Accepted Manuscript

Title: A NOVEL AND AN ECO-FRIENDLY APPROACH

FOR ORGANIC DYES DEGRADATION USING
SPIRULINA PLATENSIS CULTIVATED WATER

Authors: Sharmila J, Saravanan P, Sivasankar S,

Chamundeeswari M

PII: S0920-5861(18)31064-2
DOI: https://doi.org/10.1016/j.cattod.2018.11.044
Reference: CATTOD 11778

To appear in: Catalysis Today

Received date: 16 July 2018

Revised date: 17 November 2018
Accepted date: 20 November 2018

Please cite this article as: J S, P S, S S, M C, A NOVEL AND

AN ECO-FRIENDLY APPROACH FOR ORGANIC DYES DEGRADATION
USING SPIRULINA PLATENSIS CULTIVATED WATER, Catalysis Today (2018),
https://doi.org/10.1016/j.cattod.2018.11.044

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 A NOVEL AND AN ECO-FRIENDLY APPROACH FOR

ORGANIC DYES DEGRADATION USING SPIRULINA

PLATENSIS CULTIVATED WATER

Sharmila J1, Saravanan P1, Sivasankar S2, Chamundeeswari M 3‫٭‬

1Department of Chemistry, St. Joseph’s College of Engineering, Sholinganallur, Chennai - 600 119, Tamil Nadu, India.
2Research & Development Incharge, Shibin Exports, Old Perungalathur, Chennai- 600063, Tamil Nadu, India.

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3Department of Biotechnology, St. Joseph’s College of Engineering, Sholinganallur, Chennai - 600 119, India.

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‫٭‬Corresponding author: chamundeeswari@gmail.com

Mobile No: 91-9994752386

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Graphical abstract
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Highlights
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 Simple and eco-friendly method since waste water from one company used to treat the
waste water of the other.
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 Photo catalyst used is not synthesized by any specific method.

 No chemicals are left over after photocatalytic degradation.
 Cost effective method which promotes Green chemistry and environment.
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Abstract
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Waste water recycling may cater to the water scarcity problems which are prevailing in our

daily life. The conventional studies in photocatalytic degradation of organic dyes use some

specifically synthesized metal-oxide nanoparticles as photocatalyst which may leave its traces

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as residue in degraded water. In this prototypical study, we have reported for the first time

about light induced organic dyes degradation of methylene blue (MB), malachite green (MG)

and congo red (CR) carried using a novel photosensitizing agent, Spirulina platensis

cultivated water (Spcw). The aim of the present study is to probe a simple, novel, an eco-

friendly and a cost effective degradation of dyes without using any chemicals. It was

observed that 15 ppm MB, 70 ppm MG and 6 ppm CR got degraded to 100% within 2, 1 and

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3 h respectively. The presence of trace amount of elements such as Ca, Cu, Fe, K, Mg, Mn,

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Na, Mo, Co and Zn and phycobiliproteins found in Spcw were analyzed using Fourier

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Transform Infra-Red (FT-IR) Spectroscopy, Inductively Coupled Plasma Optical Emission

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Spectrometry (ICP-OES), Scanning Electron Microscopy - Energy Dispersive X-ray

Spectroscopy (SEM–EDX), UV-Visible

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Spectroscopy, microscopic studies and
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mineralization studies with TOC, TSS, BOD and COD analysis . The results obtained from
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various control experiments with fresh medium and dark treatment clearly confirms that the
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light induced organic dyes degradation occurs only in Spcw under sunlight irradiation. The

effect of pH was studied with the Spcw added dye solutions and in the degraded water from
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highly acidic to basic conditions to find the optimum pH. The kinetics of the light induced

organic dyes degradation follows pseudo- first order reaction and the mineralization studies
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of degraded water meets the water quality standards. The reason behind the

photosensitization activity is due to the presence of traces of metal ions and light absorbing
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phycobiliproteins released during the natural cell lysis process.

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Abbreviations

C - Control

CR - Congo red
D - Dark

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F I - III - Filtrate I - III

FM - Fresh medium

MB - Methylene blue
MG - Malachite green
Smf - Standard measuring flask

Sp - Spirulina platensis

Spcw - Spirulina platensis cultivated water

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Keywords: Spirulina platensis, organic dye, waste water, photo sensitizer, photodegradation.

