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Rinnakkaistallenteet Luonnontieteiden ja metsätieteiden tiedekunta
2019
Performance evaluation of isolated electrogenic microalga coupled with graphene oxide for decolorization of
textile dye wastewater and subsequent lipid production
Behl, Kannikka
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
© Elsevier B.V.
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.cej.2019.121950
https://erepo.uef.fi/handle/123456789/7680
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Accepted Manuscript
Performance evaluation of isolated electrogenic microalga coupled with gra- phene oxide for decolorization of textile dye wastewater and subsequent lipid production
Kannikka Behl, Monika Joshi, Mahima Sharma, Simran Tandon, Akhilesh K.
Chaurasia, Amit Bhatnagar, Subhasha Nigam
PII: S1385-8947(19)31344-0
DOI: https://doi.org/10.1016/j.cej.2019.121950 Article Number: 121950
Reference: CEJ 121950
To appear in: Chemical Engineering Journal Received Date: 1 March 2019
Revised Date: 10 May 2019 Accepted Date: 10 June 2019
Please cite this article as: K. Behl, M. Joshi, M. Sharma, S. Tandon, A.K. Chaurasia, A. Bhatnagar, S. Nigam, Performance evaluation of isolated electrogenic microalga coupled with graphene oxide for decolorization of textile dye wastewater and subsequent lipid production, Chemical Engineering Journal (2019), doi: https://doi.org/
10.1016/j.cej.2019.121950
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Performance evaluation of isolated electrogenic microalga coupled with graphene oxide for decolorization of textile dye wastewater and subsequent lipid production
Kannikka Behl 1, Monika Joshi 2, Mahima Sharma 2, Simran Tandon3, Akhilesh K.
Chaurasia4, Amit Bhatnagar 5*, Subhasha Nigam 1*
1 Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, 201313, India
2 Amity Institute of Nanotechnology, Amity University, Noida, Uttar Pradesh, 201313, India
3 Amity Institute of Molecular Medicine & Stem Cell Research, Amity University, Noida, Uttar Pradesh, 201313, India
4 Samsung Biomedical Research Institute, School of Medicine, Sungkyunkwan University, Suwon 16419, South Korea
5Department of Environmental and Biological Sciences, University of Eastern Finland, P. O.
Box 1627, FI-70211, Kuopio, Finland
* Corresponding author details:
E-mail: amit.bhatnagar@uef.fi (A. Bhatnagar), snigam@amity.edu (S. Nigam).
Full postal address: Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, 201313, India
ABSTRACT
Microalgae are photosynthetically competent organisms that utilize solar energy to perform several metabolic activities. In this study, electrogenic microalga Desmodesmus sp. was isolated from discharge site of textile dyeing mill effluent and was explored for its potential for dye degradation and lipid production. Extracellular electron transfer (EET) by microbes displays their capability to associate with its surrounding environment. To attain an efficient alternative, a nanobiotechnological approach was applied, wherein; extracellular electrons of Desmodesmus sp. was coupled with graphene oxide (GO) nanosheets on its electron-rich draper region to form a GO/algae bionanocomposite. Electrochemical tests of the bionanocomposite revealed that amalgamation of GO sheets with Desmodesmus sp. enhanced its electron availability (redox potential) without affecting its viability, demonstrating a sustainable and efficient reduction of azo dye along with enhanced lipid production to be used for biodiesel generation. The current bionanocomposite, thus, offers an eco-friendly, reusable, economical and sustainable solution towards water remediation and subsequent biofuel production.
Keywords: Desmodesmus sp.; Textile azo dye; Graphene oxide; Bionanocomposite;
Extracellular electron transfer; Lipid profile.
1. Introduction
Self-sustainable green technology is the key to reduce the environmental pollution and carbon footprints to maintain a suitable environment for the life. To develop such an eco- friendly, self-sustainable technology for clean energy and environment, photosynthetic microalgae are the first feasible preference due to their photoautotrophic production of biomass with minimum micronutrient requirement, abiotic stress tolerance and quick acclimatization property [1]. During photosynthesis, microalgae utilize solar energy to split water in photosystem II and produce oxygen and electrons [2, 3]. Photosynthetically produced electrons reduce carbon dioxide to synthesize glucose molecules, which are primary source of energy for almost all forms of living organisms [4]. Microalgae can potentially redirect photo-generated electrons towards various (intracellular/extracellular) sinks relying upon their growth status [5]. The concept of extracellular electron transfer (EET) has previously been reported in archaea [6], anaerobic bacteria [7], aerobic bacteria [8], cyanobacteria [9], and microalgae [2], which helps them in communicating with either their biotic community partners or the abiotic environment [10]. Among these organisms, microalgae are promising candidates in clean energy and environmental applications, primarily because of their self-sustainability and their ability to efficiently utilize their EET property. EET can occur either via a direct or indirect mechanism. Long-range direct electron transfer takes place via outer membrane c-type cytochromes (OMCs) or conductively proteinaceous nanowires such as bacterial pili or other cell appendages , whereas indirect electron transfer occurs via secreted mediators like flavin, oxygen etc. [11, 12].
Clean and safe drinking water is a prevalent problem worldwide [13]. Textile industrial effluents, containing azo dyes and their degraded intermediate compounds, are highly carcinogenic in nature and pollute several rivers and water-bodies, especially in many Asian countries [14]. Eukaryotic microalgae have wide applications in wastewater treatment
[13, 15]. These microbes can competently and proficiently biodegrade pollutants including azo dyes, several organic and inorganic compounds [16]. However, lower efficiency of these biological systems and the tolerance of microbes to their toxic substrate/end-products have always been one of the common inadequacies in biological applications of various microbes and autotrophs [17]. In this context, nanotechnology offers a viable solution to enhance these biological reactions [18, 19].
