Bio Degradation of Reactive Textile Dyes By

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Biodegradation of Reactive Textile Dyes by

Aspergillus ochraceus (NCIM 1146)

Document By: Bharadwaj

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KEYWORDS: Aspergillus ochraceous, Biodegradation, Decolorisation, Malachite green, Reactive textile dyes, Repeated

atch mode, Xenobiotic dyes

ABSTRACT:

spergillus ochraceous (NCIM 1146) has the ability to degrade various xenobiotic dyes. This process was demonstrated by

heir decolorisation in the culture medium. The extent of biodegradation was determined by various conditions such a

omposition of media, concentration of dye, amount of mycelia and agitation. The durability of degradation activity unde

ptimum conditions was investigated in repeated batch mode. An increase in the amount of mycelia positively affected the

oncentration in repeated batch mode. Spectrophotometric data revealed that the process involved in degradation is through

microbial metabolism but not biosorption. This study showed that fungal mycelia could effectively used as an alternative to the

aditional physico chemical process.

NTRODUCTION

ynthetic dyes are extensively used in textile dying, paper printing, color photography pharmaceuticals, food, cosmetics and

ther industries. Approximately 10,000 dyes and pigments are industrially used and over 0.7 million tonnes of synthetic dyes

re produced annually worldwide. All dyes used in the textile industry are designed to resist fading upon exposure to sweat

ght, water, many chemicals including oxidizing agents and microbial attack. During processing, up to 15% of the used

yestuffs are lost in industrial effluents. Major classes of synthetic dyes used are azo, anthraquinone and triphenylmethane. In

ddition to their visual effect and adverse impact in terms of chemical oxygen demand (COD), many synthetic dyes show their

oxic, carcinogenic and genotoxic. Conventional waste water treatment plants are unable to perform a complete dye removal,

0% of reactive textile dyes persist after activated sludge treatment. Other physico- chemical methods for waste wate

ecolorisation have shortcomings due to high costs and operational problems with less efficiency. Now a day, effective

iological processes would be of great value, due to their inexpensive, eco- friendly nature and lesser sludge producing

roperties.

A number of biotechnological approaches have been suggested by recent research as of potential interest towards combating

ollution by dyes in an eco- friendly manner. They include the use of bacteria and fungi often in combination with physico-

hemical processes. Though bacteria could utilize dyestuff under anoxic conditions, the disadvantage of this method is the
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roduction of aromatic amines by these organisms.

hese amines may be toxic and carcinogenic. Aerobic bacteria usually tend to be specific towards a particular dye. Therefore

here is a necessity to develop a new method; by the single class of microorganisms most efficient in breaking down synthetic

yes are fungi.

iological degradation of triphenylmethane dyes has been widely reported red yeasts, Rhodotorulae rubra andRhodotorulae

P. Cyathus bulleri can decolorize crystal violet, malachite green and bromophenol blue. Biotransformation of malachite green

y Cunningamella elagans and Phanerochaete chrysosporium have been reported earlier. Aspergillus ochraceous showed

ytochrome P450 mediated biotransformation viz. benzo(a)pyrene hydroxylase. The aim of the present study is to investigate

he ability ofAspergillus ochraceous to degrade triphenylmethane dyes at different concentrations.

MATERIALS AND METHODS

Dyes:

Analytical grade malachite green

Cotton blue

Crystal violet

Methyl violet

Organism and Culture Conditions:

. ochraceous was used in the experiment. The stock culture was maintained on Potato dextrose agar (PDA) slants and

ubcultured periodically to maintain its viability. Loop full of culture from PDA slants were inoculated in to 250mL

rlenmeyer flasks containing 100mL of PDA medium consisting of peeled potatoes 200g/L, glucose 20g/L and yeast extrac

0.1g/L. the mycelium was grown aerobically for 96hrs at 300 c in static conditions. The harvested fungal mycelia were used

n the degradation experiments.

