Removal of Zn(II) from electroplating effluent using yeast biofilm formed on gravels: batch and column studies
© Basak et al.; licensee BioMed Central Ltd. 2014
Received: 27 February 2013
Accepted: 6 October 2013
Published: 7 January 2014
Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels.
The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR).
Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels.
The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm.
Zn (II) is a metal ion which is released into the environment through industrial activities at concentration of physiological and ecological concern. In the Dangerous substances Directive (76/464/EEC) of the European Union, zinc has been registered as List 2 dangerous substance . It is one of the 13 metals found in the contamination list proposed by the United States Environmental Protection Agency (USEPA) . The World Health Organisation (WHO) recommends a 5.0 mg/L maximum acceptable concentration of zinc in drinking water . Zinc is phytotoxic, and the recommended level of zinc for disposal on agricultural land is 2.5 mg/g of dried sludge solids. Effluents from the industries such as manufacture of alloys, sheet metal galvanization, TV picture tubes etc. contain high concentrations of zinc. Discharging these effluents into natural systems adjoining landmasses and sewer systems is a normal practice in small and medium scale industries which poses serious problems to the environment and ecosystems. Therefore, there is a significant need regarding the removal of zinc from effluent .
Conventional methods for metal removal include precipitation, filtration, coagulation, evaporation, ion exchange, membrane separation and solvent extraction. However, application of such processes is always expensive and ineffective in terms of energy and chemical products consumption, especially at low concentrations of 1–100 mg/L. Therefore, an alternate cost effective treatment strategy is required which will be eco- friendly. Though biosorption has been regarded as a cost effective technique for removal of Zn(II) ion using microorganisms such as bacteria [5–7], fungi [8, 9] and yeast [10–12], application of biofilm may be a better choice for Zn(II) removal from effluent.
Biofilm is a kind of immobilization of microorganisms in a solid matrix and can be applied for bioremediation of effluents. During the last few decades, biofilm reactors have become a focus of interests for the researchers in the field of bioremediation of pollutants. There are reports on the application of bacterial biofilm for zinc removal using granular activated carbon [13, 14], moving bed sand filter  and combined AS- biofilm process . Reports are scanty regarding the use of yeast biofilm for Zn(II) removal.
Biofilm formation in microorganisms is closely linked with production of extracellular polymeric substance (EPS) which acts as a glue, helping the attachment of cell surface to the submerged structures . Extracellular polymeric substance are mainly composed of polysaccharides, proteins, humic substances and uronic acid  which contains several functional groups like carboxyl, phosphoric, amine and hydroxyl groups. EPS has various functions viz. induction of cell aggregation , producing a protective barrier for cell against harmful products and allowing sorption of inorganic ions from the environment . There is report on the production of a water soluble 300kDa extracellular polymeric substance produced by yeast Candida albicans made up of glucose, mannose, ramnose and N-acetyl glucosamine using gas chromatography, gel permeation chromatography, FTIR spectrophotometer, 1H and 13C NMR spectrophotometer .
For large scale effluent treatment, continuous flow operations in column mode are more useful than batch mode. However, little effort has been focused on column study using yeast biofilm for removal of Zn(II) ion from synthetic solutions.
The aim of the present study was (i) to study the biofilm forming ability of yeast isolates and monitoring of biofilm formation through microscopic analysis viz. SEM and CLSM (ii) to quantify the amount of extracellular polymeric substances (EPS) produced by yeast during biofilm formation and characterization of EPS through FT-IR analysis and (iii) to evaluate the process of Zn(II) removal by yeast biofilm in batch mode and to study the removal of Zn(II) ion from real effluent in continuous flow column reactor packed with, gravels coated with yeast biofilm.
Materials and methods
Preparation of Zn(II) solution
Zn(II) stock solution (1000 mg/L) was prepared by dissolving 4.55 g of powdered Zn(NO3)2.6H2O (Hi Media, Mumbai, India) in 1000 ml of deionised water. The working solutions of metal were prepared by diluting the stock solution to desired concentrations.
Yeast and growth condition
Two yeast species were isolated from Common Effluent Treatment Plant (CETP), Ranipet, Vellore, Tamilnadu, India. The yeasts were phenotypically characterized and identified to species levels as C. rugosa and C. laurentii by Vitek 2 Compact Yeast card reader with software version V2C 03.01 from Council for Food Research and Development (CFRD), Kerela, India. The isolates were subcultured in YEPD (yeast extract: 10 g/L; peptone: 20 g/L; dextrose: 20 g/L) agar slant and maintained at 4°C. Sugarcane bagasse extract having 24 g/L total sugar (pH 5.0) was used as media for the biofilm formation and Zn(II) removal studies.
