OPTIMIZATION OF Cr AND Cu BIOSORPTION BY GREEN MARINE ALGAE Caulerpa racemosaVar. Cylindracea & Ulva lactuca

* Krishna Y. Pandya 1,2 , Rinku V. Patel 1,2 , Rakesh T. Jasrai 3 and Nayana Brahmbhatt 2 . 1. Sophisticated Instrumentation Centre for Applied Research and Testing, Vallabh vidhyanagar-388120, Gujarat, India. 2. Department of Biology, V. P. Science College, Sardar Patel University, Vallabh vidhyanagar-388120, Gujarat, India. 3. Department of Chemistry, R. K. Parikh Arts & Science College, Sardar Patel University, Petlad-388450, Gujarat, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Biosorption potential of the Caulerpa racemosa var. cylindracea & Ulva lactuca (Chlorophyta) were studied in removal of toxic heavy metals such as chromium (Cr) & copper (Cu) from industrial reactive azo dyes effluents in batch mode. The experiments were carried out in ambient conditions at pH 7, 50 & 60 minutes of equilibrium time was obtained in Caulerpa racemosa & Ulva lactuca respectively. The maximum 20.23 % Cr and 42.91 % Cu removal was observed in Caulerpa racemosa. In Ulva lactuca 62.2 % Cr and 70.35 % Cu removal was observed. The biosorption yield towards Cu > Cr observed in both seaweed biomass. Ulva lactuca gives potential biosorption yield as compare to Caulerpa racemosa. .The data are well fitted by the Freundlich and Langmuir isotherms shows the favorable biosorption of dye effluent by green seaweed biomass. The data on the kinetic studies fitted well and shows the adsorption kinetics of dyes effluent by both green seaweed biomass followed the pseudo-second order model for biosorption of Cr and Cu, only pseudo first order model was observed in Cr treatment by Ulva lactuca. The SEM analysis shows the uneven surface and cells were damaged & swollen after treatment with effluent and FTIR study reveals the variation in functional groups and decrease in peak intensity indicates the participation of metal binding inside the cells of the biomass. Thus the present study indicates this biomass can be used as effective, eco friendly and low cost material in biosorption of highly polluted water containing heavy metals.

…………………………………………………………………………………………………….... Introduction:-
In recent years the pollution level is raised with increased human activities, the government and industries need to take action against pollution reduction at source and initiation for waste water cleanup. Among various pollutants dyes and heavy metals are very hazardous for living organisms and aquatic life if ingested inside the body they cause carcinogenic and mutagenic effect. At present there are various physical, chemical and biological process are ISSN: 2320-5407 Int. J. Adv. Res. 5 (8), 923-939 924 available for treatment such as coagulation, flocculation, sedimentation, filtration, ionization, ozonation, ionexchange, oxidation/reduction, reverse-osmosis etc. but these treatment technologies required high maintenance, high consumption of energy, high cost and toxic slurries generations. Therefore it is an urgent requirement to apply alternative treatment method which is efficient, eco-friendly and low cost [1].
Biological treatments are environment friendly and cost effective process applied using biological materials such as algae [2][3] [4], living or non-living bacteria [5][6] [7], yeast [8] [9], fungi [10] [11] and plants [12] [13]. Biosorption is a process of removal of toxic heavy metals from the waste water using biological material which is alternative treatment technology involves biosorbent containing favorable functional groups and a solvent/liquid with dissolved sorbate group. Through various mechanisms the sorbate groups are attracted and bound with the sorbent and the sorbent possessing higher affinity towards the sorbate groups. The reaction continues till the equilibrium is developed between the adsorbent bound sorbate groups and the amount left in a solution [14]. Further metal sorption mechanisms using biosorbent occurs via chelation, ion-exchange, micro-precipitation, co-ordination, complexation etc [15]. The potential of seaweeds as biosorbents for removal of heavy metal has been studied by various scientists in recent years offers continuous availability and reusability of sorbent in numerous cycles. Brown and green seaweeds are studied most for the biosorption research [16] [17]. Nasab et al., 2017 [18] reported 53 to 80 % of metal removal efficiency for 0.1 to 0.5 g/l of biomass of Gracillaria corticata. Badescu et al., 2016 [19] investigated biosorption capacity of Ulva lactuca reached 29.63 mg Zn (II)/g of biomass which was further applied as fertilizer to improve soil quality. Amongst various heavy metals such as Cu(II), Ni(II), Cd(II), Pb(II) R.S. Praveen and K. Vijayraghavan, 2014 [20] studied highest metal ions affinity in aqueous solution as Pb>Cd >Cu >Ni by red algae Kappaphycus alvarezii. In brown seaweeds the cell wall is comprised of alginate and green & red algal cell wall is comprised of cellulose, carrageenans and agar [21] however the more research is required to study the potential of seaweeds in biosorption of metals Therefore the present study investigates the potential of green algae Caulerpa racemosa and Ulva lactuca (Chlorophyta) in the biosorption of Cr and Cu as they are the largest tropical green algae with fastest growth rate [22]. Although seaweeds possess different commercial applications such as in food industry due to high carbohydrate and protein content in aquaculture waste water treatment as they uptake potassium, phosphorus, nitrogen for their growth and survival, thus successfully cleanse the aquaculture waste water. These seaweeds possess successful vegetative reproduction, high growth rate and having plastic morphology. Thus it is desirable to study the biosorption potential of green seaweeds as an alternative application. The present paper investigates the binding mechanisms and kinetics behind the biosorption of Cr and Cu by Caulerpa racemosa and Ulva lactuca.

