"Effect of some process parameters on efficiency of mixed aerobic culture for decolourization of reactive red dye"

Effect of some process parameters on efficiency of mixed aerobic culture for decolourization of reactive reddye

1. INTRODUCTION

Synthetic dyes are colouring agents mainly used in

textile industries which generate a huge amount of wastewater

in the process of dyeing. It is estimated that these

industries discharge near about 280,000 tonnes of dyes

worldwide every year into the environment. A very small amount

of dye in water (10-50 mg L-1) affects the quality,

transparency of water and gas solubility of water bodies. The

effluents from these industries are complex; contain a wide

variety of dyes and other products such as dispersants, acids,

bases, salts, detergents, humectants, oxidants, etc. Discharge

of these coloured effluents into rivers and lakes results into

reduced dissolved oxygen concentration, thus creating anoxic

conditions that are lethal to resident organisms. Many reports

indicate that textile dyes and effluents have toxic effects on

the germination rates and biomass concentration of several

plant species which play many important ecological functions

such as providing the habitat for wildlife; protecting soil

from erosion and providing bulk of organic matter that is

significant to soil fertility. The toxicity of effluent is

because of the presence of dye or its degraded products which

are mutagenic or carcinogenic. Therefore, the treatment of

industrial effluents contaminated with dye becomes necessary

prior to their final discharge to the environment. Various

kinds of physico-chemical methods are in use for the treatment

of wastewater contaminated with dye. These methods are not

environment friendly and cost-effective and hence become

commercially unattractive. Many microorganisms belonging to

School of Environmental and Earth Sciences, NMU, Jalgaon Page 1


the different taxonomic groups of bacteria, fungi,

actinomycetes and algae have been reported for their ability

to de-colourize azo dyes. Pure fungal cultures have been used

to develop bio-processes for the mineralization of azo dyes,

but the long growth cycle and moderate decolourization rate

limit the performance of fungal de-colorization system.

Textiles are among the basic needs of human being. The textile

industries therefore have great economic significance by

virtue of its contribution to overall industrial output and

employment generation. This sector has wide spectrum of

industries ranging from small scale units that use traditional

manufacturing process, to large integrated mills using modern

machineries and equipment. There are 2324 textile industries

in the country including composite and process houses. It can

be seen from these data that there are 83 composite mills in

the country. Rest 2241 is semi composite and processing units.

Tamil Nadu, Gujarat, Punjab and Maharashtra are among the

states which have large number of textile industries amounting

to 1895 i.e. about 81 per cent of total industries. Textile

industries transform fibers into yarn; convert the yarn into

fabrics or related products, and dye and finish these

materials at various stages of production. In processing of

textiles, the industry uses a number of dyes, chemicals,

auxiliary chemicals and sizing materials. As a result,

contaminated waste water is generated which can cause

environmental problems unless properly treated before its

disposal. The waste water treatment is mostly by primary and

secondary processes. However, these conventional treatment



systems are not very effective in removal of pollutants such

as dissolved solids, colour, trace metals etc. The advance

treatment methods, while reducing these pollutants also give

scope for recovery and recycling of water and chemicals.

1.1 Pollution problems in textile industry:

1.1.1 Colour

Presence of colour in the waste water is one of the main

problems in textile industry. Colours are easily visible to

human eyes even at very low concentration. Hence, colour from

textile wastes carries significant esthetic importance. Most

of the dyes are stable and has no effect of light or oxidizing

agents. They are also not easily degradable by the

conventional treatment methods. Removal of dyes from the

effluent is major problem in most of textile industries.

1.1.2 Dissolved Solids

Dissolved solids contained in the industry effluents are

also a critical parameter. Use of common salt and glauber salt

etc. in processes directly increase total dissolved solids

(TDS) level in the effluent. TDS are difficult to be treated

with conventional treatment systems. Disposal of high TDS

bearing effluents can lead to increase in TDS of ground water

and surface water. Dissolved solids in effluent may also be

harmful to vegetation and restrict its use for agricultural

purpose.

1.1.3 Toxic Metals

Waste water of textiles is not free from metal contents.

There are mainly two sources of metals. Firstly, the metals



may come as impurity with the chemicals used during processing

such as caustic soda, sodium carbonate and salts. For

instance, caustic soda may contain mercury if produced using

mercury cell processes. Secondly, the source of metal could be

dye stuffs like metalized mordent dyes. The metal complex dyes

are mostly based on chromium.

1.1.4 Residual Chlorine

The use of chlorine compounds in textile processing,

residual chlorine is found in the waste stream. The waste

water (if disposed without treatment) depletes dissolved

oxygen in the receiving water body and as such aquatic life

gets affected. Residual chlorine may also react with other

compounds in the waste water stream to form toxic substances.

1.1.5 Others

Textile effluents are often contaminated with non-

biodegradable organics termed as refractory materials.

