 g/L and for Spirogyra was 4.907 ± 1.24 g/L and 2.304 ± 1.26 g/L, respectively (Fig. 5A, B, C, D). From these results, maximum growth rate and biomass were observed in Oscillatoria (VITMA2) followed by Phormidium (VITMA3), Anabaena (VITMA1)

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Fig. 3. Phylogenetic tree of isolated VITMA01, VITMA02, VITMA03 and VITMA04 with the most similar sequences retrieved from NCBI nucleotide database.

and Spirogyra (VITMA4) treated with effluent containing chromium. It is reported that the growth of microalgae is a significant factor that influences the metal-binding efficiency [8]. Reduction in growth rate and biomass of microalgal isolates by heavy metals in tannery effluents is considered as a reasonable determinant of toxic effect of chromium in the effluent [32,33]. It has been exemplified that the increase in metal concentration in culture media results in decreased growth and biomass. Results indicate that the isolated to cyanobacteria have the ability to tolerate the heavy-metal chromium stress and hence, it can be grown in the heavy-metal–contaminated water, which attributes for bioadsorption capacity. 3.4. Total protein content Microalgal cell wall surfaces containing proteins with different functional groups, such as carboxyl, hydroxyl, sulphate and other charged groups, differ in their affinity and specificity of the heavy-metal binding [34]. In this study, the total protein content of the microalgal species in control and effluents treated with Anabaena was 15.5 μg and 11.12 μg, for Oscillatoria it was 15.5 μg and 12.37 μg, for Phormidium it was 16.37 μg and 12 μg and for Spirogyra it was 15 μg and 10.25 μg, respectively (Table 1). The maximum protein content was observed in Oscillatoria followed by Phormidium, Anabaena and Spirogyra treated with effluent containing chromium. Proteins in microalgae have the capability of binding heavy metals. These molecules are organometallic complexes and are able to store the heavy metals in the vacuoles inside the cell, thereby converting them into non-toxic forms [35]. Increased total protein content will be found in the high-tolerance microalgal species. Total protein content plays a significant role in the bioadsorption mechanism. Its contribution in the reduction of heavy metals is also

noteworthy [36]. The intracellular metal detoxification mechanisms comprise binding of metal with metal-binding peptides and proteins, binding and precipitation of metal within the cytoplasm or vacuole and sequestration of metals in the form of electron-dense polyphosphate granules [37]. 3.5. Morphological studies by scanning electron microscopy analysis Scanning electron microscopy (SEM) analysis was done to investigate the response of microalgal species towards chromium present in the effluent. SEM images are shown in Fig. 6. Based on SEM analysis, the possible mechanisms involved in the sorption of toxic substances, such as structural damage and alteration in the surface morphology due to heavy-metal exposure, were investigated. Changes observed in the morphology indicate the accumulation of chromium on the biomass surface and many irregular surface features. Morphology changes indicate the accumulation of liquid-phase concentration of charged moieties on the microalgal surfaces. Surface changes expedite the possibility of chromium ions to be adsorbed. Microalgae after adsorption the surface textures were found to be granular complex, porous and uneven [38]. The interaction of electrons with the atoms of the sample produces signals containing information about topography, morphology and composition of the sample surface [39]. 3.6. Interaction studies of biomass with heavy metals To determine the interaction of heavy-metal ions with the functional group of microalgae, the differences in the FTIR, NMR and XRD spectra of microalgal biomass in control and effluent treatment were compared. The adsorption capacity of the adsorbent depends upon the functional

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Fig. 4. Difference between the specific growth rate, doubling time of microalgae without treatment and with treatment of tannery effluents: (A, E) Anabaena, (B, F) Oscillatoria, (C, G) Phormidium and (D, H) Spirogyra.

groups at the adsorbent site [40]. The characteristics of the C–H functional group stretching alkyne that corresponds to the peak shifts from 3269 cm−1 to 3292 cm−1 (Fig. S1A), similarly from 3269 cm−1 to 3282 cm− 1 (Fig. S1B), from 3313 cm− 1 to 3344 cm−1 (Fig. S1C) and from 3369 cm−1 to 3288 cm−1 (Fig. S1D) are mainly due to carbon, hydrogen atoms and alkyne-related polysaccharides in the microalgae. The additional peaks are because of the stretching of vibrations. These peaks show the adsorption sites and surface chemical interactions of the species [36]. Shifting of peaks indicates the adsorption sites involved in the functional groups of microalgae that bind with heavy metals [41]. Metal uptake process observed in the FTIR analysis provides insights on the affinity of microalgae with metal ion, and this confirms the bioadsorption process. To investigate the involvement of functional groups present in the biomass of microalgae with metal ion binding, NMR spectroscopy provides important information. Three major peaks were observed in the spectra; the broad resonance from 1 to 5 ppm shows the possible

