Bioethics in India: Proceedings of the International Bioethics Workshop in Madras: Biomanagement of Biogeoresources, 16-19 Jan. 1997, University of Madras; Editors: Jayapaul Azariah, Hilda Azariah, & Darryl R.J. Macer, Copyright Eubios Ethics Institute 1997.
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85. Environmental Risk Caused By "Colorants"

K. Latha, A. Hilda, S. Gopinath & N.S. Kavitha,
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus; Chennai 600 025


Abstract

Industrial wastes are usually discarded into water, with or without processing. When waste substances reach such a concentration that they exert measurable effects upon ecosystems then they are said to be pollutants. Thus, the problem of environmental risk is caused by these xenobiotics.

The major environmental problem of "colorants", therefore, is the removal of dyes from effluents. The decolorization of "colorants" present in a commercially available dye Porcion Red M8B was studied since, to a certain extent, azo and reactive dyes are carcinogenic. Two fungi namely Geotrichum candidum and Paecilomyces variotii were used to decolorize the dye. Batch studies were conducted in a defined medium with pH 7.0. As decolorization progressed, there was a substantial reduction in pH to a range between 4.0-4.5, a feature common to both the test organisms. Maximum biomass growth was observed in both the test organisms on the 10th day. Maximum decolorization of Porcion Red M8B by P. variotii of up to 81.99% was seen on the 10th day only. Whereas, maximum decolorization of Porcion Red M8B by G. candidum was evidenced up to 85.76% on the 10th day of incubation. In all the cases, the mycelium of the test organisms also absorbed some amount of colour. Thus, these two potential fungi can be used for decolourising the "colorants" present in the dye which may have carcinogenic effect on humans and that they have the potentiality to minimize environmental risk.

Everything touched by King Midas turned to gold. By a sort of inversion process, pretty well everything modern men touch, including themselves, turns to a waste product sooner or later. Wastes are usually discarded into water, with or without processing. Presently, water is becoming a rare commodity, and the available water sources are inadequate to meet the essential basic needs of man, which is mainly due to increased industrialization of developing countries like India. Improper disposal methods and inadequate control of toxic effluents from different industries have led to the widespread contamination of surface as well as ground water and have made the water resources unfit for usage (Odum, 1969).

Estimation of pollution largely depends on the physical appearance of colour, odour and turbidity conditions. In many instances, colour is the quick and easy yardstick by which the pollution level is assessed. The major sources of coloured effluents are from textile, dye stuff industries, particularly where the process of bleaching, dyeing, printing and finishing in textile operations import huge amounts of coloured effluents. Textile industry is the country's largest industry. Apart from earning large amount of foreign exchange, it catches the public attention from the viewpoint of pollution. Untreated effluents from dyestuff production and dyeing mills may be highly coloured and thus particularly objectionable if discharged into open waters. Eventhough the dye concentration may be well below 1 ppm i.e., lower than many other chemicals found in waste waters, the dye will be visible even at such low concentrations (Zollinger,1987).

Physical, chemical and physicochemical methods that are available to treat these coloured effluents are expensive and do not provide satisfactory results. Biological treatment methods are cheap and offer the best alternative with proper analysis and environmental control. Almost all waste water can be treated biologically. Biologically, decolorization can be achieved by the use of a number of naturally occurring micro-organisms such as bacteria and fungi. The specific characteristics of the microbes have been exploited in the colour removal process and the enhancement through various approaches can be developed.

The mineralization or complete biodegradation of an organic molecule in water is always a consequence of microbial activity (Alexander, 1980). Ecological and public health consideration, have resulted in greater attention, being given to certain categories of microbial transformation. The initial degradation is accomplished as a result of secretion of a group of stable extracellular fungal enzymes which may include proteases, pectinases, lipases, celluloses and ligninases. The action of these extracellular fungal enzymes may provide a mechanism by which some organo-pollutants are made more accessible for biodegradation (Griffin, 1981).

To ascertain the ability of micro-organisms to degrade coloured effluents of an industrial source, two potential organisms, namely, Geotrichum candidum and Paecilomyces variotii are used as test organisms to decolourize a commercially used reactive dye, such as, Porcion Red M8B. The "Porcion" dyes are a range of reactive dyes whose fixation is achieved by chemical linkage of the dye with the fibre. By the use of Porcion dyes a wide range of shades, possessing good fastness to light and washing, can be obtained. Three distinct classes of Porcion reactive dyes are available.

Porcion H Dyes: They are the less reactive type.