Experimental:-Sample collection & characterization of collected dyes effluents:-
The survey of industrial dyes manufacturing industrial units of Gujarat state, India has been carried out and untreated effluent samples of reactive dyes industries were selected for biosorption study. The selected effluent samples entitled as E1, E4 and E6 which were comprised of mixture of reactive red dyes, reactive yellow dyes and reactive black dyes respectively. E1collected from dyes manufacturing industrial units of Padra GIDC, Vadodara district; E4 collected from Vatva GIDC, Ahmadabad district and E6 collected from industrial unit at Kalamsar village, Khambhat taluka, Anand District. As shown in our previous study [23].
The effluent samples E1, E4 & E6 were comprised of mixture of chemical components and metals; physico chemical parameters were analyzed and indicated in previous research article. In the present study the effluent samples were analyzed by ICP-OES (Model-Optima 3300 RL, Make-Perkin Elmer) for determination of heavy metal concentration of Cr and Cu followed by standard methods of APHA-AWWA, 1985 [24]. These metals are toxic to aquatic life and living organisms if discharged without prior treatments into the water bodies. The concentration of heavy metals represented in Table-

Seaweed collection & identification:-
The seaweed species Caulerpa racemosa var. cylindracea and Ulva lactuca (Chlorophyta) were collected from Beyt-dwarka near Okha coast, Gujarat, India (Longitude -68°20´ E to 70°40´ E Latitude -22°15´ N to 23°40´ N). The collected seaweeds immediately washed with seawater at source to remove unwanted debris, adhering sand particles and epiphytes then it was kept in icebox and immediately transferred to laboratory. The seaweed species washed three to four times with distilled water to remove surface salt and impurities. These seaweed species were identified at Fisheries department, Junagadh Agricultural University, Okha, Gujarat with method described by M. Umamaheswara Rao [25]. This seaweed biomass were dried by keeping it under sunlight and then crushed in mixture to get its powder form. These powdered biomasses of green seaweed were used in further study.

Heavy metal sorption by batch Expeiments:-
The batch experiment was carried out to determine dynamic biosorption of Cr & Cu from the dyes effluents by biomass of Caulerpa racemosa var. cylindracea and Ulva lactuca. The batch adsorption experiments were conducted by inoculating 2 g of seaweed biomass in 200 ml of dyes effluents in 500 ml of conical flask at pH 8. The experiments were performed in flask shaker at constant agitation speed of 80 rpm for better contact of surface area of the biomass and ion transfer at room temperature (28°C). The sample was taken at every 10 minutes of interval thus the experiment run time was prepared for 90 minutes and immediately filtered the adsorbent biomass with Whatman filter paper No. 40 to make effluent adsorbent free. The residual heavy metals in filtered effluent samples were analyzed by atomic absorption spectrophotometer (Make: shimadzu, Model: 1800). The equilibrium biosorption q e (mg/g) was calculated by Where, Co is the initial concentration (ppm) and Ct is the heavy metal concentration (ppm) in filtrate effluent taken at time t; V is the volume (ml); & W is the weight (g) of the biomass taken as adsorbent. The heavy metal removal in percentage was determined by the following equation (3) [27] Biosorption yield (%) = (3)