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1. Introduction
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Rigorous environmental pollution alarms and changes the way of recent
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chemical research approaches. Advancements in green chemistry is replacing the
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conventional chemical approaches towards synthesis and catalysis of a reaction [1-4].

Commercial and non-commercial waste degradation process is mostly attributed to the

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conventional methods which uses normal stoichiometric quantity of reagents to degrade the
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waste material [5-7]. The conventional photocatalytic methods such as laser induced,

microwave assisted, electrochemical techniques, incineration, adsorption, oxidation involving

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ozone, hydrogen peroxide, metals, metal oxides, metal hydrides create waste streams
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overloaded with inorganic salts such as TiO2, ZnO, ZnS, WO3 and Fe2O3 [8-11].

Photocatalysis with metals, metal oxides, metal hydrides, etc. necessarily generates
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stoichiometric amount of metal waste towards its conversion process. So, these conventional

photocatalysis methods have to be replaced with the greener methods which promotes better

conversion without leaving more metallic residues [12,13]. Generally solvents used in

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reactions play an important role in creating some of the deleterious pollution problems to the

environment. In such cases, an environmental friendly solvent can be substituted directly to

have a positive effects on hazardous and pollution issues. Among all possible liquids, water is

the most versatile and eco-friendly solvent which has been gifted by nature to carry out all

kinds of chemical transformations no matter whether substrates are soluble or insoluble [14-

16]. Organic dye pollutants such as MB, MG, CR, methyl orange, crystal violet, rhodamine

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B, alizarin red S etc., are released from industries such as textile, paper, pharmaceutical,

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printing, leather and cosmetic industries [17-22]. These industries very often have complaints

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of water contamination which may cause several serious health issues like skin allergy,

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vomiting, stomach problems, cancer, lung and heart dysfunctioning and environmental issues

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such as bad odors, decreased biological oxygen demand (BOD) and chemical oxygen demand
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(COD) in final transformations [23,24].
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Generally biotransformation, biodegradation, and bioremediation of organic
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compounds are carried out using various microalgae such as Chlorella, Coenochloris,

Pseudokirchneriella, Scenedesmus, Coelastrum, Synechococcus, Phormidium,

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Ankistrodesmus, Chlamydomonas, Anabaena, Selenastrum and Ochromonas species. They

not only accumulate metals by chelation and chemical transformation, but also produce
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biomineral structures and metal-oxide nanoparticles in solutions [25, 26]. Sp, a blue-green

microalgae is rich in protein source, containing all essential amino acids and gained more
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attention in the field of medical science because of its nutraceutical and pharmaceutical

importance. It is a free floating filamentous cyanobacterium algae grown worldwide naturally

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in tropical and sub-tropical lakes with high pH and high concentrations of carbonate and

bicarbonate level [27,28]. It has two different species such as Arthrospira

platensis and Arthrospira maxima which usually grows in a standard fresh medium called as

Zarrouk’s medium with proper supplement of water and nutrients. The Sp is rich in two
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important phycobiliproteins such as phycocyanin and allophycocyanin, both having same

chromophoric group which promotes light absorption [29]. For commercial purpose, open

raceway ponds are used in the cultivation of Sp which requires large quantities of water for its

growth [30]. Mostly, in case of chemical processes like hydrogenation, oxidation, etc., a

catalyst plays an important role in enhancing the rate of reaction. These reactions include

catalyst such as nickel, palladium or platinum which costs high and may leave a harmful

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residues after completion of the process. So, the selection of catalyst is an eminent task to

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carry out such research work. In most of the studies carried out so far, a photocatalyst is

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specifically synthesized and used for effective dye degradation [31-33]. This motivated us to

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plan for a novel and simple greener approach in degradation process.

