Abstract: High salt concentrations in textile industrial wastewater challenge the efficiency of microbial strains used for decolorization of dye pollutants. In this study, the decolorization of Methylene blue (a phenothiazine dye) under high salt conditions using Bacillus firmus was investigated. The bacterial isolate was obtained from textile effluent as a positive enrichment culture with dye using simulated saline dye wastewater at 4% salt content. The effects of chemical parameters inherent in real textile wastewater such as dye, salt (sodium chloride) and surfactant (sodium duodecyl sulphate) concentration on decolorization of Methylene Blue (MB) by the bacterium were assessed. The bacterium showed tolerance for high concentrations of the dye as it was able to decolorize different concentrations (25-125 mg L-1) of MB albeit at varying rates. Efficient Methylene blue decolorization in saline medium was at salt concentrations between 1 and 6% while, tolerance to the effect of surfactant was at concentrations between 0.1 and 5.0 mg L-1. The isolate had a broad spectrum decolorization efficiency being able to decolorize seven structurally different dyes: Safranin (82.6%); Serva blue (76.8%); Neutral red (63.7%); Methylene blue (94.6%); Crystal violet (67.2%); Nigrosine (41.5%) and Basic fuchsin (68.1%) to varying degrees under high salt conditions. Spectrophotometric analyses in the UV-visible domain of culture supernatants indicated color removal was due to biodegradation rather than adsorption of dyes on bacterial cells. Detoxification of MB following decolorization by Bacillus firmus was confirmed using phytotoxicity studies. Results demonstrate the ability of the isolate to eliminate MB and other dyes under high salt conditions.
INTRODUCTION
Color removal from textile wastewater has been an onerous task over the last few decades and till date; problems still exist with the conventional treatment strategies used for decolorization of dyes. Recent advances in dye synthesis, resulting in better color fastness, higher stability and resistance of dyes to degradation, have made color removal from textile wastewaters even more difficult (Kaushik and Malik, 2009). Various physical/chemical methods (such as adsorption, incineration, chemical transformation, photo-oxidation or ozonation) have been implemented by various researchers (Izadyar and Rahimi, 2007; Jayarajan et al., 2011; Rezaee et al., 2008; Tchatchueng et al., 2009) in a bid to remove color from textile wastewater but these methods have been plagued with inherent drawbacks. These drawbacks include the high cost of operating these treatment methods, the involvement of complicated procedures and their limited applicability. In addition, they create secondary pollution problems as a result of the generation of significant amounts of difficult-to-dispose sludge (Forgacs et al., 2004; Kalme et al., 2007; Singh and Arora, 2011). On the other hand, biotechnological applications using microorganisms tend to provide an ecofriendly, low-cost and efficient alternative for decolorization of dyes in textile wastewater (Rai et al., 2005; Saratale et al., 2011).
Two mechanisms are involved in dye decolorization; adsorption (or biosorption) on the microbial biomass and/or biodegradation by the cells (Zhou and Zimmermann, 1993). Biosorption involves the entrapment of dyes in the matrix of the adsorbent (microbial biomass) without destruction of the pollutant. However, in biodegradation, decolorization occurs when the original dye structure is fragmented into smaller compounds. Structural fragmentation involving the cleavage of the chromophoric centre of a dye usually results in loss of color (Kaushik and Malik, 2009).
In recent past, notable advancements have been made in the use of some microorganisms for degradation of dyes in the laboratory. These microorganisms were shown to degrade and decolorize synthetic dyes, albeit to varying degrees. They include different species of fungi (Ali et al., 2008; Youssef et al., 2008), bacteria (Kalyani et al., 2009; Oranusi and Ogugbue, 2001; Rajeswari et al., 2011), yeasts (Saratale et al., 2009) and algae (Omar, 2008; Parikh and Madamwar, 2005). Although these reported strains seemed to be efficient in dye decolorization, their application has been insufficient for color removal from textile industrial effluent due to the adverse effects of some dyeing additives (salt and surfactants) on the dye-decolorizing microbes (Kapdan and Erten, 2007; Manu and Chaudhari, 2003).