Heavy Metal Analysis:-
The effluent samples were taken for heavy metal analysis. The adsorbent free filtered effluent samples entitled as E1, E4 and E6 were digested with aqua-regia of HCl: HNO 3 (1:3 V/V) in hot plate and then diluted with double distilled water. These light yellow colored samples after digestion were analyzed for Cr & Cu by Atomic Absorption Spectroscopy (Make-Shimadzu, Model-AA 7000) where the computer desktop is attached with the instrument so the results were digitized and printed.

Scanning electron microscopy (SEM):-
The study of morphology of the seaweed biomass surface of Caulerpa racemosa var. cylindracea and Ulva lactuca were studied by Field Emission Gun-Scanning Electron Microscopy (Make: FEI Ltd., Model: Nova NanoSEM 450) for before and after treatment of effluents E1, E4 & E6.

Fourier Transform Infrared (FTIR) Analysis:-
The FT-IR spectra of dried untreated and treated seaweed biomass were taken with FT-IR spectrophotometer (Make: Perkin Elmer, U.S.A., Model: Spectrum GX). It was obtained by KBr disk method. The seaweeds samples were ground with mortar and pestle for 5 min after dried it by blow drier for 15-30 minutes. 2 mg of dried samples were taken with 200 mg of KBr (spectroscopic grade) were carried out with additional crushing and prepared pallets by hydraulic pallet press and the spectra were scanned between 4000 to 400 cm -1 range under ambient conditions.

Result and Discussion:-
In the present study, the Figure-1 indicates quantity absorbed in ppm as function of contact time by green seaweed biomass Caulerpa racemesa var. cylindracea and Ulva lactuca with the Cr and Cu. This study reveals that seaweed Caulerpa racemosa var. cylindracea and Ulva lactuca shows rapid absorption initially and reaches equilibrium at 50 926 minutes and 60 minutes time periods respectively; after that the absorption becomes constant and slowly decreased. This was occurred because of vacant space availability initially on the biomass surface therefore absorption becomes rapid as a result the concentration rise between adsorbate in the effluent and adsorbate on the seaweed biomass becomes higher. The process initially becomes rapid and reaches to equilibrium because of intra-particle diffusion in the cells of the biomass from bulk to surface and then it slowly decreased because of minimization of availability of vacant sites in surface of biomass and repulsive forces of molecules cause to slower down the absorption rate of reaction [27]. Thus biosorption process is dependent on attraction forces and diffusion process in the molecules and availability of vacant sites on the surface.    [36]. Ulva lactuca (B) shows higher affinity towards Cu > Cr and gives potential biosorption yield as compare to Caulerpa racemosa. The affinity towards metals is depending on ionic charge, ionic size and hydrolysis constant of the respective heavy metals [32][36] [37].

Biosorption isotherms:-
The adsorption isotherms (Langmuir and Freundlich) used to derive wide range of sorbent concentration to study adsorption characteristics such as equilibrium concentration of adsorbate in the bulk and gain of metals onto the surface of adsorbent. The Langmuir and Freundlich isotherms have been applied in the present study; generally they are commonly used in the sorption study. These models have been successfully applied to explain the adsorption of metals [38][39] [40]. The Langmuir equation [41] described in linerized form: The Figure Table-2. The essential characteristics of the Langmuir isotherm can be expressed using constant separation factor RL which is indicated in the following equation [42]: In the above equation the RL value indicates the character of adsorption to be either unfavorable (RL > 1), linear (RL = 1), favorable (0 <RL < 1), or irreversible (RL = 0). The Freundlich isotherm [43] is indicated by the following relation: (6) Where K f and n are Freundlich constants; it can be determined by intercept and slope of the Figure-4 (4,5,6,10,11,12) which indicates the adsorption capacity and intensity respectively represented the values in Table-2.