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Keeping in mind the above aspects, we for the first time have selected an eco-
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friendly and cost effective catalyst capable of degrading the dyes in presence of sunlight

(28oC – 37oC) without using any harmful reagents. So after harvesting Sp, the Spcw was
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collected and used as photosensitizing agent in minimal quantity for organic dyes

degradation. The major water pollutants of various dyeing industries contain dyes such as
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MB, MG and CR which are degraded effectively using the Spcw. This method may gain its

attention for re-usage in washing, cleaning, cooling and irrigation activities since they are
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devoid of toxicity. So, the main objective of the research is to adapt a novel and an eco-

friendly method which promotes the green chemistry approach towards the organic dyes
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degradation in order to reduce the water pollution. This innovative greener approach is not

reported by any one as per the literature survey and also satisfies the journal aim and
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objectives. The society will be highly benefitted by applying this novel approach for organic

dyes degradation rather than using conventional methods.

2. Experimental procedure
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2.1Materials

Dyes such as MB, MG and CR were purchased from Loba Chemie Pvt. Ltd., Chennai, India.

The Spcw was collected from 3 different companies located at various areas of Chennai. The

collected Spcw was named as filtrate I (F-I: Chengalpet), filtrate II (F-II: Singaperumal koil)

and filtrate III (F-III: Madhavaram). A fresh medium (FM) used in the growth of Sp from a

company located at Chengalpet was collected for comparative analysis and used as a control.

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2.2 Selection of dyes

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Dyes were selected based on their solubility in water and and trials in triplicates using

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different dyes such as MB, MG, CR, methyl orange and alizarin red S to check its

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degradation level. Among these dyes, commercially important 3 dyes such as MB, MG and
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CR possessing different chemical structure were selected (refer supplementary data Fig. 1 A-
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C), color (blue, green and red), functional groups (tetra-methyl thionine chloride, triphenyl

methane backbones and biphenyl-bis-amino-naphthalene sulfonic acid) and lambda (λ) max
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of 667, 616 & 498 nm respectively.

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2.3 Selection of photosensitizer

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Spcw from open raceway ponds was collected from 3 companies located at different areas of

Chennai. 3 companies was selected to bring clarity in the selection of catalyst and to prove its
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nature being almost the same though collected from different companies and areas. The Spcw

collected from different companies were green in color initially due to the presence of traces
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of single cell protein, which was then removed by filtering using ordinary filter paper

followed by A1 filter paper to remove maximum biomass. It was then transferred into clean

bottles and labeled as F I-III (refer supplementary data Fig. 2 A&B). Further the clear filtrates

was stored in refrigerator and used as a photosensitizer for degradation of the selected organic
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dyes. The photosensitizing activity of the filtrate samples was found to be efficient even after

refrigeration for about 1 month.

2.4 Degradation studies on Methylene blue, Malachite green and Congo red

45 ml of dye was measured and mixed with 5 ml of Spcw in about 6 standard measuring

flasks (smfs) for each finally selected concentrations such as 15 ppm MB, 70 ppm MG and 6

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ppm CR. For 15 ppm MB: 1 smf for FM (control), 1 smf for dark environment (control), 1

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smf with 50 ml of dye alone without adding photosensitizer (untreated- control) and 3 smf's

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for F I-III which acts as photosensitizer (treated - experimental) was labelled and used for the

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experiments. The same experimental setup was carried out for 70 ppm MG and 6 ppm CR.

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After thorough mixing of dyes with photosensitizer (Spcw) the smf's was kept in dark

(control) and sunlight (28-37oC). About 2 ml of degraded water was sampled out for every 30
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min until the complete degradation which occurs at 3 h. The OD was checked using UV-
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Visible spectrophotometer and the percentage (%) dye degradation was calculated [34]. The

same procedure was repeated 3 times and measurements also taken for all the 3 trials.
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2.5 Degradation study on the mixture of dyes

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Mixture of 3 selected dyes such as 15 ppm MB, 70 ppm MG and 6 ppm CR each of 300 ml

were taken in a 1 L beaker and mixed with 100 ml of Spcw (F-I) in the ratio of 9:1 and kept
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under sunlight for 3 h. A complete degradation of all 3 dyes was noted in large volume and

this study also shows the efficiency of the selected photosensitizer on scaling up process. The
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procedure was repeated thrice to minimize the error percentage.