Analysis of effluent
Effluent was collected from Krishna electroplating works, located at Kolkata, West Bengal, India. The physico-chemical characteristics of effluent were analyzed promptly after collection using standard analytical methods . The concentration of zinc, copper, nickel and cadmium present in effluent was analyzed using Atomic Absorption Spectrophotometer (Varian AA-240, Australia).
The gravels were collected from a local nursery at VIT University. The size of the gravels was made uniform at a size of 7.5 mm by passing through the mesh. They were dipped in 1 ml culture with 5 × 108 CFU/ml of 48 h grown yeast species, placed for 90 min of adhesion phase at 28°C and were then washed with sterilized phosphate buffered saline to remove loosely adherent cells. One millilitre of sterilized sugarcane bagasse medium was added to the washed pieces and incubated at 28°C for 48 h. The biofilm thus formed was then quantified using XTT reduction assay.
XTT reduction assay
XTT (sigma, St Louis, MO, USA) solution (1 mg/ml in PBS) was prepared, filters sterilized through a 0.22 μm-pore size filter and stored at -70°C. Menadione (sigma) solution (0.4 mM) was prepared and filter sterilized immediately before each assay. Prior to each assay, XTT solution was thawed and mixed with the menadione solution at a ratio of 5 to 1 by volume. The biofilms formed on gravels were first washed five times using 1ml of PBS, and then 1 ml of PBS and 60 μl of XTT-menadione solution were added to each of the prewashed and control tubes. The tubes were then incubated in the dark for 2 h at 28°C. Following incubation, the colour change in the solution was measured spectrophotometrically at 492 nm (Hitachi U-2800) .
Morphological characterization of yeast biofilm
Yeast biofilm formed on gravels were morphologically characterized using Scanning electron microscopy (Stereo Scan LEO, Model -400) following the method of Hawser and Doughlas  and Confocal laser scanning microscopy (Olympus FV 1000, America) following the method of Sundar et al. .
Recovery and characterization of Extracellular polymeric substance
Extracellular polymeric substance (EPS) was isolated from culture supernatants of yeast species using acetone precipitation technique . The carbohydrate composition of EPS was also determined using HPLC (chromatograph Waters, Milford, MA, USA) following the method of Simova et al. . The dialysed EPS was characterized through infrared analysis using FT-IR spectrophotometer (Perkin Elmer Spectrum 1).
Batch studies on Zn(II) removal using yeast biofilm formed on gravels
Where C i is the initial concentration of Zn(II) ion (mg/L). C f is the final concentration of Zn(II) ion (mg/L).
Removal of Zn(II) from electroplating effluent in column mode using yeast biofilm
A glass column with an internal diameter of 3 cm and height 15 cm was employed in the column experiments. The column was packed with gravels coated with yeast biofilm. Effluent collected from electroplating industry containing 85 mg/L Zn(II) ions was used in this experiment. Before passing through the column, effluent was mixed with sugarcane bagasse extract, pH was adjusted to 6.0 and fed through the column at a desired flow rate using a peristaltic pump. To study the effect of bed height on Zn(II) removal in column mode, experiments were conducted at three different bed heights viz. 4, 8 and 12 cm. The effect of flow rate on Zn(II) ion removal was studied at three different flow rates viz. 1, 3 and 5 ml/min. Samples collected from the exit of the column at different time intervals were analyzed by AAS. Effluent was passed through the column till the values reached the US EPA standard (5.0 mg/L).
Results and discussion
Yeast biofilm formation
Recovery of extracellular polymeric substance (EPS)
Characterization of EPS
FT-IR analysis of EPS
The absorption bands at 2932 cm-1 (C. rugosa) and 2936 cm-1 (C. laurentii) were intensified and assigned to the stretching vibration of the methylene group (C-H). Furthermore, a continuous absorption beginning at approximately in the region of 3339 cm-1 for C. rugosa and 3401 cm-1 for C. laurentii are the characteristic of a carbohydrate ring. In comparison with the IR spectra of polysaccharides documented in literature, a characteristic absorption band appeared at 1652 cm-1 for the exopolysaccharide produced by both the yeast species. The absorption band at 1652 cm-1 were assigned to the stretching vibration of the carboxyl group (C = O). The peaks observed at 1410 cm-1 and 1318 cm-1 for C. laurentii is the symmetric stretching vibrations of carboxylic group (-COO-). A broad stretching of C-O-C, C-O at 1000–1200 cm-1 indicated the presence of carbohydrates . Specifically, the absorption band appeared at 1088 cm-1 (C. rugosa) and 1079 cm-1 (C. laurentii) ascertains the presence of uronic acid, Ο-acetyl ester linkage bonds. The presence of acidic sugars in the EPS may be important, considering the heavy metal-binding properties of the polymers . In addition, the absorption bands in the region 900-800 cm-1 were associated to the glycosidic linkage types in polysaccharides. The absorption peaks at 910 cm-1 and 861 cm-1 in the EPS produced by C. rugosa and absorption peaks at 912 cm-1and 875 cm-1 in C. laurentii exopolysaccharide revealed the coexistence of α and β glycosidic bonds . Similar FT-IR results were reported by Ma et al. .