Our previous study revealed successful degradation of a textile azo dye, DR 31, along with enhanced lipid production [15], where sawdust derived biochar was added as a component of nutrient source for cultivation of Chlorella pyrenoidosa in an aqueous solution of DR 31 dye to analyse its impact on biomass production, dye decolorization, and lipid production. This inspired us to explore the performance of a newly isolated electrogenic microalga, Desmodesmus sp. coupled with graphene oxide nanosheets for rapid decolorization of textile azo dye and subsequent lipid production.
In the present study, we strive to develop a bionanocomposite that can efficiently and rapidly degrade azo dyes (to improve the overall quality of water) as well as induce enhanced lipid production. To design an efficient bionanocomposite, it is imperative to identify a novel organism that (a) should possess a photosynthetic, self-sustainable survival potential with abundant biomass production under stressful conditions, (b) should be resistive to azo dyes and its intermediates, (c) utilizes and exploits carcinogenic amines as nitrogen source to generate biomass, (d) has electrogenic properties (e) has a potential to couple with a high surface area, electron transporting nanomaterial (f) consumes azo-bond containing pollutants and dyes as a terminal electron acceptor in extracellular surrounding and (g) produces sufficient lipid for biofuel.
Graphene Oxide (GO) was selected as a feasible mediator as it facilitates high electron conductivity at room temperature, has high specific surface area, and is nontoxic in nature
[19-21]. The two-dimensional π-conjugation structures of GO enable effortless electron capture and rapid electron transportability [20, 22].
In this context, three microalgal species were isolated from a textile dyeing mill wastewater discharge site and characterized in terms of their electrogenic property and efficiency to reduce azo bonds. Among these isolated strains, green microalgae Desmodesmus sp. displayed the highest decolorization efficiency and demonstrated a superior EET. To develop an efficient bionanocomposite, we coupled the biological EET of Desmodesmus sp. and embellished GO nanosheets onto the electron rich draper region of microalgae. The GO/algae bionanocomposite was characterized by various advanced analytical techniques. Electrochemical characterization revealed that coupling of algae with GO provides an upgraded electron-interface for transferring biologically generated electrons to azo dye molecules that ultimately stimulates degradation of azo bonds to primary amines.
These amines are subsequently assimilated by the algae as a nitrogen source to produce biomass leading to enhanced lipid production. We have tried to fabricate an innovative, efficient, eco-friendly and self-sustainable bionanocomposite that not only degrades the toxic azo dye, but also enhances the lipid content.
2. Materials and methods 2.1. Materials
Diazo dye, Direct Red 31 (DR 31) was procured from a textile dyeing mill, located at Ghaziabad, Uttar Pradesh, India. Analytical grade natural graphite powder (325-mesh size), ammonium chloride (NH4Cl), chloroform, sodium hydroxide (NaOH), sulphuric acid (H2SO4), nitric acid (HNO3), hydrogen peroxide (H2O2), methanol, hexane, potassium chloride (KCl), potassium permanganate (KMnO4), propidium iodide (PI), sodium azide (NaN3), phosphate buffer saline (PBS), gluteraldehyde, potassium phosphate buffer (PPS),
ethanol and fluorine doped tin oxide (FTO) electrode were purchased from Sigma-Aldrich.
FTO electrodes had a thickness of 2.3 mm, with about 7 /sq. surface resistivity.Ω
2.2. Isolation and purification of microalgal genera and optimization of culture conditions
The wastewater used in the present study was collected from effluent outlet of a textile effluent treatment plant (ETP) situated right outside a local textile dying mill in Ghaziabad, Uttar Pradesh, India. Microalgal species were isolated from this effluent, serially diluted, incubated and maintained in growth chambers (28ºC, 16:8 h light: dark cycle 100 µmol photons m-2 s-1), in a nutrient rich BG-11 medium [23]. Unialgal cells were streaked and further made axenic on BG-11 supplemented agar media (1.5%) added with ampicillin (antibacterial agent) and kanamycin (antifungal agent). In the absence of any bacterial or fungal contaminant, individual microalgal colonies were cultured in liquid BG-11 media for further analysis. Purity of the isolate was confirmed under laser confocal microscope (Olympus FluoViewTM FV1000). The isolated microalga was excited at 488 nm and examined under 100-450× oil immersion magnifications. The isolate was identified morphometrically using SEM (ZEISS EVO Series, EVO 50). Samples were prepared and fixed as described by Shubert and Wilk-Wozniak [24]. Molecular analysis of the isolate was performed using 18S ribosomal DNA sequencing. Sequences were compared with GenBank database using BLAST. The isolate was phylogenetically identified by constructing phylogenetic trees (MEGA ver.7.0.21). Pairwise analysis was done using the LALIGN tool present in the ExPASy server to see the perspective places with similarity between the sequences.
2.3. Preparation and characterization
2.3.1. Lyophilized Algal cells
Lyophilized algal cells were prepared by centrifugation of fresh algal cultures that were freeze dried (ScanVac Coolsafe 55-4; temperature: -55 ; pressure: 1kPa). The lyophilized ℃ algal powder was stored at 4 until further use. FTIR analysis (Excalibur Series FTS 3000 ℃ spectrometer) of lyophilized algal cells was carried out to identify the functional groups present in the isolated microalgae.
2.3.2. Graphene oxide
GO was synthesized by modified Hummers method [20]. Surface morphology of the prepared GO nanoparticles was confirmed by SEM, FTIR and Raman spectroscopy. FTIR spectroscopy was measured from 400-4000 cm-1. Raman Spectroscopy of GO was carried out using WITEC alpha 300R microscope at 560 nm wavelength at room temperature.
