Decolorization of Mordant Orange-1 by Marinobacter algicola MO-17

Problem statement: The textile industries use different types of dyes in their processing units which are liberated in natural marine water b odies. This release of dyes in turn affect the mari ne flora and fauna very adversely. Approach: The release of textile dyes into the environment i s a matter of concern due to the toxicity, mutagenicity, carci nogenicity and xenobiotic nature. Hence, considerable attention has been given in determinin g the ability of marine microorganism in decolorization and degradation of textile dyes. Results: The present study deals with the decolorization of Mordant Orange-1 was carried out using the accli matized Marinobacter algicola MO-17 isolated from natural marine environment. The acclimatized s train was capable of decolorizing 800 μg mL of the dye at high salt concentration (12%) in 24 h. T e percent decolorization of the dye was moderated by spectroscopy at 385 nm ( λmax). Conclusion/Recommendations: The spectrophotometric analysis showed 78.78% decolorization in Nutrient Broth and 76.78% decolorization in Half Strength Nutrient Broth. The percent Chemical Oxygen Demand (COD) red uction of the dye by the strain was 90%. From the study performed, we conclude that, this ac climatized strain can prove better option for bioremediation of textile dyes in wastes containing high salts and in marine environment.


INTRODUCTION
Water is essential to human life and to the health of the environment. As a valuable natural resource, the sea is an immense and practically unexploited source of new potentially useful biologically active substances possessing great diversity. Large number of textile industries are located on the coastal areas due to ease of transport to the various places in world and help in building nations economy, but on the contrary the effluents released from these industries are proving a great problem for the marine life.
Large amount of water is required for the processing (dyeing) of the fabric in textile industry, which inturn releases large amount of effluent in the environment. Annually, approximately 2,80000 tonnes of textile dyes are discharged in industrial effluent worldwide (Jin et al., 2007). These dyes in large amount are liberated in marine environment. Dyes undergo chemical changes as well as biological changes in the aquatic system, consume dissolved O 2 and thus disturb the aquatic eco-system (Huang et al., 2008). Consequently, survival of life fishes and other organisms becomes difficult, especially in environmental water (Rahman et al., 2009). All of these dyes persist in the environment due to their high stability against light, temperature, detergents, chemicals and microbial attack (Couto, 2009). Disposal of these dyes into the environment causes serious damage, since they may significantly affect the photosynthetic activity of hydrophytes by reducing light penetration (Aksu et al., 2007) and also they may be toxic to some aquatic organisms due to their breakdown products (Hao et al., 2000). Dyes can be removed from wastewater by chemical and physical methods including adsorption, coagulation, flocculation, oxidation and electrochemical methods (Lin and Peng, 1994;1996;Supaka et al., 2004). However, both the physical and chemical methods have many shortcomings such as high-energy costs, high-sludge production. Hence, economical and biofriendly approaches are needed to remediate dye containing wastewater.
Marine fungi degrade and remineralize the lignocellulose substrates by their extracellular lignin degrading enzymes. Such fungi were isolated from decaying mangrove wood, leaves, seagrass and algae (Raghukumar et al., 1994). Marine microorganisms are of immense use in the decolorization of dye because most of the industrial effluents contain high concentrations of salts, especially chlorides and salts. Besides, marine microbial enzyme would act efficiently at neutral/alkaline pH and wide range of temperatures to combat the neutral/ alkaline dye effluents (Chandramohan, 1997).
Therefore, it may be economical to develop alternative means of dye decolorization such as bioremediation as it is an environmentally friendly, cost effective treatment technology (Hao et al., 2000;Verma and Madamwar, 2003). Microbial decolorization and degradation is an environment friendly and cost effective means to different conventional treatment technologies (Gogate and Pandit, 2004;Uddin et al., 2007).
In the present study, a bacterium was isolated from marine water capable of decolorizing Mordant Orange-1 textile dye. This strain was studied for decolorization of the dye Mordant Orange-1 in various different conditions like in complete nutrient medium, in half strength nutrient medium and in presence of different co-substrates. The decolorization of the dye was monitored spectrophotometrically (Systronics-106) at its specific absorbance maxima (λmax) 385 nm. Percent COD reduction of the dye was calculated.
Analytical Grade dye purchased from Sigma-Aldrich (USA). • Marine water from different places.

Acclimatization and isolation of microorganisms:
Soil samples from nearby salterns (salt pans), compost and textile sludge from sludge drying beds of textile industry were collected and mixed properly. The microflora from the homogenized samples were acclimatized by adding increasing concentration of NaCl (0.5-12%) and after a week, the dye in increasing concentration was added to this soil for the period of one month. One gram of acclimatized soil was inoculated in nutrient media containing 0.5-12% NaCl. Isolation of promising bacteria was carried out on nutrient agar having the same NaCl concentration and Mordant Orange-1 at a final concentration of 10,000 µg mL −1 . The colony showing zone of decolorization of the dye on the plate was designated as MO-17 and was selected for further studies.