Textile wastewaters are characterized by high salt concentrations due to the presence of high amount (40-100 g L-1) of sodium chloride used in dye baths to ensure maximum fixation of dye to fibres (Alinsafi et al., 2005; Carliell et al., 1998; Manu and Chaudhari, 2003; Verma et al., 2010). The sensitivity of many bacteria to this high salt content makes dye degradation by microorganisms difficult and is, currently, a limiting factor in the development of efficient biological processes for color removal (Carliell et al., 1994; Manu and Chaudhari, 2003). Also, posing a further challenge is the use of sodium duodecyl sulphate; a surfactant used as a wetting and emulsifying agent to achieve better fabric permeation and even dyeing of textile fabrics. Since it may be present in dye wastewater, it is pertinent to use efficient and suitably selected strains that are adapted to the conditions in saline textile industrial wastewater (Novotny et al., 2004). Currently, there are no reports on the effects of sodium duodecyl sulphate on dye decolorization by microorganisms.
Biological treatment of saline wastewater by pure halophilic and halotolerant bacteria has been studied by various authors. These organisms were utilized in the treatment of high salt containing wastewater (Abou-Elela et al., 2010; Asad et al., 2007; Nagasathya and Thajuddin, 2008) and in bioremediation of oil polluted environments (Margesin and Schinner, 2001). However, there is scant knowledge on the decolorization of dyes in saline wastewater containing especially phenothiazine dyes. Most of the current studies on decolorization and degradation of dyes have focused on azo, anthraquinone and triphenylmethane dyes. Decolorization of phenothiazine dyes has received very little attention, although they are an important group of synthetic aromatic dyes extensively used in the textile, pulp and paper industries. Their removal from textile wastewater is also important since their discharge into aqueous ecosystems without adequate treatment can result in decrease in sunlight penetration (which in turn reduces photosynthetic activity), dissolved oxygen concentration and water quality. These dyes can also affect humans and elicit acute toxic effects on aquatic flora and fauna, causing severe environmental problems worldwide (Dubey et al., 2006, 2007; Khataee et al., 2009; Li and Guthrie, 2010).
Hence, this study was aimed at investigating the effects of salt (sodium chloride) concentrations up to 8% and sodium duodecyl sulphate on the efficiency of phenothiazine dye removal by Bacillus firmus isolated from textile wastewater. The effects of dye concentration and culture agitation on dye decolorization were also determined while the toxicity of the products formed after decolorization was ascertained using seed bioassay.
MATERIALS AND METHODS
Dyes and chemicals: The dyes used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The dyes were filter sterilized and added to the growth medium at required concentrations. Figure 1 shows the chemical structure of Methylene Blue (MB). All other chemicals used were of analytical grade.
Organism and medium: Sludge and wastewater samples were collected in October, 2009 from a textile industry in Thessaloniki, Greece for enrichment culture. The samples were acclimatized for eight weeks in the dark and then used as stock cultures for isolation of dye degrading strains. After acclimation, the homogenized fluid was briefly filtered aseptically through four layers of gauze and stored in a top-sealed conical flask prior to use. The filtrate (10%, v/v) was added as an inoculum to 90 mL of sterile simulated saline dye wastewater medium and incubated with agitation on a rotary shaker running at 150 rpm at 30°C in a 250 mL top-sealed Erlenmeyer flask. The simulated saline dye wastewater medium was prepared with de-mineralized water and contained the following (g L-1); (NH4)2SO4 0.28, NH4Cl 0.23, KH2PO4 0.067, MgSO4.7H2O 0.04, CaCl2.2H2O 0.022, FeCl3.6H2O 0.005, NaCl 40.0, NaHCO3 1.0, dye 0.01 and 1 mL L-1 of a trace element solution containing (g L-1); ZnSO4.7H2O 0.01, MnCl2.4H2O 0.1, CuSO4.5H2O 0.392, CoCl2.6H2O 0.248, NaB4O7.10H2O 0.177 and NiCl2.6H2O 0.02. The pH of medium was adjusted to 7.2±0.1 with 1 M KOH. After a 5-day incubation, the resultant culture broth was added to fresh simulated saline dye wastewater medium (10% v/v) and incubated for another 5 days.
