Treatment of Dye Industry Effluent Using Free and Immobilized Cyanobacteria

In the present study, dye industry effluent was treated with Cyanobacteria for removing colour and other nutrients. Oscillatoria brevis and Westiellopsis prolifica were selected for the study based on their dominant occurrence in the effluent. Organisms were used in both free and immobilized conditions. These organisms not only removed the organic and in organic chemicals but also reduced the intensity of the colour from the effluent. The result revealed that within 30 days, more than 75% of colour has been removed. Nutrients such as nitrites, phosphates and ammonia were completely removed. Increase in Dissolved Oxygen (DO) content and reduction of Biological Oxygen demand (BOD), Chemical Oxygen demand (COD) up to 95% have been reported. Among the two conditions, immobilized cyanobacteria were more effective than that of free cells. Generally the effluent supported the growth of Oscillatoria and Westiellopsis, but the growth was not well pronounced as compared to control. It is concluded that Oscillatoria had a little edge over than Westiellopsis, can successfully be used not only to reduce pollution load but also for colour removal purposes. Journal of Bior emediation & Biodegradation J o u r n a l o f B ior em ediation & Biegra d a t i o n


Introduction
Now-a-days urban people are facing many problems and water pollution is one of them. Pollution of water and land due to hazardous toxic wastes has become a global concern. The industrial, municipal and agricultural wastes, which are legally or illegally discharged into the environment, are responsible for environmental pollution [1][2][3].
With an increased awareness about the need for a clean environment, industrialists, environmentalists and policy makers are to be forced to look for the efficient, cheap and eco friendly method for the treatment of waste water.
Physico-chemical methods of waste water treatment are inevitably cost intensive and cannot be employed in all industries especially in developing countries like India. Hence, in recent years, the importance of biological treatment systems has attracted the attention of many workers all over the world and helped in developing relatively efficient, low-cost waste treatment systems. Algal systems, more particularly the cyanobacteria are not only useful in treating the waste but also producing a variety of useful products from the biomass [4].
Dye industry effluent causes one of the major environmental problems due to its removal of colours from the effluent prior to discharge of local sewage treatment. Bright coloured water soluble dyes are problematic. Not all dyes, currently used can be degraded or removed with physical and chemical processes and sometimes the degradation products are more toxic [5]. Several combined aerobic and anaerobic microbial process, using adapted mixed populations; (marine algae) are believed to enhance the degradation of dyes [6]. Though, lot of works on dye effluent using physico-chemical method have been carried out [7,8] very little work on the degradation of dye using biological organisms are available [9,10] and particularly there is no work on treatment of dye industry effluent using natural habitats of immobilized cyanobacteria. Suspended cultivation of microalgae has been employed to remove various nutrients and inorganic chemicals. However, some difficulties limit the practical application of suspended microalgae which include monospecificity and good operation conditions are hard to be separated from efficient before discharge. Recently, the use of immobilized micro algae for the removal of nutrients from waste water shows the potential to solve the above problems [11,12]. Immobilized living cells have some advantages over free cells as they provide simple treatment for liquid, final separation of cells not required and metabolic activities remain constant for longer periods [13]. The most commonly used matrices for micro algae are agar, alginate gels, polyurethane and polyvinyl foams. A disadvantage of agar and alginate is their low mechanical stability for long term use in bioreactor; more over calcium alginate gels are disrupted by phosphate ions. Polyvinyl and polyurethane foams offer better mechanical properties and are neutral to most commonly used ions [14]. Hence, the present work was carried out to study the treating dye industry effluent using polyurethane foam for entrapment of cyanobaterial cells.

Materials and Methods
Dye industry effluent was collected from Modern Dye Industry, Tiruppur, Tamilnadu, India. Remazol and venyl sulfone dyes were used by the industry. In order to select a suitable organism for the treatment process, cyanobacterial populations were collected at different places from where the effluent was collected. Isolation and identification of cyanobateria was done by using standard manuals [15][16][17][18]. Among the cyanobacteria, Oscillatoria brevis and Westiellopsis prolifica dominantly occurred (Figure1). They responded well to the treatment of dye and industry effluent. The gene sequencing analysis was employed for confirmation and got the accession no as DQ208679, 680, 681, 682. The cultures were maintained in BG 11 medium [19]. To study the role of Immobilization of cyanobacteria in polyurethane foam (PUF) cubes was made using standard methods [20]. Scanning electron microscopic study on PUF immobilized cyanobacteria, using standard methods, was carried out [21] Physico-chemical parameters including BOD, COD were analyzed using standard methods [22]. Colour removal was studied by optical density and visual appearance.
Experiments were conducted in duplicates and repeated at least three times. The experimental data represents the mean of the ± standard deviations, ANOVA test. The experiment was conducted for a total period of 30 days under laboratory conditions. Since the effluent was dark brown in colour and the growth of Oscillatoria and Westiellopsis was very slow in the effluent, a long duration was provided to get a culture of exponential growth. Effluent samples were periodically analyzed (Every 3 rd day) for various physico-chemical parameters. Cyanobacteria were harvested at each time and growth was measured in terms of chlorophyll 'a' as a biomass component [23].

