ORANGE-G TOLERANCE , UTILIZATION AND DEGRADATION POTENTIALS OF NATIVE BACTERIAL ISOLATES

Soil and sediment sample obtained from Orange-G dye contaminated habitat was screened for heterotrophic bacterial population. Consistently, high heterotrophic bacterial density was recorded to the tune of 107 CFU/g. Six isolates comprising of three Pseudomonas sp. two Escherichia sp. and one Bacillus sp. were checked for Orange-G tolerance and utilization. All the bacterial strains were found to resist the azo dye, Orange-G up to 75 ppm. Above this concentration, only Pseudomonas sp. SACO3, Bacillus sp. SAC01 and Escherichia sp. SAC01 were able to tolerate. All the test bacterial strains were found to utilize Orange-G as a sole carbon and / or nitrogen source with distinctive decoloration. While the pH optima of these strains ranged from 8 to 10, their temperature optima was 37°C except for Escherichia sp. (44°C). In accordance with the abundant occurrence in the soil ecosystem, Pseudomonas sp. were found to decolorize orange-G more effectively that other strains.


INTRODUCTION
The textile industry in India is one of the oldest and largest industry in the country.These mills require volumes of water of high purity and generate equally large volumes of wastewaters, which is highly colored and chemically complex.The textile mill effluent contains appreciable concentration of pollutants.The BOD and TSS can be controlled by biochemical treatment (Sharma et al., 1999).Color removal from textile effluent is a major environmental problem because of the difficulty in treating such enormous quantity of water (Sharma et al., 1999) Triphenylmethane and azodyes are used extensively in textile industry for dying nylon, wool, silk and cotton (Mali et al., 1999).Azodyes constitute the largest group of synthetic dyes with great deal of structural and colour variation (Cripps et al., 1990).Azodyes are potential mutagens and carcinogens, which necessitates their proper degradation and safer disposal (Vyas and Molitoris, 1995).Microbes can degrade azodyes both aerobically and anaerobically.Decolorization of the dyes usually occur during secondary metabolism, was suppressed at high nitrogen levels, and was dependent on the concentration of atmospheric oxygen (Platt et al., 1985).Azodyes' aromatic moieties is linked together by azo (-N=N) chromophores, with different auxochromes viz.NH 2 , NR 2 OH.The release of these compounds into the environment is undesirable, not only because of the principle compound but also their breakdown products are toxic and / or mutagenic (Van der zee, 2000).
Orange-G is a monoazo dye and is sparingly in water (10.86%).This is a valuable acidic dye used in many staining methods including Papani color's 0G6 stain.It is often combined with other yellow dyes in alcoholic solution to stain erythrocytes in trichome methods and is used for demonstrating cells in the pancrease and pituitary (William et al, 1998).
Realizing their wide spread usage and subsequent risk involved as a constituent of the industrial effluent, this preliminary investigation was taken pu.In this attempts were made to

Biosciences, Biotech. Research Asia
Volume 01, No. (2) 87-91 (2003) assess the dye contaminated soil ecosystem bacteriologically and the residual bacterial strains were examined for their dye resistant and decoloration characteristics.

Sampling site
Soil samples were collected aseptically from five different azo dye contaminated sites in Rajapalayam and Nagercoil, Tamil Nadu.

Sample collection
Sampling was carried out a weekly interval from the dye contaminated site.Soil sample from a visibly polluted area with textile effluent was aseptically collected in a pre-sterilized 250 ml conical flask.The sample was placed in an ice box maintained at 4°C and was brought to the laboratory for bacteriological analysis within 4-6 hours.

Enumeration of Total Heterotrophic Bacterial Population (THBP) in the soil sample
One gram of dye contaminated soil sample was serially diluted using 9 ml sterile saline and the dilution was made upto 10 -8 .From this, 1 ml of diluted soil sample was poured into sterile petridish to which 20 ml of the sterile nutrient agar was added aseptically and mixed well.After solidification, the petriplates were incubated at 37°C for 24 hours.The plates containing enumerable bacterial colonies were selected and the THBP determined.

Isolation of Orange-G tolerant bacterial isolate
The dye contaminated soil sample was enriched in Orange-G (10 ppm) incorporated nutrient broth and kept in an orbital shaker (37°C) for five days.One ml of this culture was taken, serially diluted and pour plated on air-dried nutrient agar medium.The plates were incubated at 37°C for 24 hours.Morphologically different colonies were selected from the plates purified by quadrant streaking on air-dried nutrient agar medium and stored in nutrient agar slant.

Identification of Orange-G resistant bacterial isolates
Pure cultures of Orange-G resistant soil bacterial isolates were identified upto generic level on the basis of their microscopic, biochemical and physiological characteristics (Aiso and Simudo, 1962).For this, Bergey's manual of systemic bacteriology and the Prokaryotes were also referred.

Dye utilization assay (qualitative)
To determine the ability of the Orange-G tolerant strains to utilize the chosen dye, plate assay technique was employed.For this, MSA without carbon (C) or nitrogen (N) source was prepared and the Orange-G was incorporated into this medium at 75 ppm concentration.Also MSA was prepared with Orange-G either as the sole C and N source (50 ppm) or with C and N supplementation.On these plates, saline washed suspension of the chose bacterial isolate was single streaked and its ability to utilize the dye namely Orange-G was recorded.

Optimization studies Determination of temperature optima-
Nine ml of sterile MS broth was inoculated with one ml of saline washed bacterial culture and incubated at different temperatures (30°C, 37°C, 44°C).The OD 595 was taken at different intervals (24 hours, 48 hours, 72 hours, 96 hours) in a spectrophotometer (Systronics 118) with uninoculated medium as the blank.