Detergents are typical example of such materials. The presence

of these chemicals results in high chemical oxygen demand

(COD) value of the effluent. Organic pollutants, which

originate from organic compounds of dye stuffs, acids, sizing

materials, enzymes, tallow etc. are also found in textile

effluent, such impurities are reflected in the analysis of

bio-chemical oxygen demand (BOD) and COD. These pollutants are

controlled by use of biological treatment processes. In many

textile units, particularly engaged in synthetic processing,

low BOD/COD ratio of effluent is observed which makes even



biological treatment not a ready proposition. The waste water

of cotton based textile units is usually alkaline, whereas

synthetic and woolen fabric processing generates acidic

effluent.

1.2 Effluent treatment:

1.2.1 Primary and Secondary Treatment

The conventional treatment systems like physico-chemical

treatment and physico-chemical treatment followed by

biological treatment system are installed in majority of

textile industries. The first step in the waste water

treatment is to mix and equalize the waste water streams that

are discharged at different time, and different intervals from

different stages in the processes. Some industries also prefer

screening, oil trap prior to equalization for removal of

solids and oil and grease. Equalization ensures that the

effluent have uniform characteristics in terms of pollution

load, pH and temperature. The effluent is then subject to

flash mixing for the addition of coagulants such as lime,

alum, ferrous sulphate, ferric chloride, polyelectrolyte and

processed through clariflocculator or flocculator and settling

tank. Selection of appropriate coagulants and doses of

chemicals are determined on the basis of treatability study of

effluent samples. The chemical treatment helps in reduction of

colour and suspended solids. A significant reduction in BOD

and COD values is also observed. This physico-chemical



treatment is followed by biological treatment process, with

settling which further reduces BOD and COD values. The textile

process houses which undertake chemical processing do not have

much organic load in their effluents. In such cases, the

recent trend is to set up an activated adsorption system or an

ozonation unit instead of biological treatment process.

1.2.2 Tertiary Treatment

Textile effluents may require tertiary or advance

treatment methods to remove particular contaminant or to

prepare the treated effluent for reuse. Some common tertiary

operations are removal of residual organic colour compounds by

adsorption and removal of dissolved solids by membrane

filtration. The waste water is also treated with ozone or

other oxidizing agent to destroy many contaminants.

Evaporation and crystallization are other methods to minimize

effluent disposal problems.



Plate: Waste water discharging through textile industry insurface water

1.3 Chemical Structure of dye:



1.4 Process flow sheet of textile industry with wastewatercharacteristics:


Contains high amount of organic compounds, which contribute to BOD, & COD. Italso contains SS, which are


1.5 Origin of the research problem:


Fiber manufacturing

(Sizing)

Spinning

Weaving

Desizing

Scouring

Bleaching

Mercerizing

Dyeing

Printing

Washing

Finishing

Contains sizing agents suchas starch, polyvinylalcohol, wax, acrylic size,loose fiber etc. All thesecomponents contribute to

Contains high alkalinity

and detergent from

scouring process, sizing

chemicals resulting form

desizing process, high

alkalinity resulting from

mercerizing. These

Contains dyes, pigments,dyeing auxiliaries andchemicals used duringdyeing. It contains BOD,COD, SS, heavy metals andmost importantly the color

Contains mainly organic andinorganic chemicals e.g.resins, catalyst, softeners,stiffeners, fluorocarbon,pigments, dyes, etc. It


Textile industry plays a very important role in the

economy of the country like India and it accounts for around

one third of total export. Out of various activities in

textile industry, chemical processing contributes about 70% of

pollution. It is well known that cotton mills consume large

volume of water for various processes such as sizing,

desizing, scouring, bleaching, mercerization, dyeing,

printing, finishing and ultimately washing. Due to the nature

of various chemical processing of textiles, large volumes of

waste water with numerous pollutants are discharged. Since

these streams of water affect the aquatic eco-system in number

of ways such as depleting the dissolved oxygen content or

settlement of suspended substances in anaerobic condition, a

special attention needs to be paid. Thus a study on different

measures which can be adopted to treat the waste water

discharged from textile chemical processing industries to

protect and safeguard our surroundings from possible pollution

problem has been the focus point of many recent

investigations.

A lot of information is available on removal and bio-

degradation of Reactive Red Dye using pure strains of

bacteria, fungi, algae and yeast. The removal and degradation

of dye by various physicochemical, anaerobic and photo

catalytic methods have also been studied. However, it is very

difficult to maintain the purity of single cultures in the

large scale application and there are chances of growth of

other species of micro-organism due to different environmental

conditions. Effluent contains the different dyes but single



culture is unable to degrade all the dyes which is the major

drawback of applying single pure culture for decolorizing the

wastewater from dying industries. Therefore; the use of mixed

activated microbial culture seems to have more potential for

large scale application at field level.