| Fig. 1: | Chemical structure of Methylene blue (C.I. 52015) |
This enrichment process was repeated three times and thereafter, the culture broth was placed on simulated saline dye wastewater agar using the spread-plate technique (Prescott and Harley, 2002) for the isolation of pure cultures of halotolerant dye degrading bacteria. Representative colonies with active dye decolorizing activity, identified by a clear zone encircling the colonies, were purified by streaking thrice in succession on nutrient agar medium (Merck) and then screened for their dye degradability. The best active decolorizer in saline medium after screening was selected for subsequent studies. According to the taxonomic criteria in Bergey’s manual of determinative bacteriology (Holt et al., 1994), the isolate was identified as Bacillus firmus based on its morphological, biochemical and physiological characteristics as described by Vanderzant and Splittstoesser (1992) and Cheesbrough (2000).
Spectrum of dye decolorizing activity in saline medium: The ability of Bacillus firmus to decolorize structurally different dyes under high salt conditions was checked using Safranin, Serva blue, Neutral red, Methylene blue, Crystal violet, Nigrosine and Basic fuchsin as dye substrates. Decolorization experiments were carried out in simulated saline dye wastewater medium containing 50 mg L-1 of each dye. The flasks were inoculated with the isolate and incubated at 30°C for 24 h under shaking condition. After incubation, culture samples were withdrawn for determination of absorbance. Enumeration of bacterial counts in medium was done on plate count agar (Merck) using the spread-plate technique after 10-fold serial dilutions of withdrawn samples. Bacterial counts were presented as colony-forming units per mL (CFU mL-1). Biodegradability potential was highest with MB and was used for subsequent studies.
Effect of physicochemical parameters on dye elimination: To determine the maximum dyestuff concentration tolerated by Bacillus firmus under high salt conditions, decolorization medium was prepared in flasks by varying the dyestuff concentrations (5-125 mg L-1). The flasks were then inoculated with the bacterium and incubated at 30°C for 120 h under shaking conditions. The effect of culture agitation was also determined by incubating culture flasks under static and shake (in a rotary shaker running at 150 rpm) incubation conditions. Samples were withdrawn intermittently for determination of absorbance using the spectrophotometer.
Salt tolerance experiments were performed in simulated saline dye wastewater medium containing various concentrations of sodium chloride (1, 2, 4, 6 and 8%). The simulated saline dye wastewater containing 50 mg L-1 of MB was inoculated with freshly prepared inoculum of Bacillus firmus and subsequently incubated at 30°C for 24 h. The effect of various concentrations of sodium duodecyl sulphate (0.1, 0.5, 1, 5, 10 and 50 mg L-1) in simulated saline dye wastewater medium on the decolorization process was also determined. All assays were conducted in triplicates along with sets of control flasks consisting of cell-free medium and heat-killed cells to determine effects of abiotic factors on decolorization. The former, containing only medium components, indicated the effect of medium components on decolorization whereas, the latter showed adsorption of dyes to cells. During incubation, samples from each flask were taken at different times (0, 6, 12, 18 and 24 h) and analyzed for residual dye concentration.
Analytical methods: Withdrawn culture samples were centrifuged at 12,000 rpm for 10 min in order to remove bacterial cells or suspended particles that may interfere with absorbance measurements. Absorbance of the culture supernatants was then measured at each dye’s maximum absorption wavelength (λmax) (Table 1) using the UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). The λmax of the dyes were: Basic fuchsin 542 nm, Crystal violet 584 nm, Methylene blue 665 nm, Neutral red 540 nm, Nigrosine 570 nm, Safranin 530 nm and Serva blue 554 nm. The absorbance readings were used to obtain the residual dye concentration in samples from the calibration curve for absorbance versus dye concentration which was obtained by plotting the corresponding maximum absorbance in the UV-visible spectra at different concentrations of dye. The relationship between absorbance and concentration was not affected by pH in the range of 5-10 and sodium chloride concentration in the range of 0-8%. The % color removal was calculated using the following equation:
| (1) |
where, A0 and A1 represent the initial and residual concentrations of dye, respectively. Data obtained were means of triplicate determinations.