2.3.3. GO/algae bionanocomposite
GO nanosheets (0.01 g) were mixed with deionized water (25 ml). The mixture was homogenized and sonicated for 15 min at room temperature. Further, lyophilized algae were added into the GO suspension according to the ratio GO: algae (wt. /wt.), (1:2, 1:4, 1:6, 1:8, and 1:10) respectively. The mixture was stirred overnight at 120 rpm. Finally, the mixture was centrifuged and pellet was then subjected to drying at room temperature (25-27 ) till the ℃ GO/algae bionanocomposite was completely dried. Surface morphology of the prepared GO/algae bionanocomposite was determined using SEM and confirmed by FTIR.
2.4. Fluorescence-activated cell sorting (FACS)
Cell viability of the prepared ratios of GO/algae bionanocomposite was assessed by a flow cytometer assembled with an argon ion at excitation wavelength of 448 nm. Data was
attained through an Accuri-C6 flow cytometer (BD Sciences), and investigations were carried out by BD Cell Quest Pro Software. Flow rate was fixed at 35 µL min-1 and a minimum of 10,000 events (algal cells) were considered. Scattered light was amassed at two angles: Side scatter (SSC) and Forward Scatter (FSC). Mean florescence intensity for florescence channel FL2-A was measured for control (algae) and varying ratios of GO/algae bionanocomposite.
PI was used as the florescent probe to evaluate cell viability. Samples were washed thrice with 1 mL of PBS. PI was added to the GO/algae bionanocomposite at a concentration of 10 µg mL-1 and kept for 2 min in dark. Later, PI florescence emission was collected in the FL2- A channel (564-606 nm) [25].
2.5. Catalytic activities
Photodegradation of DR 31 dye was conducted to appraise the photocatalytic efficiency of Desmodesmus sp., GO and the GO/algae bionanocomposite. A 500 W halogen lamp (Philips) equipped with a UV filter (λ> 400 nm) was employed as a visible light source and a 350 W Xenon lamp (Philips) was employed as source of UV light. Each material (Desmodesmus sp., GO and the GO/algae bionanocomposite) (0.1 g) was added to DR 31 dye solution (50 mL, 40 mg L-1). Before irradiation, mixtures were agitated for 30 min in dark to achieve desorption-adsorption equilibrium. The mixtures were then kept under light irradiation, where sample suspensions (4 mL) were periodically collected and centrifuged.
Supernatant was analysed by UV-Vis spectrophotometer at 520 nm. Percent decolorization of DR 31 dye was calculated as:
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑑𝑒𝑐𝑜𝑙𝑜𝑟𝑖𝑧𝑎𝑡𝑖𝑜𝑛 (%) = 𝐶𝑜𝐶‒ 𝐶
𝑜 × 100 (1)
Where, Co and C represent the concentration of DR 31 dye at time (0) and time (t), respectively.
Mechanisms involved in dye decolorization (adsorption or biodegradation) by algae and GO/algae bionanocomposite were determined by sodium azide (NaN3) treatment (detailed in Supplementary S4). NaN3 is a cell metabolism inhibitor, and its addition to the algal cells stops the metabolic activity of the cells, thus, causing the decolorization only by adsorption process through cell wall.
2.6. Electrochemical analysis
Cyclic voltammetry (CV) experiments were conducted using an Auto lab PGSTAT-10 workstation in a 3-electrode system using platinum wire (counter electrode), Ag/AgCl (reference electrode) and FTO glass electrodes (working electrode). CV scans were observed from -1.0 to 1.0 Volt potential at 100 mVs-1 scan rate in KCl (0.1M, pH 3.2; consisting 5 mM Fe(CN)3-,4-) as electrolyte source.
To evaluate the electrogenic property of the three isolated microalgal species in terms of redox potential, CV studies were conducted for GTE-1, GTE-2 and GTE-3, grown for 15 days on FTO electrode under visible light irradiation. FTO electrodes were placed in 0.5 OD of exponential phase culture (100 ml) in an incubator at 24 , illuminated with a 1500 lux for ℃ the formation of algal biofilm on the FTO slides. To find if EET occurred via direct or indirect mechanism, an anaerobic environment was provided. Nitrogen gas was purged for 35 min to the electrolyte buffer before and during the measurement.
To evaluate the redox behaviour of dye on bare FTO, GO, algae, and GO/algae bionanocomposite modified FTO electrode, CV measurements were carried out under visible light irradiation under the above-mentioned anaerobic condition.
2.7. Lipid estimation and fatty acid profiling
Lipid content, produced by Desmodesmus sp. and GO/algae bionanocomposite, was analysed using altered Bligh and Dyer method [26]. Chloroform and methanol were added to dried and ruptured biomasses in 1:2 (v/v) and shaken overnight in dark. This suspension was centrifuged at 4000 rpm for 15 min and the supernatant was extracted. The procedure was reciprocated twice. Supernatants were mixed for phase separation; making the concentration as 2:1:1 (methanol: chloroform: water). Lower organic layer was extracted and dried. Total lipid content was estimated by using Eq. (2) as follows:
Lipid content (%) = mass of lipid obtained (g)
mass of algal culture used (g) (2)
As each experiment was done in triplicates, the mean result is presented. The comparison between the lipid content of algae and GO/algae bionanocomposite was analysed statistically using student’s t-test and the results with p value <0.05 were considered significant.
Generated lipid was esterified under acidic environment [27]. Dried oil was recovered by adding chloroform (25 ml) and NaOH (0.5 mol L-1) added in methanol (50 ml). This mixture was heated on a reflux for 2 h at 65 . Transesterification reagents (18 ml) (2 g ℃ NH4Cl, 60 mL of methanol and 3 mL of H2SO4) were then added. The mixture was again refluxed for 2 h and transferred to a separating funnel. Hexane and distilled water were added in the ratio of 2:1 (v/v) for phase segregation. An organic (clear yellowish) layer containing fatty acid methyl esters (FAMEs) was observed. Finally, the segregated organic layer was dried. FAMEs profile was analyzed using a Gas-Chromatography (GC) supported by a split injector and silica capillary column on Rtx-5.