2.6 Effect of pH on the degraded water

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Duplicates of 45 ml of 3 dyes were separately mixed with 5 ml of Spcw each and pH was

adjusted in the range 3-10 using dilute acid and base solutions. The varying pH solutions was

then kept under sunlight for 3 h for complete degradation. The samples was collected and

checked for its absorbance values using UV-Visible spectrophotometer at wavelengths 667,

616 and 498 cm-1 for before and after degradation respectively. 100 ml of dye was degraded

and divided into two equal parts: one part was checked for acidic pH and the other part for

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basic pH in order to find out whether there is any change of color with respect to pH. The

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initial pH was measured and the effect of color change with respect to pH was monitored

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with an increment of pH 1 for all the 4 degraded waters such as 15 ppm MB, 70 ppm MG, 6

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ppm CR and for the mixture of dyes (MX). The procedure was repeated thrice to analyze the

effect of pH on complete degradation of the dyes.

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2.7 Protein content analysis
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The presence of protein in FM, F I-III was monitored qualitatively and quantitatively to
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identify whether any protein was involved in enhancing the degradation process. 2 ml of FM,

FI-III was used to check the presence of protein by measuring the OD at 280 nm using UV-
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Visible double beam spectrophotometer. The amount of protein present in FM, F-I, II, III and

crude extract from Spirulina platensis was estimated using Bradford’s reagent. 100 mg/ml of
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bovine serum albumin was prepared using double distilled water and this solution was used

as a standard protein solution. 200, 400, 600, 800, and 1000 µl of standard solution was taken
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in different test tubes and the volume was made up to 1 ml with double distilled water. Test

solutions such as FM, F-I, II, III and crude extract was taken (200 µl each solution) and the
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volume was made to 1 ml. To all the above solutions 2 ml of Bradford’s reagent was added,

mixed well and kept for 10 min at 37º C. The absorbance was recorded at 595 nm against the

reagent blank. The experiment was done in duplicates and the amount of protein present was

calculated using formula given below:

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 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ×𝑇𝑒𝑠𝑡 𝑂𝐷
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 = --------------------------------Eq. (A. 1)
𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑂𝐷

2.8 Procedure for TOC study to be included

Duplicates of 1 L samples were prepared for all the 3 dyes in the ratio of 9:1 (Dye: Photo

sensitizer) and kept under sunlight irradiation for 3 h to promote complete degradation. The

samples were then analyzed using Shimadzu TOC analyzer to check the degradation before

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and after irradiation in sunlight.

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2.9 Characterization
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The presence of protein in FM and Spcw (F I-III), the percentage of degradation of dyes and
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effect of pH on the degraded water (optical density) was measured using Systronics UV-
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Visible double beam spectrophotometer: 2202 (bandwidth – 2.0 nm). The amount of elements

such as Ca, Cu, Fe, K, Mg, Mn, Na, Mo, Co and Zn (elements in Zarrouk’s medium) present
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in the FM and F-I was identified using ICP-OES of Perkin Elmer Optima 5300 DV. The

surface morphology and the presence of elemental composition was carried using SEM (Carl
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Zeiss MA15/EVO 18 Scanning Electron Microscope; resolution 3.0 nm at 30 kV in low

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vacuum mode by BSD) and EDX (Oxford Instruments Nano Analysis INCA energy 250
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microanalysis system with 130eVINCAx - at Peltier cooled analytic detector with PentaFET

with tungsten hairpin filament and magnification - 5x to 1,000,000x) respectively.

Furthermore, the presence of protein functional groups and metal ions present in the FM and

F-I was also confirmed using FT-IR spectrum one: FT-IR Spectrometer (Perkin Elmer

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Spectrum), Scan Range: MIR 450-4000 cm-1, Resolution: 1.0 cm-1. The Total Organic

Carbon (TOC) analysis was determined using a Shimadzu TOC analyzer (model 5050A) for

degraded water. The effect of pH on degradation was carried out with an increment of 1 with

the pH ranging from 3-10 using portable pH meter.