contributions of alkynes (C–H) and aromatic groups (Figs. S2A,B and S3A,B). The binding of these groups to chromium metal ions caused the reduction and the absence of corresponding peaks in the effluenttreated microalgal species (Figs. S2B,D and S3B,D). The alkynes and aromatic functional groups have been reduced in the effluent-treated microalgae. Binding indicates the strong affinity of microalgal species with chromium through these functional groups [42]. The affinity is because of the potential involvement of microalgal functional groups towards adsorption [43]. XRD intensities were recorded as a function of 2 U at a scan speed of 1.2°/min. XRD patterns were recorded from 10° to 80° for the microalgal samples before and after biosorption (Fig. S4A,B,C,D). The microalgal species without effluent treatment show sharp peaks, which indicate the crystalline nature of algal biomass. After effluent treatment, the microalgal species have a low-intensity XRD peak, which indicates the poor crystallinity of microalgal biomass. These observations revealed that there was a change in crystallinity of microalgal biomass. This

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Fig. 5. Difference between the biomass of microalgae without treatment and with treatment of tannery effluents: (A) Anabaena, (B) Oscillatoria, (C) Phormidium and (D) Spirogyra.

information indicates the adsorption of metal ions to the microalgal surface [44]. FTIR and NMR studies support the strong affinity of alkynes and aromatic groups in microalgae with heavy metals that possess high binding capacity. XRD supports the adsorption of metal ions. The recognition of chemical structures of microalgal adsorbents is important to predict their affinities against metal ions to know the complexation properties [42]. This interaction study examines chemical functionalities that are responsible for metal ion binding in the microalgal species and provides meaningful knowledge to back the use of microalgal species as an efficient bioadsorbent [43]. 3.7. Biosorption efficiency Heavy-metal–removal ability of the four different microalgal species, namely Anabaena (VITMA1), Oscillatoria (VITMA2), Phormidium (VITMA3) and Spirogyra (VITMA4), against Cr (VI) was analysed by

Table 1 Total protein content of Anabaena, Oscillatoria, Phormidium and Spirogyra with tannery effluents. Species

Days

Anabaena

Control Effluent Control Effluent Control Effluent Control Effluent

Oscillatoria Phormidium Spirogyra

3

7

10

12

3.0 ± 0.22 2.0 ± 0.14 3.0 ± 0.13 2.0 ± 0.15 4.0 ± 0.14 2.0 ± 0.23 3.0 ± 0.22 2.0 ± 0.18

16.0 ± 0.18 13.0 ± 0.22 14.5 ± 0.18 16.0 ± 0.12 17.0 ± 0.22 12.0 ± 0.22 16.0 ± 0.18 10.0 ± 0.22

20.0 ± 0.22 14.5 ± 0.23 19.0 ± 0.12 20.0 ± 0.21 20.5 ± 0.12 14.0 ± 0.16 20.0 ± 0.16 11.0 ± 0.16

23.0 ± 0.16 20.0 ± 0.14 22.0 ± 0.24 23.0 ± 0.18 24.0 ± 0.18 20.0 ± 0.18 23.0 ± 0.14 18.0 ± 0.24

atomic absorption spectroscopy. Based on the results, the biosorption efficiency was calculated and was found to be 65% for Anabaena (VITMA1), 90% for Oscillatoria (VITMA2), 80% for Phormidium (VITMA3) and 55% for Spirogyra (VITMA4), respectively. The high removal biosorption capacity in Oscillatoria might be due to more chemical interactions with chromium present in the effluent, which greatly influences the biosorption process. Similar results were reported by Vankar and Bajpai for the biosorption study of heavy metals [45]. 4. Conclusion In the present study, four different microalgal species possessing phycoremediation activity have been isolated from the estuaries receiving tannery effluents. Morphological and molecular studies revealed the identity of microalgal species such as Anabaena (VITMA1), O. acuminate (VITMA2), P. irriguum (VITMA3) and S. maxima (VITMA4). From the results, it is clear that O. acuminate (VITMA2) has high biosorption efficiency (90%) and is thus a potential candidate for phycoremediation of Cr (VI). FTIR, NMR and XRD results also confirm the interaction of microalgal species with the heavy-metal chromium present in the tannery effluent. This investigation on chemical functionalities responsible for heavy-metal binding with microalgal species provides important information to support the bioadsorbent ability of the microalgal species. Further work will include the development of a lab-scale reactor to monitor the efficiency of microalgae for the treatment of Cr (VI)—contaminated tannery effluents. Abbreviations Fourier transform infrared spectroscopy FTIR NMR nuclear magnetic resonance AAS atomic absorption spectroscopy XRD X-Ray diffraction study SEM scanning electron microscopy

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Fig. 6. Scanning electron microscopy images of microalgae: (A, B) Anabaena, (C, D) Oscillatoria, (E, F) Phormidium and (G, H) Spirogyra.

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2015.12.016.

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