Batch studies on Zn(II) removal using yeast biofilm formed on gravels
Removal of Zn(II) ion using biofilm formed by C. rugosa and C. laurentii was tested in growth restricted (without sugarcane bagasse extract) and growth supportive (with sugarcane bagasse extract) media at pH 5 and temperature 28°C for 12 h. It was found that Zn(II) removal efficiency by yeast biofilm in growth supportive media was more compared to growth restricted media. In the growth supportive medium, Zn removal was 35% and 29% by C. rugosa and C. laurentii biofilms respectively, whereas in growth restricted medium 25% and 21% Zn(II) ions was removed by C. rugosa and C. laurentii biofilms respectively. Therefore, further experiments were carried out with growth supportive media. In the growth supportive media, the doubling times of C. rugosa and C. laurentii were 90 min and 120 min respectively. Yeast is simple, economical, and rapid, with a doubling time in rich medium of approx 90 min to 140 mins. In addition to sugars, the aqueous extract of sugarcane bagasse also contained nitrogen compounds and other nutrients including sulphates, chlorides, phosphates, potassium, sodium, calcium, iron and copper  that enhanced the doubling time of the yeast species. The specific growth rate of C. rugosa and C. laurentii were found to be 0.135 h-1 and 0.129 h-1 respectively.
The temperature of sorption medium is important for energy dependent mechanisms in metal sorption by microorganisms . The optimum temperature for Zn(II) removal by yeast biofilm biomass was found to be 28°C at 12 h. (Figure 10B). At low temperature, the yeast biofilm biomass dry weight and Zn(II) removal was found to be less because the binding of Zn(II) onto yeast biofilm biomass took place only by passive uptake. Biofilm biomass dry weight and Zn(II) removal was found to be minimum at higher temperature. Higher temperatures inhibited the yeast growth due to reduced enzymatic activity .
Experiments were performed to study the effects of initial metal concentration on Zn(II) removal by yeast biofilm. Zinc removal efficiency was increased with increase in initial Zn(II)concentration ranging from 10 mg/L to 90 mg/L (Figure 10C). Maximum removal of Zn(II) ion by yeast biofilm occurred at 90 mg/L. The increase in initial Zn concentration increased the driving force to overcome mass transfer resistance of metal ion between aqueous and solid phases . In this study, Zn(II) removal efficiency of C. rugosa and C. laurentii biofilm was found to be 88% and 74.2% of 90 mg/L respectively. The maximum biofilm biomass (dry wt) produced by C. rugosa was 1.42 g/m2 and C. laurentii was 1.34 g/m2 at 12 h.
Treatment of effluent in column mode using yeast biofilm formed on gravels
Physico-chemical analysis of electroplating effluent
7.63 ± 0.17
6.4 ± 0.63
5.68 ± 0.20
2.1 ± 0.02
1310 ± 3.7
615 ± 5.2
1137 ± 4.1
550 ± 4.5
61 ± 1.2
23 ± 0.64
85 ± 0.64
4 ± 0.4
10 ± 0.54
1.8 ± 0.09
21 ± 0.76
2.8 ± 0.17
20 ± 0.43
2.6 ± 0.15
The present study showed the capability of the yeast species to form biofilm onto natural substrate like gravels. The EPS characterization study showed that extrapolymeric substances produced by the yeast species are made up of glucose, mannose and glucuronic acid subunits which would protect the cells from environmental chemical toxicity. This study gives an insight about the ability of artificially formed yeast biofilms on gravels to remove Zn(II) ions from aqueous solution as an inexpensive and alternative method to traditional techniques for removal of Zn(II) from waste waters. The potentiality of C. rugosa biofilm for removal of Zn(II) ions from real effluent was also studied in column mode.
Authors of this article would like to thank VIT University for providing Lab facility and financial support for the smooth conduct of the work. We also extend our gratitude towards Pondicherry University for the assistance in SEM analysis. The efforts provided by Sastra University for CLSM analysis is greatly acknowledged.
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