2.10 Statistical analysis

In order to minimize the error percentage and to evaluate the reproducibility of this

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photosensitizing activity by Spcw, all the experimental procedure and measurements were

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triplicated. In FT-IR, each sample was scanned under the same conditions with three different

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pellets and these replicates were averaged. Similarly averaged spectra were used in plotting

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UV graph. The ICP-OES analysis also taken for three trials and the statistical analysis were

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performed using SPSS 16.0 software. Correlation matrix was performed to access the
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influence of each metal concentration over the other metal.
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3. Results and Discussion

3.1 Percentage (%) of dye degradation

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The percentage of dye degradation for MB, MG and CR were clearly shown in the Fig. 1 A -

C as determined at regular time interval of 30 min. The linear square fitting was plotted for
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percentage degradation of dyes which shows that the best fit was obtained for CR. In case of

MB and MG, maximum degradation was obtained before 60 min and hence beyond 60 min
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there was no significant variation obtained in degradation profile of MB and MG. The

percentage degradation in CR varies linearly with time from 30 min to a maximum of 180
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min. The maximum degradation was obtained at 180 min. This study implies that MB and

MG are significantly degraded at lower time (1-2 h), whereas the degradation of CR

consumes more time (3 h). There was no dye degradation incase of C, FM and D which are

used as control for these experiments as shown in Fig. 5A – C (IV a-f) and discussed under
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supplementary data 1.4 Standardization of dye degradation. The OD was checked using UV-

Visible double beam spectrophotometer and the % of dye degradation was calculated using

the formulae (2) [34].

𝐴0 −𝐴𝑡
% 𝑜𝑓𝑑𝑦𝑒 𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = − − − − − − − − − − − − − −Eq. (A. 2)
𝐴0

A0– absorbance at initial time and At – absorbance at different time intervals.

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3.2 Protein content analysis

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As per the literature survey phycocyanin, a phycobiliprotein is capable of absorbing sunlight

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and helps during photosynthesis of the microalgae by promoting electron transfer to

cholorophyll [35-38]. This property made us to think about phycocyanin as a photosensitizer

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and an initial trial for degradation of dyes such as MB, CR and MG was done using 100 µl
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crude extract of phycocyanin. After exposure to sunlight for 6 h, the degradation of dyes was
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by 50% in MB, 100% in CR and MG respectively (refer supplementary data Fig. 3).
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Similarly, as per earlier studies reported the presence of transition metal ions such as Cu(2+),
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Mn(2+), Zn(2+), Fe(3+), Co(2+), Ni(2+), Mg(2+), TiO2, Al3+, and Cd2+ [36-39] promotes

catalytic degradation. These ions when bound to the phycobiliprotein will form metal-protein
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complexes and promotes faster degradation when compared to the dye alone. So, F I-III

solution was collected and checked for the presence of phycocyanin using Bradford's
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reagent. Quantitative estimation of protein content using Bradford's reagent was carried out
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after checking the presence of protein at 280 nm using UV-Vis spectrophotometer (refer

supplementary data Table 1; Fig. 4). FM showed 15 mg/ml; F-I 92 mg/ml; F-II 79 mg/ml; F-
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III 90 mg/ml & crude extract of Sp showed 140 mg/ml respectively (Fig. 2). This analysis

carried out in duplicates also clearly shows that 6 times greater amount of phycobiliproteins

was present in F I-III than in FM. A crude extract sample protein was analyzed as a positive

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control for the test. Though the concentration of protein content is high in crude extract when

compared to F I-III the formation of metal-protein complexes has reduced the concentration

of phycocyanin protein and time of photosensitizing activity. Later, we standardised the dye

degradation procedure using the F I-III as a photosensitizer in the presence of metal ion left

unused during the growth. This metal ion complex produced a good degradation when

compared to the crude extract. Hence, we selected the Spcw as a suitable photosensitizer for

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dye degradation. This proposed mechanism of study can be understood from the two basic

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laws of photochemistry i.e. Grotthuss–Draper & Stark–Einstein law of photochemical

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reaction which is shown below in Scheme 1[39]

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Scheme 1 Mechanism involved in light induced organic dyes degradation using Photosensitizer

3.3 Effect of photo catalyst on the complete degradation of MB, MG and CR

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Fig. 3. A-C shows the effect of F I-III on the complete degradation of dyes such as MB, MG

and CR. The change in absorbance with respect to time was monitored at a regular intervals

of every 30 min. The changes in absorbance for initial and final experiments was carried out

till 3 h to prove that a complete degradation occurs due to the presence of photosensitizer

(Spcw).