Decolorization of dye in nutrient broth:
The selected culture was then inoculated in 25 mL nutrient medium (Peptone -1.0 g, NaCl -0.5 g, Beef Extract -0.3 g, Distilled Water -100 mL, pH -7.2) containing 12% NaCl concentration and 10,000 µg mL −1 concentration of dye. These tubes were then incubated at ambient temperature for 24 h and observed for decolorization of the dye. Percent decolorization was determined.
Decolorization of dye in half strength nutrient broth: Isolated culture was then inoculated in 25 mL half strength nutrient medium (Peptone -0.5 g, NaCl -0.25 g, Beef Extract -0.15 g Distilled Water -100 mL, pH -7.2) containing 12% NaCl concentration and 1ml of 10,000 µg mL −1 concentration of dye. These tubes were then incubated at ambient temperature for 24 h and observed for decolorization of the dye.

Decolorization studies in presence of different Cosubstrates:
The isolate was inoculated in 25 mL nutrient broth (Peptone -1.0 g, NaCl -0.5g, Beef Extract -0.3 g, Distilled Water -100 mL, pH -7.2) containing 12% NaCl concentration, 1ml of 10,000 µg mL −1 concentration of dye and 1% Glucose. Tube was then incubated at ambient temperature for 24 h and observed for decolorization of the dye. Additionally, nutrient medium containing 1% Starch and 1% Yeast Extract with the same dye and NaCl concentration were also used to test the ability of the isolate MO-17 to decolorize the dye Mordant Orange-1.

Percent decolorization study:
The percent decolorization studies were done by using spectrophotometer. The percent decolorization was calculated by using the formula as: Initialabsorbance final absorbance Percent decolorization= 100 Initialabsorbance − × Percent COD reduction: Percent COD reduction was determined by reflux method using Potassium dichromate as a strong oxidizing agent.

Isolation and identification:
The organism was isolated from marine environment on nutrient agar with 12% salt concentration. It was then identified by using biochemical tests and 16s rRNA analysis technique. The biochemical results showed, Oxidase, Catalase positive but unable to ferment Glucose, Sucrose, Lactose and Maltose. The phylogenetic tree was developed by using Neighbor joining method by Kimura-2-parameter with 1000 replicates in MEGA 4.0 (Fig. 1).

Percent decolorization of dye in nutrient medium:
The promising isolate MO-17 was studied for its ability to decolorize the dye Mordant Orange-1 in nutrient medium containing 12% NaCl concentration and 1ml of 10,000 µg mL −1 concentration of dye. The percent decolorization results of dye Mordant Orange-1 in nutrient broth is given in Table 1.

Percent Decolorization of Dye in Half (½) strength nutrient medium:
The promising isolate MO-17 was studied for its ability to decolorize the dye Mordant Orange-1 in half strength nutrient medium containing 12% NaCl concentration and 1 mL of 10,000 µg mL −1 concentration of dye.  The results of percent decolorization of dye Mordant Orange-1 in nutrient broth is given in Table 1.

Percent decolorization of dye in presence of different co-substrates:
The promising isolate MO-17 was further studied for its ability to decolorize the dye Mordant Orange-1 in nutrient medium containing 12% NaCl concentration, 1% Glucose, 1% Starch and 1% Yeast extract as co-substrates and 1ml of 10,000 µg mL −1 concentration of dye. The results of percent decolorization of dye Mordant Orange-1 in presence of different co-substrates is given in Table 2.
Percent COD reduction of dye: The Percent COD reduction of the dye Mordant Orange-1 after decolorization by Marinobacter algicola MO-17 was 90%.

DISCUSSION
The isolate decolorize the dye substrate and decolorizing efficiency was dependent on the growth of the isolate in the tubes. There was neither growth nor decolorization in the control tubes. This showed that the decolorization was due to the metabolic activity of the organism.
Halophiles have been reported to be involved in the dye decolorization (Khalid et al., 2008a). It has been investigated that the decolorization of azo dyes by a member of the genus Shewanella: Shewanella putrefaciens strain AS96 under hypersaline conditions (Khalid et al., 2008b;Ammozegar et al., 2010).
It has also been reported that reported halophilic microorganism Shewanella putrefaciens to be capable of the complete removal of Reactive Black-5, Direct Red-81, Acid Red-88 and Disperse Orange-3 (all 100 mg L −1 ) within 8 h in presence of 40 g L −1 NaCl (Ammozegar et al., 2010). In the present study, the extreme halophilic Marinobacter algicola MO-17 isolated from acclimatized soil decolorized the dye Mordant Orange-1 to an extent of 78.88% in nutrient medium, 76.78% in half strength nutrient medium. This rate of decolorization may be due to the high metabolic diversity being observed in the halophiles due to their extremophilic nature (Oren et al., 1992;Ventosa et al., 1998). Decolorization in marine water medium; indicate that the organism can be used for effluents containing high salt content.

CONCLUSION
The isolated strain MO-17 was identified as a member of the genus Marinobacter by 16 s rRNA sequence analysis and biochemical tests. The decolorization of the dye in half strength nutrient broth was comparatively less than in Nutrient broth but this method will be cost effective. The COD of the dye was reduced considerably after the treatment of Marinobacter algicola  The results of the present study indicate that the isolate from Marine environment Marinobacter algicola could decolorize the dye Mordant Orange-1 efficiently and the organism can be used for the bioremediation of textile dye Mordant Orange-1 in Marine Environment.