Decolorisation Experiments:

Decolorisation of dyes was determined as relative decrease in absorbance for each dye at their absorbance maximum a

articular time interval. In an attempt to solubilize any bound dye, the mycelia were homogenized in methanol and the

omogenate was centrifuged and absorbance of supernatant was then determined. Results are mean of three experiments

erformed at different times. Various media viz. normal water, potassium phosphate buffer (50mM, pH 7.4), potato dextrose

roth, distilled water with yeast extract (0.1g/L) and Czapek dox medium were autoclaved separately at 1210c for 20minutes

Dye solution was added aseptically to the respective media. 10g (fresh weight) of 96h grown mycelia of A. ochraceous were

ransferred to Erlenmeyer flasks containing various media (100mL) along with malachite green(0.5g/L) dye solution. The


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asks are kept on shaker(150rpm) at room temperature (300 c) decolorisation of media was measured by taking optical density

malachite green 620 nm, crystal violet -592 nm and methyl violet- 583 nm) at different time intervals. Degradation activity

s calculated as follows:

Decolorisation (%) = Initial Absorbance Observed Absorbance * 100

________________________________________ ----------- (1)

Initial Absorbance

he effect of various concentrations of malachite green (0.5 -10 g/L) and amount of mycelia (10 and 20g/ 100mL) on

egradation activity of dye using A. ochraceous was studied. Flasks containing only dye and normal water with put mycelia

were used as control. In another set of experiment, the degradation pattern of various dyes (cotton blue 0.5g/L, crystal viole

0.2g/L methyl violet 0.2g/L) byA. ochraceous was studied. Percent degradation was estimated as described for malachit

reen. Concentrations of dyes were selected which obeys Lambert Beer law.

Repeated use of Mycelium for Dye Degradation:

A fresh test dye solution (normal water, 100mL) was first inoculated with 10g of 96h grown A. ochraceous mycelia. The

egraded medium was replaced with 100mL fresh test solution for the next cycle of degradation by the same mycelia, after

ncubation period of 24hr for 0.5g/L dye concentration and 48hr for other concentrations (1.0 2.5g/L). Five such cycles were

epeated. The experiments were done for various mycelia amounts (10 and 20g fresh weight / 100mL) and dye concentration

malachite green 0.5 2.5g/L). To study the effect of phosphate on durability of malachite green dye decolorisation, the

xperiment was performed using phosphate buffer (50mM, pH 7.4) as a nutrient media.

RESULTS AND DISCUSSION:

Degradation of various dyes by A. ochraceous (NCIM- 1146): four triphenylmethane dyes were selected and decolorisation

attern was measured byA. ochraceous mycelia in normal water, without adding any inorganic compounds. This organism

howed significant ability to degrade all the dyes tested viz. malachite green (98%), cotton blue (92%), methyl violet (61%)

nd crystal violet (57%) in 24hr incubation.

Concentration of the dyes present in the media are

a= 0.5g/L c= 0.2g/Lb= 0.5g/L d= 0.2g/L

Table 1: Percentage of decolorisation of various dyes (%) byAspergillus Ochraceus (NCIM 1146)

Under the conditions at 300c and 150rpm)

Time Malachite Cotton Crystal Methyl violet


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(hr) green(a) blue(b) violet(c) (d)

0 0 0 0 0

6 57.5 47 27 22

12 72.5 63 42 40

18 90.0 75 51 54

24 98.0 92 57 61

Percentage decolorisation of textile dyes

0

20

40

60

80

100

120

0 6 12 18 24

Time (hr)

RemovalEfficiency(%)

Seri

Seri

Seri

Seri

Figure 1 Degradation of textile dyes at different time intervals

ome of the dyes appeared to be bound to the mycelium. However, even the bound dye was degraded at the end of the

ncubation period (24h). Spectophotometric analysis of methanol extracts ofA. ochraceous showed that the mycelia contained

nly 3% to 5% of the dye after 24hr incubation. When autoclaved, mycelia were tested for their ability to degrade malachite

reen. Autoclaved mycelia absorbed about 5% of malachite green and did not show degradation activity within 24hr. this

ndicates that a high percentage of degradation of malachite green is mainly due to fungal metabolism and not due to

iosorption. Similar results were also reported earlier in the case of orange II and crystal violet metabolism by fungus F29 and

hanerichaete chrysosporium.

Effect of conditions on Decolorisation pattern:

All types of media showed decolorisation activity in the range of 90% - 98%, except potato dextrose broth (82%) by A

chraceous. The best results were obtained when normal water and 50mM Phosphate buffer used as medium. Rate o

egradation was highest in the case of phosphate buffer compared to other media. Further, experiments were done using

ormal water throughout the study since it will be more useful for bioremediation process.

o test the effect of agitation on the malachite green degradation process, mycelia ofA. ochraceous (10g/100mL) were used

oth in static and shaken conditions. The highest degradation activity was obtained at 150rpm (96%) in 24hr. lowe

egradation rate (72%) was recorded under static conditions after 24hr. Knapp et al reported only degradation of orange II in


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tatic condition (45%) and shaken condition (97.5%) using wood rotting furniture. High degradation activities in shaken

onditions become advantageous over static conditions, for the development of practical process.