2.8. Reusability of GO/algae bionanocomposite
Reusability or recyclability is an essential parameter for practical (economic/commercial) application of a photocatalyst. To examine the effect of desorption on the dye uptake capacity of the GO/algae bionanocomposite, reusability tests were carried out for 3 consecutive cycles.
After each decolorization cycle, the GO/algae bionanocomposite was centrifuged and washed with HNO3 (0.1N) as a desorbing agent before using it for the next cycle.
3. Results and discussion
3.1. Isolation and selection of microalgal species
Three green microalgal species, initially labelled as Ghaziabad Textile effluent 1-3, (GTE-1 to GTE-3), were applied in terms of their azo dye removal efficiency and comparative electrochemical properties (Fig. 1). GTE-1 showed maximum (36%) reduction of DR 31 dye in 150 min, followed by GTE-3 (28.92%) and GTE-2 (18.43%). These results demonstrated that GTE-1 had superior dye removal efficiency among the three isolated microalgal strains (Fig. 1a).
Comparative CV scans are depicted in Fig. 1b. GTE-3/FTO recorded an anodic current (Ipa) of 0.80 mA at a voltage (Vpa) of 0.75V and cathodic current (Ipc) of -0.79 mA at a voltage (Vpc) of -0.29V, estimating potential difference (∆V) at current positions to be ∆V=Vpa- Vpc=1.04 V. As GTE-3/FTO was oxidized at a high voltage, a probability that other interfering compounds might also have been oxidized, cannot be ruled out. No anodic or cathodic peaks were observed for GTE-2/FTO indicating the absence of an electrogenic property of the isolated microalgae. However, sharp anodic and cathodic peaks [Ipa (0.52 mA) at Vpa 0.36V and Ipc (0.42 mA) at Vpc 0.14V] were recorded with a very low change in potential, ∆V = 0.22V for GTE-1/FTO. Heights of anodic and cathodic peaks were almost identical, indicating the process to be completely reversible. No peaks were observed for control (Bare-FTO electrode). These results demonstrated that GTE-1 had a superior
electrogenic property among the three isolated species as it had the lowest ∆V. Since no oxidation/reduction peaks were observed in the supernatant (BG-11) collected at the end of batch experiment, results indicated that exoelectrogens were present on the surface of GTE-1, which were absent in the supernatant. These results suggests that OMCs or conductively proteinaceous nanowires, present on cell membrane, are responsible for direct electron transfer [12].
3.2. Morphological identification of selected microalga
As GTE-1 displayed maximum decolorization efficiency as well as highest electrogenic potential under anaerobic conditions, thus, it was selected for further experimental studies.
GTE-1 was identified as Desmodesmus sp. Confocal light microscopy revealed Desmodesmus cells 8-16 µm and 6-9 µm in length and width, respectively. Walls of these cells were ornamented with spines present at apices of end cells and small spikes were also seen in some cells. Presence of two different phenotypes (Fig. 2a and 2b) is frequently observed as a survival strategy in response to multiple environmental selection pressures [28].
Further, for phylogenetic analysis, Neighbour-joining, Maximum Likelihood, and Minimum Evolution were used to compute phylogenetic trees (Supplementary Fig. S1 (a-c)).
Based on these results, GTE-1 was phylogenetically identified as Desmodesmus sp. from the family scenedesmasceae.
As we have attempted to synthesize an efficient bionanocomposite, it was reasonable to investigate the surface morphology of Desmodesmus sp., GO and GO/algae bionanocomposite by SEM (Fig. 3). Desmodesmus sp. exhibited a striking warty, granulated appearance on its cell wall (Fig. 3a), and cells were of 8-11 µm and 3-8 µm in length and width, respectively. Two-celled coenobiums were more frequent, but unicellular coenobium
was also observed. Cells were also ornamented with other epic structural elements like spines, spikelets, and ridges. These spikelets and spines constitute a tightly clustered crystalline layer enclosing fibrillar electron transparent core [29, 30]. These cell appendages (evidently observed in Fig. 3a) facilitate an electron communication between algae and its outer environment [30]. Desmodesmus sp. also showed round openings.
GO sheets appeared as long crumpled silk waves with a multilayer appearance (Fig. 3b).
GO nanosheets were few μm in length. For GO/algae bionanocomposite (Fig. 3c), it was observed that algal cells were randomly distributed onto GO nanosheets affirming its formation. This was further corroborated by Energy dispersive X-ray spectrum (EDX) (Fig.
3d (i-ii). The EDX results of GO/algae bionanocomposite confirmed the co-existence of elements like carbon (C), oxygen (O), magnesium (Mg), potassium (K), calcium (Ca), phosphorous (P) present in algae and GO. EDX elemental analysis of algae and GO/algae bionanocomposite are depicted in Table 1.
3.3. Raman spectroscopy analysis
The synthesis of graphene oxide was confirmed by Raman spectroscopy, which exhibited two prominent characteristic peak positions at 1591 cm-1 and 1328 cm-1, representing G and D bands, respectively (Fig. S2).
3.4. FT-IR analysis
Structural information of the Desmodesmus sp., GO, GO/algae bionanocomposite before and after treatment as well as treated and untreated DR 31 dye solution were inferred using FT-IR as shown in Fig. 4 and Table 2.