| Table 1: | Characteristics of synthetic dyes used in the study |
Toxicity study: Phytotoxicity studies were carried out using seeds of Triticum aestivum, Hordeum vulgare and Lens esculenta. The seeds were selected for uniform size, surface-sterilized for 5 min in a solution of 1.2% sodium hypochlorite to discourage fungal growth and then rinsed three to five times with distilled water. Fifteen seeds of each plant species were sown in Petri dishes (9 cm diameter) in sets and irrigated separately with 5 mL samples of MB or its degradation product per day. Controls consisted of seeds irrigated with water for bioassay. The Petri dishes were kept in the dark and observed for germination. Seeds with radicle (>1 mm) were considered germinated (Wu et al., 2007). The germinated seeds were then exposed to day and night cycle length of 10/14 h respectively at 28±2°C. Seed germination was assessed daily while the length of plumule (shoot) and radical (root) of seeds after germination was recorded after 7 days. All experiments were carried out in triplicate.
Statistical analyses: Data obtained were subjected to statistical analysis to determine means, standard deviations of means and Pearson product moment correlation coefficient. Significant differences between treatment means and controls were determined by Analysis of Variance (ANOVA) and Duncan’s multiple range test. A significance level of 0.05 was chosen.
RESULTS AND DISCUSSION
Spectrum of activity under high salt conditions: A dye decolorizing and halotolerant bacterial strain was isolated from a textile industrial effluent in Thessaloniki, Greece after selective enrichment with dye at 4% salt content. The bacterium was a gram positive rod, a spore former, catalase test positive and oxidase test negative. The phenotypic, physiological and biochemical characteristics of the bacterium are as presented in Table 2. Preliminary experiments showed the isolate was capable of decolorizing chemically different dyes (phenothiazine, azine and triphenylmethane) tested to varying degrees within 24 h under high salt conditions. Figure 2 depicts the extent of decolorization of the different dyes by this bacterium. A maximum % decolorization of 95.20 was obtained with MB under shake-flask culture condition within 24 h while values ranging from 63.7 to 82.6% were recorded for Safranin, Serva blue, Neutral red, Crystal violet and Basic fuchsin under same conditions. Nigrosine showed the least decolorization extent at 41.5%. The relatively lower color removal obtained for Nigrosine may be attributed to the presence of sulphate groups on its phenazine molecule. Sulphate groups had been implicated in the inhibition of enzymatic attack on dye molecules as a result of the stearic effect induced by these ions or the resultant increased electronegativity which causes repulsion between dye’s negative ions and the negatively charged bacterial surface (Suzuki et al., 2001).
| Table 2: | The morphological, physiological and biochemical characteristics of the halotolerant bacterial isolate |
| Fig. 2: | Decolorization assay of various synthetic dyes under high salt conditions |
No significant changes in absorbance spectra were obtained in both controls (sterile and heat-killed cells) suggesting color removal was due to bacterial metabolism. A previous study by Li et al. (2009) had shown that only 11.30% of MB was decolorized by Pseudomonas sp. MDB1 after 48 h even though the bacterium showed high decolorization rates for triphenylmethane dyes (Crystal violet and Basic fuchsin) tested thus, suggesting its substrate specificity. In contrast, no preference of any class of dye for decolorization by Bacillus firmus was observed. This indicates its lack of substrate specificity and broad spectrum of activity which could be exploited for the biotechnological treatment of wastewater containing multiple dyes simultaneously. The varying decolorization extents obtained for the different dyes might be ascribed to the differences in the structural configuration of these dyes (Kalyani et al., 2009) as changes in the chemical structures of dyes may significantly affect microbial decolorization capability (Saratale et al., 2010).