Porcion Supra Dyes: They have reactivity and stability and are improved over "H" type.

Porcion M Dyes: These are the most reactive type and are, therefore, more versatile in application.

Geotrichum candidum and Paecilomyces variotii had the capability of decolourizing the colourants present in the Porcion dye. The strains were maintained in Potato-Dextrose-Agar medium consisting of: Potatoes 200 g; Dextrose 20 g; Agar 20 g in 1 L of distilled water (pH 7.0). In order to find out their maximum decolourisation potential they were grown in a basal medium, Czapex-Dox medium, consisting of: sodium nitrate 2 g; dipotassium hydrogen phosphate 1 g; magnesium sulphate 0.5 g; potassium chloride 0.5 g; ferrous sulphate 0.01 g; sucrose 30 g in 1 L of distilled water (pH 7.0). Basal medium was prepared, sterilized and raised in 250 ml conical flask containing 50 ml, which included 1 ml of spore suspension and 1 ml of dye. Sterile control, without inoculum was also maintained under similar conditions.

Analysis of samples

Decolorization: The dye - Porcion Red M8B - was prepared to give a final concentration of 100 um (10 fold concentration) and filter sterilized (using 0.45um pore size Millipore membrane filter). The dye was added to conical flasks containing 50 ml basal medium & 1 ml of 5-day old spore suspensions of the test organisms, and incubated in a rotary shaker at 110 rev/min. at 30¯C. The experiment was conducted in duplicate. The decolorization efficiency exhibited by the test organisms was estimated.

During the incubation period, samples were drawn at 48 hours interval for 10 days. They were then analyzed for decolorization, after centrifuging the samples at 10,000 ug for 20 minutes.

Initial - Final

absorbance absorbance

Value Value Decolorization % = ------ x 100 Initial absorb. value

The degradation of the dye by the organisms was monitored in a Spectrophotometer (Philips 8470) by scanning the culture supernatant. Sterile control was maintained throughout the incubation period and was taken as the blank value in the absorption peaks.

Absorbance of dye bound to the Mycelium: During incubation, the intensity of the dye in solution was reduced due to fungal adsorption as well as by fungal transformation. Hence, it is necessary to find the dye bound to the mycelium. For this reason, the absorbed dye was solubilized with 10 ml of water and the mycelium was centrifuged. Again the pellet was suspended with 5ml of distilled water and recentrifuged. The supernatant collected by these two operations were combined and the absorbance values were measured. From this the degree of absorption of dye bound to the mycelium was determined.

Wet mycelial pellet, separated by centrifugation, was dried at 80¯C for 5 hours in hot air oven and the dry weight was taken. The pH of the supernatant was recorded using Orion EA 940.

Results And Discussion

Spectral characteristics

The decolorization was monitored by scanning the absorbance at wavelength ranging from 200-700 nm. The scan profile recorded the highest peak for Porcion red M8B at 543 nm. The absorbance was assayed at every 48 hrs. throughout the incubation period. As the decolorization proceeded the concentration of dye decreased in terms of their absorbance value. While, P. variotii showed complete decolorization of 81.99% Porcion red M8B on the 10th day , the decolorization of Porcion red M8B was 85.76% on the 10th day of incubation by G. candidum (Table 1 &2) . The spectral characteristics showed that dye decolorization was in progress due to decrease in peak value i.e., reduction of the dye concentration.

In all the cases, the mycelium of the test organisms also absorbed some amount of colour. There was a general decrease in the percentage adsorption to mycelium as the decolorization of dye proceeded, thus, indicating that there was an initial transformation of dye to the mycelium. Much of the initial, rapid loss of dye from the culture fluid appears to be due to adsorption by the mycelia. The results of the study coincided to that reported by Cripps et al.(1990), who had reported that much of the initial, rapid loss of dye from the culture fluid appears to be primarily due to adsorption by the mycelia. They showed that biodegradation and adsorption bound to mycelia are important processes in the removal of dyes from the incubation media.

Biomass

Biomass loading plays an important role in the decolorization of `colourants', since, it enhances the degradation of the dye. G.candidum on Porcion Red M8B showed a maximum value of 0.2014 g/50 ml on the 10th day of incubation with a decolorization efficiency of 85.76% (Table 1). While, P.variotii showed a biomass value of 0.3573 g/50 ml on the 10th day of incubation with a decolorization efficiency of 81.99% (Table 2). Thus, there is a correlation between the biomass value and the decolorization efficiency percentage thereby indicating the significance of the biomass.

pH

Batch studies were conducted in a defined medium with pH 7.0, for both the test organisms, namely, G. candidum and P.variotii. As the decolorization progressed there was a substantial reduction of pH to a range between 4.0 - 4.5 exhibited by both the test organisms (Table 1 & 2).

