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M. Girilal1*, Abraham Varghese2, V. Krishnakumar3 and P. T. Kalaichelvan4
1Department of Biotechnology, SB College, Changanacherry- 686 101 , Kottayam, Kerala.
2George Clinical, # 333, Nova Miller, Thimmaiah Road, Bangalore- 560052, Karnataka.
3Department of Zoology, University of Madras, Guindy Campus, Chennai - 600 025, Tamilnadu.
4Alka Research Foundation, Anna Nagar, Maruthamalai Adivaaram, Coimbatore - 641 046, Tamilnadu.
e-mail: girilal@gmail.com
*For correspondence
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Abstract
Silver nanoparticles were synthesized biologically from the fungal species Trichoderma viridae. The synthesized biogenic silver nanoparticles were immobilized using yeast cells as well as sodium alginate beads and were used to decolourize dye. The rate of decolourization was effective using silver nano particles modified yeast cells, Saccharomyces cerevisiae. Decolourization was due to adsorption of textile dyes by silver nanoparticles modified yeast cells and adsorption capacity was enhanced up to 500 ppm within 2 hours for selected test dyes. Silver nanoparticles modified yeast cells were immobilized in sodium alginate and can be introduced to the dye treatment plant when going for a large scale dye treatment applications. Since the dye is properly attached by ionic interaction with the immobilized modified yeast cells, it can be removed easily, releasing back the treated waste water. Adsorption was mainly due to the ionic interaction of silver nanoparticles modified yeast cells with dyes. A schematic diagram is proposed explaining the ionic interactions between silver nanoparticles modified yeast cells and dye.
Keywords
Silver nanoparticles, Saccharomyces cerevisiae, adsorption, textile dyes.
Introduction
Pollution control is one of the prime concerns of today’s society. With economic constraints on pollution, affordable and effective methods have become a necessity to control the pollution. Untreated or partially treated wastewaters and industrial effluents are discharged into natural ecosystems that pose serious problems to the ecosystem and life forms. Among the various types of organic pollutants present in the ecosystems, chemical colouring agents or dyes are the most difficult to decompose and may act as carcinogens. Textile industries are the main source of dye that pollutes the environment. As the production of textile industry increase, volume of wastewater containing processed textile dyes also increases steadily (Bhole et al., 2004; Mohamed , 2004).
Different approaches have been used to reduce the textile dye pollution in aquatic medium. Generally chemical and physical treatment procedures are used to treat the textile pollution (Hao et al., 2000); but they are less effective and highly expensive. Among various adsorbents used for the removal of dyes from polluted waters, the use of biological materials, (live or dead microorganisms) are more efficient due to their biosorption and biodegradation properties and cost effectiveness.
Although dye decolourization can be achieved by bacterial (Kumar et al., 1997) and fungal (Sathiyamoorthi et al., 2006) degradation, increasing demands for effective and economical technologies have led to research into a biosorption-based process and have been used effectively in the effluent treatment processes mainly for heavy metals and dyes. In this study, adsorption of some common textile dyes were carried out using biogenic silver nanoparticles modified Saccharomyces cerevisiae immobilized in sodium alginate.
Materials and Methods
Silver nanoparticles (AgNPs) were synthesized from fungus Trichoderma viride, as per the optimized protocol by Fayaz et al. (2009). To prepare the biomass for biosynthesis of AgNPs, the fungus was grown aerobically and the fungal biomass was mixed with 100 ml of sterile double distilled water and agitated on an orbital shaker at 150 rpm for 48h at 27oC. After incubation, the cell filtrate was filtered through Whatman filter paper no. 1 and to 100ml of cell filtrate AgNO3 was added and kept undisturbed for 24 h in dark conditions to get an overall Ag+ ion concentration of 10-3 M.
The yeast, Saccharomyces cerevisiae was procured from Rhibozen chemicals, India and it was modified by the following optimized procedure as given below. The yeast cell pellets (1.5 g) were suspended in 2 ml of de-ionised water and centrifuged at 5000 rpm. The pellet was resuspended with 2 ml of 0.1M acetate buffer (pH 4.6). The suspension was centrifuged and finally the pellet was dissolved in acetate buffer. Yeast cell suspension was kept in boiling water bath and the killing time was optimized at 5 minutes interval (5 to 20 min). The silver nanoparticle and heat killed yeast cell suspension ratio was optimized. The suspension was vortexed and incubated at room temperature (27oC) for one hour in an orbital shaker. After incubation, the modified yeast cells were washed with de-ionized water and it can be used for further experimental works.
The textile dye stock solution was prepared from the selected anionic dyes like Reactive Black 5, Remazol Brilliant Blue R, Reactive Navy Blue and Acid Red at the concentration of 100 – 500ppm.
The modified yeast cells were immobilized using calcium alginate beads and blended in 2 % (w/v) sodium alginate solution. Calcium alginate beads were prepared using 0.2 M calcium chloride. The beads were allowed for curing in calcium chloride solution (one hour). The hardened beads were washed with sterile de-ionised water in order to remove excess CaCl2. The treated dye solutions were incubated in orbital shaker at 120 rpm.