Figure 3 shows the time courses of growth and decolorization of MB by Bacillus firmus in medium containing 4% salt under shaking-incubation condition. Cell proliferation was inversely proportional to dye concentration (r = -0.997, p<0.000) thus, giving credence to the earlier suggestion that MB decolorization might be due to cellular metabolic activities. Proliferation of cells with time in saline medium also accentuates the halotolerance of this isolate. Cells obtained after centrifugation following decolorization showed elimination of dyes from culture broth was due to biodegradation rather than adsorption, as the cells appeared colorless when viewed under the microscope. Similar observations have been made by Chen et al. (2003) who stated that dye adsorption on cells can be easily ascertained by an evidently colored cell pellet, whereas those retaining their original colors are accompanied by the occurrence of biodegradation.
| Fig. 3: | Time course proliferation of Bacillus firmus and decolorization of MB under high salt conditions |
Effect of dye concentration on decolorization in saline medium: Appraisal of the decolorizing ability of Bacillus firmus at different concentrations of MB was studied by assessing the time-dependent color reduction in simulated saline dye wastewater medium containing different initial concentrations of MB. Results obtained show the fate of the dye in medium was dependent on its initial concentration. Complete color removal was evident in culture broth containing 25 and 50 mg L-1 dye concentrations within 25 h of incubation. However, with higher initial MB concentrations, complete decolorization was obtained much later in medium containing 75 mg L-1 (48 h), 100 mg L-1 (72 h) and 125 mg L-1 (116 h) of dye (Fig. 4). Although the decolorization extent was slower at higher dye concentrations (75-125 mg L-1) when compared to the lower concentrations (25-50 mg L-1), the bacterium showed an appreciably high decolorizing performance at these high initial dyestuff concentrations. Similar trends have been reported by many authors who showed that dye decolorization can be strongly inhibited when a high concentration of dyestuff was used to examine the effect of dye on the degrading microorganisms (Gopinath et al., 2009; Kalme et al., 2007; Khehra et al., 2005). According to Jirasripongpun et al. (2007), Enterobacter sp. was unable to grow in higher dye concentrations, as it was dead when 50 and 100 mg L-1 concentrations of Reactive Red 195 were used to test its decolorizing activity. The dye was considered toxic to the cells at such high dye concentrations. The concentration of dyes in a typical industrial effluent can vary between 10 and 50 mg L-1 (Padamavathy et al., 2003). Since, Bacillus firmus demonstrated decolorizing ability at concentrations above those reported in wastewaters, the isolate can be successfully employed for treatment of dye-bearing textile wastewaters.
Data presented in Fig. 5 indicate a general decrease in rate of MB decolorization with time during decolorization of different initial concentrations of dye by Bacillus firmus under high salt conditions. At all concentrations, decay rates increased up till a certain time before a decline in rate proceeded. For 25-75 mg L-1 initial concentrations, peak decay rates were observed between 6-12 h whereas for the higher concentrations (100-125 mg L-1), decay rates peaked between 12-16 h. The highest decay rate (2.67 mg/L/h) was obtained between 6 and 12 h in simulated saline dye wastewater medium containing 50 mg L-1 MB. Increasing dye concentration slowed decay rates probably due to accumulation of inhibitory cellular metabolites in the medium or in cells, toxic effect of dyes with regard to individual bacterial cells as well as blockage of active sites of degrading enzymes by dye molecules (Jadhav et al., 2008; Saratale et al., 2009; Tony et al., 2009).
| Fig. 4: | Time course decolorization of MB by Bacillus firmus at various initial concentrations of dye under high salt conditions (insert: residence time for complete decolorization of different concentrations of MB) |
| Fig. 5: | Time course decay rate obtained for various initial concentrations of MB under high salt conditions |
An exception was the slight increase in decay rate obtained with increase in dye concentration from 25 to 50 mg L-1 (Fig. 6). Ali et al. (2008) had made similar observations which was attributed to the fact that substantial amount of substrate (dye) concentration sometimes is required for stimulating biological activity in an organism. In addition, careful examination of cells obtained at 6 h from 25 mg L-1 MB flasks had shown they were less intensely colored than those obtained from higher concentrations when viewed under the microscope. This suggests that increasing dye concentration may have been an important driving force to overcome all mass transfer resistance between dye molecules and bacterial cells.