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3.4 Degradation kinetics on the different dyes

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The degradation kinetic studies of MB, MG and CR have been carried out at different time

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intervals and concentrations. The degradation rate of dyes were analyzed for every 30 min of

reaction time and calculated from linear regression ln(C0/Ct) versus time plots with all

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regression coefficients greater than 0.98. Hence, the degradation of the dyes follows pseudo-
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first order kinetic model.
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ln(C0/Ct )= kt------------------------------------------------Eq. (A.3)
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where k is the pseudo-first-order rate constant, Ct is the concentration of dye at t minute and t

is the reaction time. The degradation rate (Table 1) for different F I-III of 15 ppm MB, 70
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ppm MG and 6 ppm CR were obtained from Fig. 4.The photocatalytic catalytic degradation

rate results of dyes was in the order MB > MG > CR. The results showed that the chemical
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structure of the dyes also influenced the reactive rate.

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Table 1 Represents the degradation rate for MB, MG and CR

Dyes Concentration K(min-1)

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(ppm)
MB FI (15) 0.064
F II (15) 0.055
FIII (15) 0.056
MG FI (70) 0.020
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 F II (70) 0.019
FIII (70) 0.021
CR FI (6) 0.004
F II (6) 0.003
FIII (6) 0.005

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3.5 ICP-OES analysis

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Table 2 represents the presence of various elements in trace amounts in FM and F-I carried

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out through ICP-OES in order to identify whether the presence of traces of any metallic salts

enhance the photosensitizing activity. Both FM and F-I shows the presence of metallic salts

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with slight change in its concentration. The presence of Ca, Fe, Na, Mg and Zn are
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comparatively high and Co is less in case of F-I when compared to FM. The variation may be
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due to the continuous supply of nutrients to the Sp growth for its everyday cultivation and
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release of the total cell content by natural cell lysis of Sp. But elements like Cu, K, Mn, Mo
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show decrease in its concentration in F-I when compared to FM which may be due to the

utilization of such nutrient elements during its growth in presence of sunlight. The presence
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of metallic ions in the F-I has shown an enhanced photocatalytic activity in case of dyes

degradation along with the release of some light absorbing phycobiliproteins and facilitate
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photosensitizing degradation [40-43]. Though the metal ions are present in FM it could not
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favor the dye degradation due to the absence of light absorbing protein pigments. The

elements analyzed are selected based on the composition of Zarrouk’s medium widely used
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for the cultivation of Sp in an open raceway pond.

Table 2 ICP-OES data analysis of FM and F-I

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 Elements Concentration in mg/L
S. Wavelength
Symbol
No. (nm)
FM F-I
1 Ca 317.933 1.747 ± 0.023 12.08 ± 0.852
2 Cu 327.393 0.116 ± 0.009 0.034 ± 0.010
3 Fe 238.204 0.820 ± 0.107 1.108 ± 0.092
4 K 766.490 964.5 ± 2.711 391.9 ± 3.045
5 Mg 285.213 32.79 ± 2.732 64.11 ± 2.221
6 Mn 257.610 0.527 ± 0.077 0.321 ± 0.042

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7 Na 589.592 6695 ± 5.173 13200 ± 4.609

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8 Zn 206.200 0.18 ± 0.006 7.456 ± 0.188
9 Co 228.616 0.046 ± 0.005 0.064 ± 0.003

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10 Mo 202.031 7.910 ± 1.007 2.359 ± 0.754

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± Standard deviation (no. of samples = 3)

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3.6 Correlation matrix
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The correlation matrix was performed using SPSS 16.0 software to evaluate the impact of
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each metal concentration over the other metals. The correlation was significant at the 0.01

level (2-tailed) and 0.01 level (2-tailed). Most of the correlations were significant at 0.01
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level for correlation coefficient values above 0.9177 and for correlation coefficient values

between 0.8932 to 0.9086 were significant at 0.05 level (two tailed). The correlation matrix
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was presented in Table 4 provided in supplementary file. A strong positive correlation