Dye decolorization rate was measured in a Spectrophotometer (Milton Roy Spectronic 601) at 498nm for Reactive Black 5, 630nm for Remazol Brilliant Blue R, 607nm for Reactive Navy Blue and at 507nm for Acid Red. The readings were taken every 10 minutes interval up to 120 minutes. The rate of decolorization was calculated by the following formula:
Where A0 is the absorbance of untreated dye, A is absorbance after treatment
All values are expressed as means ± standard deviation. The results were analyzed using one-way analysis of variance (ANOVA) and the differences of the decolorization percentage among the dye were analyzed using the Tukey-Kramer multiple comparison test. P value <0.001 was considered as significant. The software GraphPad InStat was employed for the statistical analysis.
Results and discussion
Although various adsorbents and materials have been tested and used for the removal of dyes from polluted water, biological based procedures (living or dead microorganisms) found to be cost effective and eco-friendly. Both adsorption on the surface of the cell (Mahmoud, 2014; Nilanjana and Charumathi, 2002) and exploitation of cell’s enzymes for biodegradation (Sathiyamoorthi et al., 2006) were used for removal of textile dyes from wastewater.
Nanoparticles were also reported to be used in dye treatment (Poedji et al., 2013) but had the problem of more environmental pollution due to the discharge of particles in nanoscale dimensions and it is unpredictable how these nanoparticles will react once it comes to the outer environment.
Since many biodegradation processes proved to be cumbersome and difficult to operate at a large scale because of lack of proper optimization and stringent conditions. The current work was an attempt to remove the textile dye using silver nanoparticles modified yeast cells immobilized in sodium alginate, by optimizing various parameters.
For getting stabilized modified yeast cells and to avoid the problem of colonizing the yeast cell in the waste water, dead yeast cells were used in the experiment. The heat killing time of yeast cell was optimized and a significant effect was found to be 15 minutes for all the dyes tested (P<0.001) (Table-1).
Table - 1. Different heat killing time of yeast cells on percent decolourization. Values (x±s.d) with different letters are significantly different (P<0.001) for the same day.
The heat killed yeast cell suspension and silver nanoparticle ratio was optimized and significant result was obtained from the ratio of 2:1 (Table-2).
Table - 2. Ratio of heat killed yeast vs nanoparticle on percent decolourization. Values (X±s.d) with different letters are significantly different (P<0.001) for the same day.
The experiments were carried out with heat killed yeast cells immobilized in sodium alginate (Control), silver nanoparticles modified yeast cells immobilized in sodium alginate against the four different test dyes and the results were compared. All the dyes were tested from 100 – 500 ppm concentrations. Significant results were obtained from all the four dyes treated with modified yeast cells when compared to yeast cells without nanoparticles. In all the dyes tested, faster dye adsorption rate was obtained in the lower dye concentrations (100- 200ppm) when compared to higher dye concentration (400- 500ppm). But 75% of dye adsorption was achieved within 40 minutes of incubation for all the yeast cell treatments even at the concentration of 500 ppm. In the previously reported works, only 56.57% and 52.57 % of decolourization were achieved by treating with Pleuortus florida and Trametes hirsuta and that too on the fifth day of incubation (Sathiyamoorthi et al., 2006) and by the addition of 2% glucose on the dye effluent.
The results obtained for control and silver nanoparticles modified yeast cells at 500 ppm of the test dye were plotted as graph and discussed. The maximum decolourization rate was observed in the Reactive Black 5, showed 98.89% with modified yeast cells and the control 66.34% (500 ppm) within 120 minutes of incubation (Fig.1).
Figure 1. Rate of decolourization of reactive black dye using silver nanoparticles modified yeast cell and control yeast cells
In the case of Remazol Brilliant Blue R, 86.2% and 66.47% of decolourization was obtained in 500 ppm by modified yeast cell and control respectively (Fig.2).
Figure 2. Rate of decolourization of Remazol Brilliant Blue R dye using silver nanoparticles modified yeast cell and control yeast cells.
In case of Reactive Navy Blue, an effective dye adsorption was found to be 87.21% by modified yeast cells and control was 66.76% in 500 ppm (Fig.3).
Figure 3. Rate of decolourization of Reactive Navy Blue dye using silver nanoparticles modified yeast cell and control yeast cells.
The maximum dye adsorption was recorded in Acid Orange dye treatment as 91.09% and 66.13% by modified yeast cell and the control respectively at 500ppm concentration (Fig.4).
Figure 4. Rate of decolourization of Acid Orange dye using silver nanoparticles modified yeast cell and control yeast cells.
The ionic interaction of biogenic silver nanoparticles and the dyes played a pivotal role in the efficient dye adsorption. Sliver nanoparticles were synthesized biologically from T. viridae and the proteins plays a critical role in synthesizing and stabilizing the silver nanoparticles and thus giving a net negative charge to the nanoparticles (Fayaz et al., 2011). These negatively charged silver nanoparticles form an ionic binding with the positively charged dye particles and make the adsorption efficient. A possible mechanism of interaction of the silver nanoparticles modified yeast cells and dye is illustrated as a schematic diagram (Fig.5)
Figure 5. Schematic diagram illustrating the ionic interaction between silver nanoparticles modified yeast cell with dye molecules.
The significance of the present work is that decolourization of all the selected dye were achieved in very short course of time (120 mins) for the concentration of 500ppm, using modified yeast cells. Since the modified yeast cells are immobilized using sodium alginate beads, it will settle after incubation time with adsorbed dye and will remain settled in the bottom of the tank. The beads along with the dye can be easily removed without disturbing the clear water and the treated water can be released back to water sources.
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