FTIR spectrum of untreated DR 31 aqueous dye solution (Fig 4(a)) revealed peaks at 1738 cm-1 indicating the presence of C=N group (amide (I)) and at 1598 cm-1 contributed by
N=N stretching. The appearance of peak at 1481 cm-1 was credited to aromatic groups and the peak at 1378 cm-1 was assigned to –CH2 wagging and twisting vibrations [RSC] [31].
Peaks at 1102 cm-1 and 1042 cm-1 corresponded to stretching vibration band of –C-O group [32]. Appearance of peaks at 991 cm-1 and 750 cm-1 confirmed the presence of sulphonated groups in DR 31[33].
For microalga Desmodesmus sp. (Fig. 4(b)), peaks observed at 3437 cm-1 suggested the presence of -OH stretching vibrations of amino and carboxylic groups of cellulose and proteins present in cell walls [33]. The peaks detected at 2930 cm-1, 2856 cm-1 and 1536 cm-1 corresponded to the presence of aliphatic C-H stretching vibrations of -CH, -CH2 and -CH3
groups present in cellulose [34]. Occurrence of peak at 2366 cm-1 was attributed to dibasic phosphate (HPO42-) [33]. Presence of peaks at 1656 cm-1 and 1463 cm-1 depicted asymmetrical and symmetrical stretching vibration of carboxylate ions of amino acids, respectively [35]. The peak at 1536 cm-1 corresponded to existence of N=N groups (amide (II)) [33] . Detection of peak at 1463 cm-1 denoted symmetric stretching vibration of –O-C-O groups of amino acid. Peak obtained at 1270 cm-1 was attributed to vibration bands of ester sulphates [36]. The intense peak recorded at 1050 cm-1 was possibly due to stretching vibration of –C-O-C bonds [37]. The peak generated at 875 cm-1 was contributed by sulphate groups present in D-galactose [37].
The FTIR pattern of graphene oxide displayed a characteristic peak at 1610 cm-1 due to the aromatic C-C skeletal vibrations as shown in Fig. 4(c). Moreover, other peaks observed at 1028 cm-1, 1175 cm-1, 1398 cm-1 and 1740 cm-1, correspond to C-O groups, C-O-C stretching vibrations, C-OH stretching, and C=O stretching mode carboxylic acid group, respectively;
while peaks located at 2854 cm-1, 2924 cm-1 and 3410 cm-1 correspond to -CH2/CH3 groups, C-H stretching vibrations and O-H stretching vibration, respectively, confirming successful synthesis of graphene oxide [38].
To validate the successful amalgamation of the GO nanosheets to the algae, the GO/algae bionanocomposite was analysed using FTIR analysis (Fig 4(d)). The narrowing at 3391 cm-1 (–OH stretching vibrations) and at 2936 cm-1 (aliphatic C-H stretching vibrations) was observed. The intensity of peaks generated at 2318 cm-1 (–NH stretching) and 1658 cm-1 (stretching vibration of carboxylate ions) diminished. Additionally, detection of different peaks at 2852 cm-1, 1326 cm-1, 1121 cm-1 and 1050 cm-1 confirmed successful interactions between algae and GO nanosheets. These results confirm the successful formation of algae/GO bionanocomposite. Their interactions may cause a strong impact on the electron transfer leading to a superior photocatalytic activity of the GO/algae bionanocomposite.
FTIR analysis was also performed with GO/algae bionanocomposite after DR 31 dye decolorization (Fig. 4(e)). Broadening of peaks was recorded at 3285 cm-1, 2916 cm-1, 2848 cm-1, and 1637 cm-1, demonstrating the stability of GO/algae bionanocomposite even after dye decolorization. Additional peaks generated at 1377 cm-1, 1227 cm-1, and 1078 cm-1 suggested the attachment of DR 31 to the GO/algae bionanocomposite [39]. Presence of a new peak at 1531 cm-1 revealed that GO/algae bionanocomposite could successfully degrade the diazo (N=N) bond of DR 31. Appearance of additional peaks at 1029 cm-1 and 871 cm-1 in the GO/algae bionanocomposite after DR 31 dye decolorization displayed successful interaction of DR 31 dye molecules with –C-O and –C-O-S groups, present in the GO/algae bionanocomposite.
To evaluate the degradation of DR 31 dye by GO/algae bionanocomposite, the FTIR spectrum of treated DR 31 dye solution was performed (Fig. 4(f)). The treated DR 31 dye solution exhibited a peak at 3347 cm-1 which corresponded to –OH stretching vibrations.
Disappearance of characteristics peaks of sulphonated groups of DR 31 at 991 cm-1 and 750 cm-1, confirmed its degradation by GO/algae bionanocomposite. However, it is noteworthy to
mention the observance peaks at 1101 cm-1 and 828 cm-1. Appearance of these peaks can be due to the production of sulphonated exopolysaccharides (EPSs), released from algae [40].
3.5. FACS analysis
Toxicity of GO nanosheets on Desmodesmus sp. was assessed using flow cytometry for successful fabrication of a nontoxic bionanocomposite. Cytommetric analysis revealed viability of the Desmodesmus cells to be 85.6% in control (no GO). A cell viability of 62.1%
Desmodesmus sp. was found living at 1:2, while 72.4% of the cells were living at 1:4, 79.1%
82.4% and 82.6% cells were found viable at 1:6, 1:8 and 1:10 respectively (Fig. 5). The minimum viability was observed at 1:2, whereas the maximum viability was observed at 1:10. The decrease in viability can be attributed to generation of reactive oxygen species (ROS) by GO which exhibited catalytic effects. GO disrupts the algal cell wall through lipid peroxidation by interacting with metabolic biomolecules (DNA), resulting in the toxicity to algal cells [41].