| Fig. 6: | Concentration-decay rate plot of MB at different incubation times during biodegradation by Bacillus firmus under high salt conditions |
Effect of culture agitation on dye elimination in saline medium: MB decolorization was more evident under shake rather than static-incubation condition (Fig. 7). Under shake-incubation condition, decrease in MB concentration was from 50 to 2.4 mg L-1 whereas; decrease in concentration from 50 to 39.5 mg L-1 was obtained under static-incubation condition. This suggests that the presence of oxygen was a prerequisite for efficient decolorization of MB by Bacillus firmus. The bacterial cells grew more actively in culture medium when incubated under shaking condition than in static condition (data not shown) though changes in pH of culture medium under both conditions were insignificant (p>0.05) and generally ranged from 6.8 to 7.9 (Fig. 7).
| Table 3: | Mean degradation rates during decolorization of MB by Bacillus firmus in medium containing various concentrations of salt (sodium chloride) and surfactant (sodium duodecyl sulphate ) |
| Values followed by the same letter(s) are not significantly different (p = 0.05) according to the Duncan multiple range test | |
| Fig. 7: | Decolorization profile and changes in pH during biodegradation of MB under shake and static-incubation conditions |
Despite being a facultative anaerobe, the presence of oxygen was obviously favorable to the yield process of the isolate’s degradative enzymes. There are contradictory reports about the effect of shaking/agitation on decolorization of synthetic dyes by microorganisms. According to some authors, decolorization of dyes is enhanced by static condition (Kalyani et al., 2009; Lodato et al., 2007; Steffan et al., 2005) whereas other authors, in agreement with results of this study, have reported more efficient decolorization of similar structurally complex dyes under shaking condition than when left static (An et al., 2002; Kaushik and Malik, 2009). Higher color removal in shake cultures had been attributed to better oxygen transfer and nutrient distribution as compared to the stationary cultures (Kaushik and Malik, 2009).
Effect of salt on dye decolorization: Textile industrial wastewater contains not only dyes but also substantial concentrations of other inorganic ions. Dyeing auxiliaries (sodium chloride and sodium duodecyl sulphate) which are usually applied in combination with textile dyes, were investigated for their potential inhibitory effects on biological dye decolorization. Table 3 shows the decay rates at various salt concentrations during MB decolorization by Bacillus firmus. Decay rates were higher in medium containing 5-10 mg L-1 sodium chloride when compared to medium with no sodium chloride content thus, suggesting its requirement by the bacterium for efficient decolorization. However, with increasing sodium chloride concentration beyond 10 mg L-1, a gradual decline in bacterial activity and decolorization was obtained. No significant differences (p>0.05) in decay rates were obtained at various sodium chloride concentrations ranging from 5-60 mg L-1 (Table 3). The least decay rate (0.175 mg L-1h-1) was obtained in medium containing 80 mg L-1 sodium chloride. This salt inhibition effect may be attributed to plasmolysis and/or loss of activity of bacterial cells, inactivation and precipitation of enzymes caused by increased surface tension and hydrophobic interactions (Abadulla et al., 2000). Competition for binding sites on bacterial cell wall between dye and salt molecules, resulting in salt ions masking a repelling charge, may also be responsible for the inhibition effect. Previous reports have made similar observations in other high salt wastewater (Boonyakamol et al., 2009; Guo et al., 2005). These results show that Bacillus firmus is a moderately halotolerant bacterium with dye decolorizing activity over a broad range of salt concentrations (0-60 mg L-1). The salt tolerance of this isolate may have been facilitated by the natural selective pressure exerted by its host habitat. Textile industrial wastewaters in the study area, from where the bacterium was isolated, had been reported to have a sodium chloride concentration in the range of 0.01-1.0 mol L-1 (Dafnopatidou and Lazaridis, 2008).