Degradation pattern of malachite green by A. ochraceous mycelia (10g/100mL) was tested at different initial dy

oncentrations (0.5 to 10g/L). Fungal mycelia could decolorize malachite green by 96% within 24hr when initial dye

oncentration was 0.5g/L. Increased dye concentrations (1.0 to 2.5g/L) require more time (48hr) to decolorize 82% to 92%

High dye concentration (5 to 10g/L) showed a toxic effect that adversely affected the degradation performance. The results o

his study indicate that the degradation depends on the initial concentration of the dye.

Repeated Batch Experiment:

he durability of decolorisation activity by A. ochraceous mycelia was investigated in repeated batch degradation tests

Mycelia were used in repeated batch mode under optimal conditions five times with residence time of 24hr.The experiments

were done for various concentrations of mycelia and dye. It is apparent that an increase in the amount of mycelia affects the

urability of the degradation activity. On the other hand, a decrease in the dye degradation activity ofA. ochraceous occurred

with increasing dye concentration during repeated bath mode. This suggests that high concentration of the dye shows its toxic

ffect resulting in to decrease in degradation activity.

A significant change in the degradation activity was observed when 20g/100mL mycelia were used. 77% degradation was

ecorded in 24hr against 48hr required for 70% degradation, when 10g/100mL mycelia were used in first cycle at higher dye

oncentration (2.5g/L). It indicates that time course of degradation decreased with increasing mycelial quantity. In the firs

ycle, the degradation ability was highest at various dye concentration; however, it declined sharply on reuse of mycelia

10g/100mL) especially at high concentration with increase in time course.

Table 2: Effect of malachite green dye concentration on decolorisation performance (%)

Time

(hr)

Removal efficiency of malachite green in normal water (%) (g/L)

0.5 1.0 1.5 2.0 2.5 5.0 10

0 0 0 0 0 0 0 0

6 63 44 27 16 19 18 12

12 75 53 38 31 29 22 17

18 93 71 49 42 39 24 19

24 96 82 60 59 52 24 20

48 96 96 92 86 82 25 22

hese results show that percentage of color elimination appears to be dependent on the amount of mycelia used. Increased dye

ontent leads to decrease in the degradation efficiency has also been reported in the case of degrading activity of F29 and

Funalia troggi mycelia during the repeated use.


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Effect of Malachite green concentration on removal efficiency

0

20

40

60

80

100

120

0 6 12 18 24 48

Time (hr)

Remov

alEfficiency(%)

Figure 2 Effect of Malachite green

No positive effect on decolorisation performance was observed when glucose and nitrogen source added in repeated batch

mode. Degradation performance and stability of mycelia were significantly enhanced in 50mM Phosphate buffer was retained

fter 6 7 days operation at high dye concentration (1g/L) during which retention time is 24hr.

Table 3: Effect of number of mycelia used, on decolorisation

Number of times mycelia used (cycle) Decolorisation (%)

First 97

Second 97

Third 90

Fourth 84

Fifth 79

Sixth 71

Seventh 68

Eighth 54


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Effect of number of times mycelia used on decolorisation (%)

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

Number of mycelia used

PercentageDegradation

Seri

Figure 3 Effect of times of mycelia on degradation of dyes

CONCLUSION:

he results showed that Aspergillus ochraceous (NCIM - 1146) could effectively degrade various textile dyes viz. malachite

reen and cotton blue. However, crystal violet and methyl violet were found to be lesser amenable to biodegradation and

ecolorisation. The degradation activity was due to microbial metabolism and not due to biosorption. These results suggest the

otential ofA. ochraceous (NCIM - 1146) strain in bioremediation of wastewater containing either malachite green or cotton

lue.

REFERENCES:

1. Vaidya A. A & Datye KV, (1982) Environmental Pollution during chemical processing of synthetic fibres

Colourage14, pp 3- 10.

2. Pierce J, (1994) Colour in textile effluents the origins of the problem,J Soc Dyers & colourists110, pp131- 134.

3. Meyer U, (1981) Biodegradation of synthetic organic colourant,FEMS symp12, pp371- 385.

4. William N, Guthrie J & Nelson G, V (1998) The Biotechnology approach to colour removal from textile effluent,J

Soc Dyers colouristspp38 41.

5. Azmi W. Sani RK & Benarjee UG, (1998) Biodegradation of triphenylmethane dyes,Enzyme microbe technology22

pp185- 191.

6. Reddy CA, (1995) The potential for white rot fungi in the treatment of pollutants, Curr Opin Biotechnology6, pp269

272.


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Document By: Bharadwaj

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