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significant at 0.01 level were found between Ca-Fe, Ca-Mg, Ca-Na, Ca-Zn, Cu-K, Cu-Mn,
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Cu-Mo, Fe-Mg, Fe-Na, Fe-Zn, K-Mo, Mg-Na, Mg-Zn, and Na-Zn whereas positive

correlation significant at 0.05 level found between K-Mn and Mo-Mn. These positive
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correlation shows that the metal concentration of one metal increases with the increase in

metal concentration of the other metal. On the other hand negative correlation shows that

increase in metal concentration one metal decreases the metal concentration of the other

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metal ion. The negative correlation significant at 0.01 level exist between Ca-Cu, Ca-K, Ca-

Mo, Cu-Fe, Cu-Mg, Cu-Na, Cu-Zn, Fe-K, Fe-Mo, K-Mg, K-Zn, Mg-Mn, Mg-Mo, Na-Mo

and Zn-Mo and negative correlation significant at 0.05 level exist between Ca-Mn, Fe-Mn,

Na-Mn, and Zn-Mn. Cobalt is the only metal ion which do not have any correlation with

other metal ions which attributes that the metal concentration of cobalt does not have any

impact over other metal ions.

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3.7 SEM - EDX analysis

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SEM-EDX analysis provides the topographical aspects and elemental constituents for

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samples of FM and F-I. SEM pictures (Fig. 5 A & B) of FM and F-I clearly shows the

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surface morphology differences. The fiber like proteinaceous structure was present only in F-
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I and was not observed in FM. The results of EDX analysis of FM and F-I are shown in Table
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2 given in the supplementary data. The results presented the following elements such as O,
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Na, Mg, P, S, Cl, K, Ca, Mn, Fe, Co, Cu, Zn and Mo are reported in k lines. Except for Na,

Mo and Cu, most of the elements detected from the FM were also found in the F-I. The
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concentration of most elements in the FM are greater than the concentration of elements in

the F-I. The concentration of Mn, Ni, Zn, and Al from FM and F-I were comparable to one
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another. The level of Mg in the F-I was very high compared to FM which has very high

concentration of Cl compared to F-I. Though these elements are present in both (FM and F-I)
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only F-I is capable of acting as a photosensitizer due to the presence of metallic ions along

with light harvesting photo pigments.

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3.8 FT-IR analysis

The FT-IR spectra of FM and F-I was studied in the range of 400 - 4000 cm-1. The results of

FT-IR spectroscopic analysis was used in the determination of functional groups which gives
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a qualitative description. The frequency assignment of FT-IR spectra for the Spcw for FM

and F-I were displayed in Table 3 shown in supplementary data. The Fig. 6 shows the FT-IR

image of the samples under study. The band at 3407, 3460 and 3464 cm-1 in FM and F-I are

assigned to the presence of O–H or N–H stretching of amide A protein. The amide I protein

was present in all the samples, which was observed from the band at 1655, 1675 and 1691

cm-1 in Sp, FM and F-I respectively [44]. The amide II protein band observed in Sp sample at

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1546 cm-1 which was assigned to N-H bending and C-N stretching of proteins. All samples

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exhibit a band at 1451-1458 cm-1 assigned to C-N stretching/ in-plane -OH bending. The -

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CH2- asymmetric stretching and -CH2- symmetric stretching of lipid and protein band occurs

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at 2925 cm-1 (strong) and 2876 cm-1 (medium) in Sp sample and these bands were absent in

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FM and F-I. This implies, the absence of lipids in FM and F-I and the proteins present mostly

promotes dyes degradation. The medium to strong bands from ~1186 to ~1029 cm-1 indicates
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the variation of carbohydrates in all the 3 samples. The carbonate asymmetric stretching and -

CH out of plane bending (carbohydrate) appear at 877 and 849 cm-1 in FM and F-I
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respectively [45-47]. The FT-IR results also supports that only the light absorbing
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phycobiliproteins released due to natural lysis are only responsible for photocatalytic

degradation as compared with that of the Sp and FM used as a control for data analysis.
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3.9 Effect of pH
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The effect of pH on the degradation of organic dyes shown in Fig. 7 I was examined under

using a portable pH meter. A maximum degradation of MB, MG and CR was observed in

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optimum pH of 10, 8 and 5 respectively. The pH of the degraded water was checked and the

behavior was noticed for the change in color with respect to both acidic and basic conditions

using dil. HCl and dil. NaOH to analyze whether complete degradation of the dyes has

occurred or not with an increment of pH 1 in case of basic conditions and a decrement of pH

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1 in case of acidic conditions. It was seen that all the degraded dye solutions were observed to

be colorless at all pH conditions as shown in Fig. 7 II.