Determination of pH optima-
The pH of the MS broth was adjusted to 6, 8, 10 with 1N HCl or 1N NaOH and one ml of saline washed bacterial culture was inoculated and incubated at their respective optimal temperature.The OD 595 was taken at different intervals (24 hours, 48 hours, 72 hours, 96 hours) in a spectrophotometer (Systeronics 118) with uninoculated medium as the blank.

RESULTS AND DISCUSSION
Coloring agent has become the integral part of human development.Apart from contributing aesthetics sense, dyes also provide multiple applications, which includes scientific and other fields.Most of the coloring agent used in the textile industry are azodyes that have characteristic -N=N, which is stubborn molecule that resist natural degradation.Probably, this stubbornness makes these dyes as choice coloring agent in most industries.On the contrary, contamination of these dyes and their subsequent accumulation in an ecosystem pose serious threat to the environment.While the recalcitrant dye and its metabolites alter the soil porosity, and chelate with ions, they were also observed to exert negative impact on the soil micro and macro flora.
Azo dyes constitute the largest group of synthetic dyes with a great deal of structural and color variety (Cripps et al, 1990).They are extensively used in textile, leather, food, cosmetics, pharmaceuticals and paper industries and eventually more than 7 x 10 5 tons of these dyes are produced annually world wide.Realizing the importance of selecting suitable bacteria for dye degradation and also to understand the impact of the dye industry effluent on soil microflora, textile mill effluent contaminated soil was enumerated and the residual bacterial load was recorded to be in the range of 10 7 CFU/g (Table 1).Appreciable THBP in a soil ecosystem inspite of continued exposure to azodyes is suggestive either of the enrichment of dye degradable population or the dilution of the dye, which had eventually lowered the toxicity of the dye.Sudhakar et al, 2002 had reported similar load of THBP in sediment samples collected from a dye contaminated habitat.
From THBP, 6 strains were chosen on the basis of their morphological dissimilarity and their generic identity was determined by employing standard bacterial identical techniques.As it is well known, soil is predominantly inhabited by the gram negative Pseudomonas sp. and gram positive Bacillus sp.Bacteria belonging to these genera are known for their abilities to utilize the variety of simple and complex nutrients and hence could survive even in hostile conditions.While Pseudomonas sp. by virtue of the presence of a wide variety of extra chromosomal genetic material survive environmental hostilities, Bacillus sp. has the ability to switch to dormancy in the event of physical, chemical or biological threats.Hence, occurrence of bacteria belonging to the genera of Pseudomonas and Bacillus in the dye contaminated soil is rather an anticipated one.But isolation of an enteric organism, Escherichia sp. is suggestive of the probable contamination of domestic sewage in the textile effluent that might have contributed to the entry of this strain.
As a presumptive to decolorization experiments, the chose bacteria were examined for their ability to tolerate the dye namely, Orange-G.Even though all the isolates were able to resist the stain up to 75 ppm, only three strains namely, Bacillus sp.SAC01, Escherichia sp.SAC01 and Pseudomonas sp.SAC01 were found to tolerate Orange-G even at high concentration of 100 ppm.The inability of the native isolate to tolerate high concentration of Orange-G confirms the possible negative effect of the accumulation of the Orange-G on the native micro flora.Further, these strains were examined for their ability to utilize orange-G as sole nutrient, which could indicate their degrade potentials.Examining the data presented in Table 3 it could be inferred that the chosen soil bacterial isolates not only have ability to utilize Orange-G as the carbon or nitrogen source, but also as a sole carbon and nitrogen source.Their ability of dye utilization was evident from the appearance of zone of decoloration around the growth.Sudhakar et al (2002)    similar concentration dependent tolerance and utilization of azo dyes by the native bacterial strains such as Pseudomonas sp.
In order to employ these organisms for Orange-G decolourization, their growth conditions were optimized.In spite of having co-existed in the effluent contaminated habitat, a sharp difference in pH and temperature optima was found among the isolates.While Pseudomonas sp.SAC01 required 37°C and pH 8 at its optima, Bacillus sp.SAC01 required pH 10.Even though Escherichia strains were found to prefer pH 8 for their growth, Escherichia sp.SAC02 grew maximally at 44°C.In fact this observation is suggestive of its faecal origin.While Pseudomonas sp.SAC02 grew comparably well at both 37°C and 44°C in alkaline pH Pseudomonas sp.SAC03 preferred more neutral pH and 30°C for its maximal growth.
The metabolic potentials of the members of Pseudomonas sp. was clear in the present study.Of the three Pseudomonas strains examined, two strains namely, Pseudomonas sp.SAC03, Pseudomonas sp.SAC01 had exhibited extremely high potential in the decolourization (92.93% and 91.14% respectively) of orange G (Table 4).Zimmermann et al (1982) recorded such potentials in Pseudomonas KL46 with respect to Orange II.Observations of Coughlin et al (1997) regarding the aerobic degradation of azodye correlates with the data presented in this study with respect to the rate and percentage of dye degradation by the chosen bacterium.Even though Escherichia sp. is observed to be a very good dye degrading strain, its enteric origin discourages its The present investigation clearly demonstrates the relevance of native bacteria in bio-remedial applications.Further research on the analytical aspects of dye degradation, the fate of metabolites and molecular aspects would make it possible to assemble bacteria for the bioremediation of Orange-G polluted habitat.
had reported

Table - 4 : Rate of decoloration of Orange G by the native bacterial isolates (75 ppm)
Bacillus sp.despite of being popular in pollution management had very low efficiency in Orange-G decolourization in the present study.