Chloride 4699
Except pH, all the values are expressed in mg l -1 .   The result of the initial physico-chemical analysis of the effluent is given in the Table 1. Higher amount of oxidizable organic matter, traces of dissolved oxygen, considerable amount of calcium, phosphates, nitrates and alkaline pH were probably the factors favoring the growth of cyanophyceae. The findings of the present investigation are also supported by Shashirekha et al. [4].
Inoculated cyanobacteria are known to grow fairly well in different types of effluents [14,[24][25][26][27][28][29][30][31]. Among the various cyanobacteria, Oscillatoria and Westiellopsis were able to grow well in dye effluent ( Figure 2). But the growth was well pronounced in control (BG 11 medium) as compared with effluent. Immobilization of cyanobacteria, in polyurethane foam, is a very mild and convenient method of cell entrapment as they show to have good mechanical properties for continuous use in photobioreactors. They allow high cell loading and are translucent to light [3,32]. The mechanism of adsorption of cyanobacteria inside the foam was confirmed from the studies on mammalian cell adsorption and it was concluded that a primary, reversible interaction between the cell and the solid must somehow induce a secondary irreversible [33]. It is likely that in the present study, a primary reversible charge interaction between cells and matrix surface induces a secondary related to the formation of mucilaginous envelope observed by SEM and which appears to create filamentous connections between cell and matrix surface ( Figure 3). A similar formation of hydrated mucilage was observed [21] with plant cells immobilized in a polyurethane or nylon matrix.
In the present investigation, both Oscillatoria and Westiellopsis, significantly reduced the colour from the dye effluent in both free and immobilized conditions. More than 74% removal of colour was recorded in all the treatment with the incubation period of 30 days; though the immobilized cells performed marginally well over free cells (More than 80%) (Figure 4). Among the cynobateria Oscillatoria was more efficient in colour removal than Westiellopsis. Hence the effluents were used in 100% without dilution and very dark colour to achive more than 80% colour removal as the cod of both day ( Figure 4) similar observations were made in textile dyes by using various fungi [10]. They reported that Aspergillus niger and Trichoderma viride proved to be efficient in decolourization of scarlet red upto 80%. Other fungal isolates showed more than 70% decolourization of brillian violet. There are reports on the oxidation of different azo dyes by certain deuteromycetes. Also on the characteristics of important degradative enzymes involved in the process [34][35][36] recent observations were made that immobilized white rot fungus, Irpex lacteus on polyurethane foam and pine-wood was found to decolourize the dyes and effluents effectively than free mycelium [37]. The above observations were made mostly with different fungi and bacteria, but in the present study cyanobacteria were used to achieve more than 80% colour removal from the dye effluent.
The ability of the cyanobacteria to reduce pollution load in industrial waste water has been studied by different workers all over the world [3,4,30,37,38,39]. In the present study also, nearly 90 to 100% reduction of various nutrients including BOD and COD were achieved by immobilized cyanobacteria than that of free cells when treated with Oscillatoria and Westiellopsis ( Table 2). Efficiency of immobilized cyanobacteria in removing BOD and COD over free cells has also been reported [12,14]. Nutrients, such as nitrate, nitrite, and ammonia, total inorganic and organic phosphates are essential requirements for the growth of cyanobacteria. However, these organisms were effectively all forms of inorganic nitrogen in both free and immobilized conditions ( Table 2). Among the nutrients ammonia was completely removed from the effluent followed by nitrite. Though nitrate removal was also observed, the cyanobacteria could not remove nitrate completely from the effluent. Under such conditions, cyanobacteria utilized first ammonium, nitrite and nitrate. This is mainly because, ammonium is required to be least processed to incorporate into cell constituents, where as nitrate requires special enzyme for transport of nitrate into cell and then converter to nitrate and ammonium by the sequential action of nitrate and nitrite reductase. Hence, there was a complete removal of these nutrients except nitrate observed in the dye effluent when treated with free and immobilized cyanobacteria. In general, a gradual reduction of nitrate level was noticed from 9 th day onwards. On 30 th day the percentage removal of nitrate was maximum (62) in effluent with PUF immobilized Oscillatoria and minimum [40] with free cells of Westiellopsis (Table 2). Of the two cyanobacteria, Oscillatoria was more efficient in removing nitrate than Westiellopsis. It was found that there was 50% of nitrate and 100% removal of ammonia and nitrite [4,41]. Normally a gradual reduction of phosphates was noticed from 6 th day onwards. On the 30 th day, complete removal of total, organic and inorganic phosphates was observed ( Table 2). The capacity of cyanobacteria to remove large amount of phosphorus from waste water was demonstrated by several workers [4,14,25,29,38].
In the present study, there was a gradual increase in DO content    (Table 2). Increase in DO level when treated with different cyanobacteria with different effluents has already been reported [4,6,24,25,41,42]. The correlation between an increase in DO and removal of BOD and COD observed in this study agree with the observations made already [4,9].
It is very difficult to remove calcium, magnesium and chloride successfully by the conventional systems consisting of settlement tanks and aerobic digester. In the present study, the dye effluent containing these wastes was successfully treated with free and immobilized cyanobacteria. Nearly 50% reduction of chloride and more than 90% removal of calcium and magnesium were removed ( Table  2). [4,6,26,27,43 and 39] also reported similar observations while investigating the feasibility study using cyanobacteria. Similarly [40,44] reported 70% reduction of calcium and 35% reduction of chloride in paper mill effluent and sugar mill effluent respectively when treated with cyanobacteria, and particularly there is no report on dye effluents using cyanobateria for removing chloride, calcium, magnesium.

Conclusion
From the above discussion it is cleared that cyanobacteria (both free and immobilized condition) can successfully be used not only colour removal but also reduced pollution load from the effluent. Hence, the present investigation concluded that PUF immobilized cyanobacteria are potential value for removal of various chemicals including nitrogen and phosphorous. Therefore, the Oscillatoria and Westiellopsis are investigated in this study are highly recommended for beneficial bioremediation applications for in-situ and off-site removal of pollutants. The most promising species should help in the low-cost and naturally renewable technology.