Currently, treatment of wastewater from textile processing and dyestuff manufacturing industries using biological systems results in low color removal due to the substantial amount of salts in addition to dye residues contained in such wastewaters (Kaushik and Malik, 2009; Khalid et al., 2008). As a result, researchers have recommended dilution of such effluents (Wesenberg et al., 2002) before biological treatment or the use of microbial species capable of tolerating salt stress (Khalid et al., 2008). The present study has shown that colored wastewater with varying salt content (10-60 mg L-1 sodium chloride) could be decolorized effectively by Bacillus firmus thus, minimizing the need for dilution.
Effect of surfactant on dye elimination in saline medium: Sodium duodecyl sulphate is a surfactant used as a wetting and emulsifying agent in the dyeing process and its impact on dye decolorization by Bacillus firmus was investigated. Generally, increase in concentration of sodium duodecyl sulphate (0.1-50 mg L-1) in medium resulted in decrease in MB decay rate (Table 3) though, no significant differences at the 0.05 level were obtained between decay rates in control experiment and medium containing 0.1-5.0 mg L-1 sodium duodecyl sulphate concentrations. The mean decay rate in medium containing 5 mg L-1 of sodium duodecyl sulphate was 1.563 mg L-1 h-1. However, in medium containing 50 mg L-1 sodium duodecyl sulphate, the mean decay rate dropped to 0.283 mg L-1 h-1. This is the first demonstration of the effect of sodium duodecyl sulphate on decolorization of phenothiazine dye by bacteria and results indicate the capability of Bacillus firmus to tolerate moderate levels of sodium duodecyl sulphate in medium. A similar study had been carried out to determine the effect of merpol on decolorization of Reactive blue 19 by laccase (Champagne et al., 2010). The authors attributed the inhibition effect of merpol surfactant to substrate depletion in which dye molecules bind to surfactant molecules, thereby reducing the amount of free dye available to decolorizing enzymes. Surfactants, because of their amphipathic nature, are also known to alter the structure and function of cellular membranes, induce cellular lysis (Glover et al., 1999) and alter the structure and functions of important bacterial enzymes (Dong et al., 1997; Goncalves et al., 2003). From the foregoing, having a knowledge not only about dye substrate specificities but also about the effect of dyeing auxiliaries is important in selecting suitable biotechnological agents for dye decolorization under industrial conditions. Hence, microorganisms used for treatment of colored wastewater containing surfactants should, as a pre-requisite, exhibit tolerance to adverse effects of surfactants.
UV-visible spectrum analysis: Figure 8 shows the UV-visible spectral scan obtained before and after decolorization of MB by Bacillus firmus. The absorbance signature of MB dye consisted of two major peaks in the visible region (at 610 and 665 nm) within the range of wavelength scanned (300-700 nm). These two peaks in the original dye solution completely disappeared after treatment for 24 h with Bacillus firmus while a minor peak, denoting a metabolite from MB biodegradation, was obtained in the UV region of the decolorized solution spectrum. MB is a heterocyclic, planar ring aromatic compound with a phenothiazinium chromophore.
| Fig. 8: | UV-visible spectra showing absorption signatures of MB before and after decolorization by Bacillus firmus |
The obvious changes obtained in the UV and visible spectra indicate an alteration in the molecular structure of MB after decolorization. The phenothiazinium chromophore is responsible for the blue color of MB and its cleavage resulted in the change in color of medium from blue to colorless. The culture medium was incubated under shaking condition and further incubation or air bubbling of decolorized medium did not result in re-emergence of the blue color of dye. The disappearance of the absorption peaks of MB and the emergence of a new metabolite peak provided obvious evidence of biodegradation of MB by Bacillus firmus and gave credence to the earlier conclusion that decolorization by bacteria was due to biodegradation, rather than surface adsorption. Similar observations have been made elsewhere (An et al., 2002; Asad et al., 2007; Fan et al., 2009).