Effect of different ratios of GO/algae bionanocomposite on degradation of DR 31 dye (40 mg L-1) was investigated under visible light. The fabricated GO/algae bionanocomposite of 1:4 (w/w) was selected for further investigations, as this ratio showed sufficient decolorization of DR 31 with minimum toxicity to the algal cells (28.9%) (Detailed in Fig. S3). Mechanisms involved in DR 31 dye decolorization by algae and GO/algae bionanocomposite were confirmed using sodium azide (NaN3) treatment (Fig. S4).
3.6. Photocatalytic activity and kinetic modeling studies
The photocatalytic efficiency of the GO/algae bionanocomposite, GO and algae under visible light irradiation is illustrated in Fig. 6a. GO/algae bionanocomposite successfully
reduced 90% of DR 31 dye under visible light in 150 min as compared to only algae (36%) and only GO (67%), indicating its superior photocatalytic activity. Linear degradation curves (Fig. 6b) followed pseudo-first-order kinetics (Langmuir-Hinshelwood kinetic model) [20]
which can be given as:
ln
(
𝐶𝐶0)
= ‒ 𝑘𝑡 (3)Where, C0 represents DR 31 dye concentration at time (0), C represents DR 31 dye concentration at time t and k represents pseudo-first order kinetic rate constant. These results clearly indicate that GO/algae bionanocomposite (1:4) has superior photocatalytic efficiency.
3.7. Electrochemical analysis using CV
To evaluate the redox behaviour of DR 31 dye on bare FTO, GO, algae, and GO/algae bionanocomposite modified FTO electrode, CV measurements were carried out under visible light irradiation to explicit its photocatalytic/photo-switching ability through the involvement of photo-generated electrons during photosynthesis in algae. CV profiles of GO, algae and GO/algae bionanocomposite immobilized electrodes were measured.
As shown in Fig. 7, anodic current (Ipa: 0.52 mA; Vpa: 0.36V) and cathodic current (Ipc: 0.41 mA; Vpc: 0.14V) were recorded for algae/FTO. Although no anodic current peak was observed, only a cathodic current peak (Ipc: 0.26 mA; Vpc: -0.39V) was recorded for GO modified FTO. However, a sharp increase in the anodic (Ipa: 0.894 mA; Vpa: 0.44V) and the cathodic (Ipc: 0.751 mA; Vpc: -0.01 V) current peaks were recorded for the GO/algae bionanocomposite as compared to GO and algae alone. The higher value of anodic current recorded by the GO/algae bionanocomposite in a similar potential difference demonstrates its
alone. The amplified redox behaviour of the GO/algae bionanocomposite is presumably due to synergistic effects of both algae and GO, which stipulates its improved electron transfer efficiency. Thus, appearance of an electron-abundant graphene backbone and electrogenic alga resulted in fabrication of a bio-nanomaterial that can find application in improving the water quality.
3.8. Lipid production and fatty acid distribution
Lipid production and fatty acid profiling of algae (Desmodesmus sp.) and GO/algae bionanocomposite were investigated to evaluate the lipid production after DR 31 dye decolorization. FAMEs profile of algae was compared with GO/algae bionanocomposite to probe the effect of GO in lipid production. Total lipid content of Desmodesmus sp. (after DR 31 dye decolorization) was recorded as 9% (dry cell weight) which increased to 11% (dry cell weight) and the difference was statistically significant (p value < 0.000447) with amalgamation of GO in GO/algae bionanocomposite (Fig. 8a). Fatty acid profiles of both GO/algae bionanocomposite and Desmodesmus sp. were compared. Six similar FAMEs profiles that differed in relative concentration were observed in both profiles (Fig. 8b). Chain lengths of these FAMEs profiles were between C10 and C20. An indicative pattern constituting C16-C18 fatty acids facilitated by 1 degree of unsaturation (16-18 < 2);
commonly present in numerous fresh water species was recorded. FAMEs profile of Desmodesmus sp. and GO/algae bionanocomposite is depicted in Table 4. Total fatty acids in Desmodesmus sp. (after DR 31 dye decolorization) constituted of 58.97% saturated fatty acids (SFAs), 31.64% monounsaturated fatty acids (MUFAs) and 9.39% polyunsaturated fatty acids (PUFAs). On the other hand, when GO/algae bionanocomposite was used for DR 31 dye decolorization, the total fatty acid content changed to 53.41% SFAs, 34.9% MUFAs and 11.69% PUFAs (Fig. 8c). Lipid and fatty acid contents in microalgae depend on the
culture conditions. Microalgal species, when grown under different conditions, exhibit different fatty acids profiles [15]. The fatty acid contents in Desmodesmus sp. deviate when factors like nutrient availability, light intensity, temperature, culture conditions, etc., are altered. In the presence of GO, metabolic activities such as ROS production may lead to more nutrient acquisition as compared to only Desmodesmus sp. cells as reflected in the fatty acid profiles. Similar results were reported by Ouyang et al. [42] and by Cheng et al. [43], wherein, graphene oxide nano sheets effectively increased the lipid content in the microalgae Chlorella vulgaris and Chlorella pyrenoidosa, respectively.
Literature review has confirmed C16 and C18 fatty acids as essential fatty acids for biodiesel production [44-46]. Fatty acids extricated from biomasses of Desmodesmus sp. and GO/algae bionanocomposite conferred hexadecanoic acid (C16:0) to be the dominant fatty acid persuaded by octadecenoic acid (C18:1). Total percentages of C16-C18 chain fatty acids were observed to be higher than 50% in the present study. Since, SFAs composition of GO/algae bionanocomposite was found higher than unsaturated fatty acids (MUFAs and PUFAs); it can be subsequently used as a potential biodiesel feedstock [47]. High SFAs content in biodiesel accounts for more stability and a higher cetane number [48]. MUFAs can effectively reduce the freezing point and intensifies biodiesel properties at lower temperatures. PUFAs are responsible for oxidative stability of biodiesel [48, 49].