Equillibrium studies:-
The values of Freundlich and Langmuir parameters are represented in Table-2. The plots of Langmuir isotherm and Freundlich isotherm shown in Fig-3 and Fig-4 for biomass of Caulerpa racemosa and Ulva lactuca which shows adsorption of heavy metals on the adsorbent biomass. The straight lines satisfactory describes Langmuir model for equilibrium adsorption process. The Langmuir and Freundlich model was found favorable for all the effluent samples and metals as indicated in Table- [44]: ln (qe -qt) = ln (qe -K 1 t) (7) The pseudo second order model in linerized form is given by the Ho [45] is: (8) Where, the K is pseudo-second order rate constant for adsorption (g/mg/time) & q e & q t were the content of metals (mg/g) adsorbed at equilibrium & time t respectively. The linear plot of t vs t/qt was obtained which indicates the kinetic data fitted best in pseudo-second order model.
The linear plot of ln (qe-qt) Vs t for pseudo-first order reaction and t/qt vs t for pseudo-second order reaction of the adsorption of metals onto Caulerpa racemosa and Ulva lactuca are shown in the Fig-5-8. The correlation coefficients are closer to the correlation coefficient of second-order kinetics than the pseudo-first order kinetics which suggests that the pseudo second order model followed by both the biomass, only pseudo first order model was observed in Cr treatment by Ulva lactuca.
(31) (32) Figure 8:-Pseudo-first order & Pseudo-second order plot for Cu by Ulva lactuca (29)(30)(31)(32) 931 The Image-1 and Image-2 represents untreated original seaweed biomass of Caulerpa racemosa and Ulva lactuca respectively and E1, E4 and E6 represents after effluent treatment images. This study shows that before exposure of the effluent the surface of the biomass cells were found smooth and uniform, having certain dimensions, after the exposure of effluent the surface of the biomass represents uneven surface and the cells were found swollen and damaged which represents the accumulation of dyes functional groups inside the surface of cells and linked with their functional groups. The heavy metals and component structures occupies the free binding sites inside the cell wall which exchange cations represents strong cross linking of the metals and complexes due to ion exchange mechanism [46].
The recorded spectra give information regarding functional groups variations for before and after treatment with effluent containing heavy metals (Fig-9 to 16 Table-3. The IR spectra of Caulerpa racemosa and Ulva lactuca indicates minor changes in the peaks frequencies because of the binding with heavy metals & dyes effluent functional groups resulted in decline in adsorption frequencies. These binding taken places due to presence of active sites of the biomass. Thus the analytical study of FTIR indicates the presence of ionizabe functional groups inside the biomass which has ability to interact with cations and other functional groups [47][48] [49]. Thus it can be concluded that functional groups play very important role in the removal of cations and breakdown or transformation of the molecules of the dyes effluent.

Conclusion:-
The present study concludes that green seaweed biomass of Caulerpa racemosa var. cylindracea and Ulva lactuca has high biosorption capacity for heavy metals. The biosorption of Cu is found maximum than Cr in both seaweed biomass. Ulva lactuca shows higher affinity towards Cu > Cr and gives potential biosorption yield as compare to Caulerpa racemosa. The highest 20.23 % Cr removal and 42.91 % Cu removal was observed in Caulerpa racemosa and in Ulva lactuca 62.2 % Cr and 70.35 % Cu removal was observed. The data are well fitted by the Freundlich and Langmuir isotherms show the favorable biosorption of dye effluent by green seaweed biomass. The data on the kinetic studies fitted well and shows the adsorption kinetics of dyes effluent by both green seaweed biomass followed the pseudo-second order model for biosorption of Cr and Cu, only pseudo first order model was observed in Cr treatment by Ulva lactuca. The SEM and FTIR analysis indicates the uneven surface, cells were damaged & swollen after treatment with effluent and the variation in functional groups, and decrease in peak intensity indicates the participation of metal binding inside the cells of the biomass. Thus the study indicates considerable potential of green seaweed biomass as biosorbent for the removal of heavy metal from effluent which can be applicable on field level.