Reduction in pH of the medium might be due to the presence of conversion products. Therefore, it is suggestive of the fact that while the fungus was biologically active, the degradation process gives rise to metabolites and the accumulation of organic acids may be considered as the most probable reason for the pH decrease. The findings of the present study agree with the observation made earlier by Livernoche et al. (1983). According to them, there was a correlation between the variation of pH and colour removal.

Table 1: Decolourisation of Porcion Red M8B by Geotrichum candidum
Incubation Time(Days)
Biomass g/50ml
Absorbance of Supernatant
Absorbance of dye bound
Decolourisation %
Adsorption to mycelium %
pHvariation during incubation
Control- 1.194 - - - 7.00
20.0360 0.975 0.716 18.34 59.975.91
40.1487 0.708 0.494 40.70 41.375.63
60.1776 0.633 0.282 66.38 23.624.69
80.1983 0.308 0.205 74.20 17.174.51
100.2014 0.170 0.045 85.76 3.774.01

Table 2: Decolourisation of Porcion Red M8B by Paecilomyces variotii
Incubation Time(Days)
Biomass g/50ml
Absorbance of Supernatant
Absorbance of dye bound
Decolourisation %
Adsorption to mycelium %
pHvariation during incubation
Control- 1.194 - - - 7.00
20.2023 0.642 0.579 46.23 48.496.89
40.2500 0.459 0.432 61.56 36.186.22
60.2744 0.389 0.331 67.42 27.725.63
80.2979 0.301 0.270 74.79 22.615.12
100.3573 0.215 0.201 81.99 16.834.50

In this study, the aerobic biodegradation of dye by the test organisms namely, G.candidum and P.variotii have been described. Based on the results obtained during the studies, the following conclusions were drawn:

a) Decolorization is an important step in the degradation of the dye and the complete decolorization was achieved by the organisms when the pH of the medium was in a range of 4.0 -4.5.

b) During the incubation period in the decolorization process by the micro-organisms, namely, G. candidum and P. variotii, the pH of the medium always showed a decrease from the initial conditions indicating the release of the degradation products.

c) Both the processes, degradation as well as transformation of dye colour bound to the mycelium are involved in the decolorization of the dye by the test organisms.

The findings in the present study can serve as an important base for the development of a economical/ecoethical as well as a simplified biological treatment system using micro-organisms for providing reusable clean water for industrial as well as for recreational use, besides the upkeep of a generally more healthy and enjoyable environment.

Finally, it is to be remembered that pollution and contamination exist as the end-products of resource processes and cannot sensibly be viewed outside this context; attempts to "cure" environmental risks without considering the whole of the relevant resource process are as useful as trying to cure lung cancer with aspirins. Hence the primary importance of recycling wastes as sources of raw materials. The alternative is clear: man, a material-using organism, must also make moral choices and try to aim for a preferred limiting.

Acknowledgement

The authors thank Prof. A. Mahadevan, Director, Centre for Advanced Studies in Botany, University of Madras, Chennai - 25, for his encouragement. The financial support by the U.G.C. and DoEn are greatly acknowledged.


References
Alexander, M. and Lustigman,B.K. 1966. Effect of chemical structures on microbial degradation of substituted benzenes. J.Agr.Food Chem. 14: 410-413.
Crips, C. Bumpus, J.A. and Aust, S.D. 1990. Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 56: 1114-1118.
Griffin, D.H. 1981. In : Fungal Physiology. Wiley, New York. 336- 339.
Livernoche ,D. Jurasek, L. Desrocheers, M. and Dorica, J. 1983. Removal of colour from Kraft mill waste waters with cultures of white rot fungi and with immobilized mycelium of Coriolus versicolor. Biotechnology & Bioengineering. 25: 2055-2065.
Longstaff, E. 1983. Dyes and Pigments. 4: 243.
Odum, E. P. 1969. In: Fungal Physiology. 2nd Edition. Saunders, Philadelphia.
Zollinger, H. 1987. In: Colour Chemistry - synthesis, properties and applications of organic dyes & pigments. VCH Publishers, New York. 92-102.

Please send comments to Email < Macer@biol.tsukuba.ac.jp >.

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