Since, slight deviation in fatty acid profiles of Desmodesmus sp. was recorded with the amalgamation of GO; it can be inferred that GO/algae bionanocomposite is suitable for lipid production without any lethal effect on algal growth and preserves the diversity of the microalgae.
3.9. Possible mechanism proposed for enhanced EET
Oxygen evolving complex (OEC), a water oxidising enzyme participates in the photo oxidation of water during light-dependent photosynthetic reactions. Thus, OECs are sustained sources of electrons and oxygen [50]. Algae, when exposed to unfavourable environmental conditions, suffer from increased oxidative stress [51, 52]. Generation of ROS species, like singlet oxygen (1O2), hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and superoxide radicals (O2•) are inevitable events in normal cell metabolism [53, 54].
Chloroplast is a major sight for ROS generation as O2 operates as an electron acceptor at photosystem I (PS I) reducing site. Photo-reduction of oxygen i.e., Mehler reaction is mediated by ferredoxin and yields O2• [54, 55]. This reaction occurs concurrently with NADPH production, signifying that oxygen competes with NADP+ for electrons from PS I [56]. Thus, they create oxidative stress. The protection of chloroplast by powerful anti- oxidative system is therefore indispensable [54]. Thus, for the down regulation of the electron flux, the electron transport enzymes are essential for the oxidative degeneration of endogenous substrates [57].
In this context, the azo-reductase enzyme is induced for the reduction of toxic dyes [14].
Azo-reductase acts on nitrogenous compounds as a donor with NAD+ or NADP+ as acceptor.
Cytochrome proteins, also, partake in this down regulation of electron flux. The extracellular electrons get extracted from algae and are transferred to GO nanosheets through cytochromes present on the cell membrane [58]. GO nanosheets act as trapping centres/electron sinks and suppress electron-hole pair recombination of algae resulting in the generation of free charge particles (e- and h+). These free charged particles are in-turn, captured by surface-adsorbed O2
to produce ROS (•OH; O2•). Eventually, DR 31 dye gets degraded by the rapid formation of ROS.
Additionally, GO nanosheets have numerous oxygenic groups and endorse large specific surface area granting access to DR 31 dye on its surfaces in the photocatalytic system [38].
Such EET enhanced by GO as an electron collector and transporter accounts for the improved photocatalytic performance of GO/algae bionanocomposite under visible light.
3.10. Reusability of bionanocomposite
Reusability of the photo-switching bionanocomposite is vital criterion for DR 31 dye removal. GO/algae bionanocomposite was investigated for its photocatalytic performance under visible and UV light irradiation. The GO/algae bionanocomposite could be competently reused up to three times for 40 mg L-1 aqueous solution of DR 31 dye (Fig. S5).
4. Conclusions
Over-exploitation of synthetic dyes and its untreated discharge into the environment is deteriorating water bodies at an alarming rate. We have attempted to design an efficient, self- sustainable nano-biotechnological alternative by coupling electrogenic property of green algae (Desmodesmus sp.) electron transporter GO for efficient DR 31 textile dye decolorization and subsequent lipid generation as a potential biofuel feedstock. Newly isolated Desmodesmus sp. displayed maximum decolorization efficiency (36%) and best bioelectric capacity [Ipa 0.52 mA at Vpa 0.36V and ∆V = 0.22V] under anaerobic conditions.
On the other hand, successful amalgamation of GO nanosheets on to Desmodesmus sp.
further enhanced its decolorization efficiency and electrogenic property. GO/algae bionanocomposite could successfully remove 90% and 92% of DR 31 dye in 150 mins under visible light and UV light, respectively. An evident enhancement (71%) in the bioelectric capacity (Ipa: 0.894 mA; Vpa: 0.44V) was observed due to synergistic effects of both Desmodesmus sp. and GO that improved its electron transfer efficiency by 1.71 folds over algae alone. Enhancement in lipid content from 9% (only algae) to 11% in GO/algae bionanocomposite after DR 31 dye decolorization was observed. Total percentage of SFAs
and MUFAs, that are essential for feasible biofuel production, was more than 50%. GO/algae bionanocomposite could be competently reused up to three times for 40 mg L-1 aqueous solution of DR 31 dye. The fabricated GO/algal bionanocomposite, thus, proved to be an eco- friendly, reusable, economical and sustainable solution towards water treatment.
Acknowledgments
Authors are grateful to the Directors of Amity Institute of Biotechnology and Amity Institute of Nanotechnology, Noida for providing the laboratory facilities. The authors thank Mr.
Kuldeep Sharma, IIT-Delhi for the SEM analysis, Mr. Ashok Kumar Sahu, Mr. Manu Vashist and Dr. Manoj Pratap Singh, AIRF, Jawaharlal Nehru University, for Laser Confocal Microscopy, and FTIR analysis, respectively. This work was financially supported by a project grant from Department of Science and Technology, Govt. of India, New-Delhi (Sanction no.: DST/TM/WTI/2K16/265).
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Figure Files
Fig. 1: The isolated microalga GTE-1, GTE-2 and GTE-3 illustrating comparative decolorization and the electrogenic activity. (a) Percent Decolourization efficiency (algal inoculum: 0.4 OD; λmax = 520 nm), and (b) Electrogenic capacity under anaerobic conditions.
To find if extracellular electron transfer occurred via direct or indirect mechanism, an anaerobic environment was created. Nitrogen gas was purged for 35 min to the electrolyte buffer before and during the measurement.