3.10 Mineralization study for organic dyes MB, MG and CR

The complete mineralization of MB, MG and CR was shown in Table 1. The total organic

carbon, biological and chemical oxygen demand and total suspended solids for all the 3 dyes

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were found to be high in before degradation samples and a drastic decrease was observed in

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after degradation samples. This study clearly infers that the light induced photosensitization

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reaction has occurred in all the 3 dyes using Spcw as a photosensitizer ensuring complete

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mineralization of all the 3 dyes. This indicates that the water quality was improved to the

standard limits which can be used for any purpose.

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Table 3 Represents complete mineralization of MB, MG and CR

Methylene blue Malachite green Congo red

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Parameters
Before After Before After Before After
(in ppm)
Degradation Degradation Degradation Degradation Degradation Degradation
TOC 38 6 30 9 42 8
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BOD 27 10 41 12 35 5
COD 113 15 94 21 89 4
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TSS 54 7 46 9 62 <2
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4. Conclusion

Complete decolorization and degradation of aqueous solutions of MB, MG and CR was

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achieved within 3 h at higher efficiency rates with minimal amount of Spcw as

photosensitizer. The photodegradation rate of dyes are good, strongly depends on the

intensity of sunlight irradiation and does not involve any complicated mechanisms, harmful

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chemicals, methodology and experimental set up. From the control experiments carried out

such as with only dye, FM and in dark conditions, we clearly understand that only

phycobiliproteins along with traces of metal ions left in the Spcw (F I-III) has acted as a

photosensitizer to promote the degradation of selected organic dyes by satisfying the

fundamental laws of photochemistry to produce activated metal-protein complexes which aid

in photosensitizing activity. This novel, cost effective, simple and an eco-friendly light

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induced organic dyes degradation method in future may also open new doors for the sewage

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treatment plants at reduced costs.

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Acknowledgement

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This research did not receive any specific grant from funding agencies in the public,
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commercial, or not-for-profit sectors. We, acknowledge our gratitude to Shibin Exports,
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Chengelpet, Natura Overseas, Singaperumal Koil and Tamilnadu Fisheries University, near
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Madhavaram for their constant support in providing us Spcw.

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Conflicts of interest

There is no financial conflict of interest or other interests that could be perceived to influence
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the outcomes of the research here presented.

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Statement of informed consent, human/animal rights

No conflicts, informed consent, human or animal rights applicable.

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Legends for figures
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Fig. 1 A–C Representing the % of dye degradation for triplicates of MB, MG and CR
after treating with F I- III and exposing to the sunlight for 3 h. MB, MG and
CR completely degrades at 2, 1 and 3 h respectively.
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Fig. 2 Quantitative estimation of protein content using Bradford's reagent indicating

the amount of protein in FM - 15 mg/ml; crude extract (t) of Sp 140 mg/ml; F
I-III - 92, 79 & 90 mg/ml respectively.
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Fig. 3A-C Represents the effect of F I-III on the degradation of dyes such as MB, MG
and CR. A complete degradation occurs at 2, 1 and 3 h respectively which
are sampled out at regular intervals of about every 30 min.
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Fig. 4 Represents the kinetics study of the dyes degradation.

Fig.5 A&B SEM - EDX analysis of FM (A) and F - I showing fiber like structure (B)
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Fig. 6 FT-IR spectra of Sp, F-I and FM showing various functional groups.

Fig. 7 I & II. 6 I - Represents the optimum pH for MB, MG and CR as 10, 8 and 5
respectively for maximum degradation.
6 II - Effect of pH on degraded dyes showing no change in color which
indicates complete degradation.

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