Phytotoxicity studies: In most areas, dye effluents are discharged with or without treatment into water bodies which are sometimes used for irrigation purposes. Thus, it was pertinent to assess the relative sensitivity of plant species used as bioassay to the dye solution before and after decolorization by B. firmus. Results of seed germination studies carried out using bio-treated and untreated dye solutions on three plant species (Triticum aestivum, Hordeum vulgare and Lens esculenta) indicated irrigation of seeds with untreated dye solution (50 mg L-1) resulted in significantly (p<0.05) lower germination of plant seeds when compared to the control and the bio-treated dye solution (Fig. 9a).
| Fig. 9 (a-c): | Effects of untreated and bio-treated MB solutions on growth parameters of Triticum aestivum, Hordeum vulgare and Lens esculenta. Values are means of triplicate determinations. (a) seed germination, (b) shoot length and (c) root length |
Seeds in control plates showed more than 98% germination for all three species whereas, irrigation with untreated dye solution gave significantly lower (p<0.05) seed germination percentages with all test plant species; T. aestivum (52) and H. vulgare (65) and L. esculenta (45). On the other hand, irrigation of seeds with bio-treated dye solution showed relatively higher seed germination percentages for T. aestivum (87), H. vulgare (90) and L. esculenta (82) when compared to results for untreated dye solution. There were no significant differences (p>0.05) between seed germination percentages obtained for control and bio-treated dye solution treatment. A similar trend had been reported by Parshetti et al. (2006) who demonstrated that germination of T. aestivum was less with Malachite green treatment as compared to its degradation product and control.
Similarly, irrigation with untreated MB solution caused inhibition of shoot growth (Fig. 9b) of the three plant seedlings. Mean shoot length of H. vulgare seedlings was 6.1 cm (control, 13.5 cm) after seven days when irrigated with untreated dye solution. For L. esculenta, the mean shoot length was 6.3 cm (control, 14.2 cm) after irrigation with untreated dye solution. In contrast, when H. vulgare and L. esculenta seedlings were irrigated with bio-treated dye solution, the mean shoot lengths obtained were 11.7 and 12.2 cm, respectively.
Figure 9c presents the effects of untreated and bio-treated dye solution on root elongation of the plants. Irrigation with untreated dye solution resulted in stunted root growth of the three seedlings. Mean root length of H. vulgare was 4.8 cm when irrigated with untreated dye solution whereas, the mean root length recorded for the control plant was 8.6 cm. However, with the bio-treated dye solution, lesser toxic effect was observed as the mean root length for H. vulgare was 7.8 cm. A similar trend was obtained with T. aestivum and L. esculenta when irrigated with treated and bio-treated dye solutions.
The inhibitory effects on plant growth parameters obtained with the untreated dye solution indicated the dye was toxic to these plants. In contrast, data obtained for bio-treated dye solution in all treatments did not vary significantly (p>0.05) with results obtained for control treatments. This shows the dye was rendered less toxic after decolorization by Bacillus firmus thus, suggesting the suitability of the treated dye solution for ferti-irrigation.
CONCLUSIONS
In this study, an acclimated bacterial strain, Bacillus firmus, isolated from textile industrial wastewater was shown to decolorize MB and other structurally different synthetic dyes under high salt conditions. Dye decolorization by this bacterium was more efficient under shake-flask condition rather than static-incubation condition. The isolate could tolerate a range of dye, salt and sodium duodecyl sulphate concentrations suggesting its suitability for biotechnological application. Decolorizing activity of the isolate was shown to be as a result of degradation of dyes rather than adsorption on cells. Phytotoxicity studies indicate detoxification of MB following its bio elimination by the isolate. This study has shown that Bacillus firmus has a practical potential for application in the efficient biological treatment of saline textile wastewater containing different dyes.
ACKNOWLEDGMENT
The authors wish to thank the Coimbra Group, Europe for granting a research scholarship to Ogugbue, C.J. during his stay at the Aristotle University of Thessaloniki. We also thank Dr. Dimitris Bellos for technical and logistic assistance.