Fig. 2: Photomicrograph of GTE-1 microalga identified as Desmodesmus sp. (a) two- celled coenobia (450× magnification), and (b) four-celled coenobia (400× magnification).
Fig. 3: Morphological and elemental analysis of GO/algae bionanocomposite using Scanning Electron Micrographs (SEM) (a) Isolated Desmodesmus sp. (10K×
magnification), (b) Graphene oxide sheets (30.9K× magnification), (c) GO/algae bionanocomposite, (10K× magnification), and (d) Energy dispersive X-ray spectroscopy (EDX) showing the elemental analysis of (i) GTE-1 alga, and (ii) GO/algae bionanocomposite.
Fig. 4: FTIR spectra representing structural information and presence of various functional groups. (a) DR 31 dye solution, (b) algae, (c) GO, (d) GO/algae bionanocomposite, (e) GO/algae bionanocomposite after decolorization, and (f) DR 31 solution after decolorization.
Fig. 5: Analysis of algal viability in GO/algae bionanocomposite using flow cytometry measurements. Various compositions of GO/algae bionanocomposites were made by mixing different ratios of GO and algal cells (w/w). The flow-cytogram showing the bionanocomposites labelled with propidium iodide (PI) fluorescent dye wherein all axes are plotted at logarithmic scale. Flow cytogram showing red coloured alive cells (M1) vs. green coloured dead cells (M2) of GO/algae bionanocomposite at (a) Control, (b) 1:2, (c) 1:4, (d) 1:6, (e) 1:8 and (f) 1:10.
Fig. 6: Photocatalytic activity and kinetic model studies of GO/algae bionanocomposite under visible light irradiation. (a) Percent decolorization efficiency, and (b) Linear degradation curves following pseudo-first order kinetics (Langmuir-Hinshelwood kinetic model) (Co is initial concentration, and C is concentration at time t).
Fig 7: Cyclic Voltammetric scans of algae, GO and GO/algae bionanocomposite illustrating electrogenic capacity under anaerobic conditions. Nitrogen gas purging was provided for 35 min to the electrolyte buffer before and during the measurement.
Fig. 8: Lipid production and fatty acid distribution of algae and GO/algae bionanocomposite after DR 31 dye decolorization. (a) Enhancement in total lipid content (Percent dry cell weight), (b) Fatty acid methyl esters (FAMEs) analysis profiles, and (c) FAMEs distribution depicting various fatty acids percentage.
Tables Table 1
EDX elemental analysis of algae and GO/algae bionanocomposite
Algae (Desmodesmus sp.) GO/Algae bionanocomposite
Element Weight% Atomic% Weight% Atomic%
C K 50.48 60.24 59.91 68.92
O K 39.64 35.52 32.14 27.76
Mg K 1.96 1.15 1.48 0.84
P K 2.86 1.32 2.92 1.30
K K 1.33 0.49 0.56 0.20
Ca K 3.18 1.14 2.48 0.86
Fe K 0.55 0.14 0.51 0.13
Total 100.00 100.0
Table 2
Major IR transmittance bands and possible assignment in the FTIR spectra of algae, GO, and GO/algae bionanocomposite before and after DR 31 dye decolorization.
Transmittance
band (cm-1) Possible assignment
3437-3285 Stretching vibration of -OH group
2936-2848 Aliphatic -CH stretching vibration of -CH, -CH2 and -CH3 groups 2366 Stretching vibration of dibasic phosphate (HPO4 2-)
1740-1638 Stretching vibration of -C=O of carboxylate group 1598-1531 Stretching vibration of -N=N group
1481-1398 Vibration of aromatic groups 1270 Vibration of ester sulphate group 1175-1050 Stretching vibration of -C-O-C of group 991, 871 and 750 Vibration of sulphonate group
Table 3
Kinetic parameters of Pseudo-first-order model for photocatalytic activity of DR 31 dye under UV and visible light.
Pseudo-first-order model
Visible Light UV Light
Photocatalyst
R2 k (min-1) R2 k (min-1) GO/Algae Bionanocomposite 0.9598 0.0059 0.9755 0.007
GO 0.7619 0.003 0.8805 0.004
Algae 0.7693 0.0021 0.8891 0.003
Table 4
Fatty acid methyl esters (FAMEs) profiles of algae and GO/algae bionanocomposite after DR 31 dye decolorization.
Fatty acid Algae
(Desmodesmus sp.)
GO/Algae
bionanocomposite
Lipid Numbers
Systematic name Common name Total Percentage (%) Saturated Fatty acids (SFAs)
C9:0 Nonanoic acid Pelargonic acid 0.91 -
C10:0 Decanoic acid Capric acid 0.73 0.44
C14:0 Tetradecanoic acid
Myristic acid 2.50
C16:0 Hexadecanoic
acid Palmitic acid 49.09 42.07
C17:0 Heptadecanoic acid
Margaric acid 7.39 8.4
Monounsaturated fatty acids (MUFAs) C16:1 (9Z)-Hexadec-9-
enoic acid
Palmitoleic acid 2.69 14.23
C18:1 (9Z)-Octadec-9-
enoic acid Linolenic acid 28.95 20.67
Polyunsaturated fatty acids (PUFAs) C20:2 (11Z,14Z)-icosa-
11,14,-dienoic acid
Eicodienoic acid 9.39 11.21
C20:3 (11Z, 14Z, 17Z)-
icosatrienoic acid Eicotrienoic acid - 0.48
∑SFAs
∑MUFAs
∑PUFAs
58.97 31.64 9.39
53.41 34.9 11.69
Total 100.00 100.00
Highlights
Graphene oxide and microalgae bionanocomposite was synthesized.
Enhanced dye degradation and lipid generation was achieved by bionanocomposite.
GO/algae bionanocomposite was rich in C16 and C18 fatty acids.