2. LITERATURE REVIEW

An attempt was made to examine the potential of aerobic

mixed culture for decolourization of Remazol Black B dye in

batch reactors. The effect of pH, temperature, inoculum,

initial concentration of dye and initial concentration of

glucose was studied with an aim to determine the optimal

conditions required for maximum decolourization and

degradation. The culture exhibited maximum decolourization

ability at pH between 7-8 and at 30°C. A 10% (v/v) inoculum

and 1% (w/v) glucose concentration were found to be the

optimum for decolourization. A maximum of 98% decolourization

was observed at 25 ppm initial concentration of dye after 18

hours of incubation period. At higher dye concentration of 300

ppm, the removal in colour was found to be 75% in 48 hours of

incubation period. The results show that the enriched mixed

culture from activated sludge has good potential in removal of

Remazol Black B dye from wastewater under aerobic conditions.(

Kapil Kumar, M. G. Dastidar, T. R. Sreekrishnan– “Effect of



process parameters on aerobic decolourization of reactive azo

dye using mixed culture: World Academy of Science, Engineering

and Technology, 2009)

A bacterial culture was isolated from soil in the

vicinity of textile industry and was identified as

Bacillus subtilis SPR 42. It was found to be the most

active azo dye degrader using submerged fermentation

technology. B. subtilis SPR 42 was able to decolourize

azo dyes: Vaxent Red HE7B (Reactive Red 141) and Vaxent

Yellow HEGR at a concentration of 100 mg/L upto 73% and 92%,

respectively within 24 hr at 37oC (pH 8.5) during static

conditions. (Baljeet Singh Saharan and Poonam Rang-

“Optimization of cultural conditions for decolourization of

textile azo dyes by bacillus subtilis spr42 under submerged

fermentation”: International Journal of Advanced Biotechnology

and Research, 2011)

Photocatalytic decoloration kinetics of triazine

(Reactive Red 11, Reactive Red 2, and Reactive Orange 84) and

vinylsulfone type (Reactive Orange 16 and Reactive Black 5) of

reactive dyes have been studied spectrophotometrically by

following the decrease in dye concentration with time. At

ambient conditions, over 90–95% decoloration of above dyes

have been observed upon prolonged illumination (15 h) of the

reacting system with a 150 W xenon lamp. It was found that the

decoloration reaction followed first-order kinetics. The

values of observed rate constants were found to be dependent

of the structure of dyes at low dye concentration, but

independent at higher concentration. It also reports for the



first time the decoloration of two different dyes together in

a binary dye mixture using visible light-irradiated

TiO2photocatalyst. Rate of decoloration of two different dyes

together in a binary dye mixture using visible light-

irradiated TiO2photocatalyst is governed by the absorptivity

of the particular dye onto the surface of the

TiO2photocatalyst. (Debabrata Chatterjee et.al 2007- Kinetics

of the decoloration of reactive dyes over visible light-

irradiated TiO2semiconductor photocatalyst: Journal of

Hazardous Materials 156, 2008)

The decolourization of azo dye RR2 in semicontinuous

anaerobic reactors was studied. After an initial adsorption of

dye on the biomass subsequent degradation of dye occurred. The

primary mechanism of dye removal appears to be adsorption of

dye on biomass followed by biodegradation. Under steady state

conditions, the parameters ORP, methane produced, COD and

colour removal attained nearly stable values. RR2

decolourization of up to 78% could be achieved. The kinetic

studies showed a first order behavior for the two dye

concentrations evaluated. The decolourization of dyes was

initiated only when the bulk phase redox potential attained a

value lower than−150 mV. Based on the total methane produced

during the feeding cycles, no toxic effect of dyes was

observed on methanogenesis. However, based on the TMA

estimates the toxicity effect was evident. (Robert Maas,

Sanjeev Chaudhari- Adsorption and biological decolourization

of azo dye Reactive Red 2 in semicontinuous anaerobic



reactors: Centre for Environmental Science and Engineering,

Indian Institute of Technology Bombay, 2006)



3. OBJECTIVES

1. To study the effects of temperature and pH for de-colorization of Reactive Red dye by using aerobicdigestion (biological) method.

2. To study the effects of temperature and pH for theremoval of Chemical Oxygen Demand by using aerobicdigestion (biological) method.

3. Optimization of selected parameters for effective de-colorization of Reactive Red dye by using aerobicdigestion (biological) method by using aerobic digestion(biological) method.

4. Optimization of selected parameters for effective removalof Chemical Oxygen Demand by using aerobic digestion(biological) method.

5. To find out cost effective and Eco-friendly method forde-colorization of Reactive Red dye and removal of COD intextile wastewater treatment.

3.1 Significance of the study:

In the present study, an attempt was made for

the degradation and de-colorization of Reactive Red Dye and

COD removal using aerobic mixed culture in batch reactors.

Effect of various process parameters like pH, temperature,

inoculums concentration, glucose concentration and initial

concentration of dye was studied. Finally, finding out the

suitable environmental conditions for effective removal of

reactive red dye and COD, which is cheap and feasible method



for the dye and COD removal as compared to the chemical

treatment method.



4. MATERIALS AND METHODS

4.1 Primary data collection from local Industry:

Study visit of textile industry from Jalgaon MIDC area

was done for studying the different types of waste water/dye

effluent and the problems associated with dye removal from

waste water effluent and to study various factors/process

parameters influencing/ important in concerned to dye removal

techniques.

4.2 Collection of Sample:

The Sample of Activated Sludge was been collected from

textile industry for preparation and development of mixed

culture for the purpose of the removal/degradation of dye from

waste water during this study.

4.3 Source of inoculums and Acclimatization of mix microbial culture:

Activated sludge collected from an effluent treatment

plant of a textile industry from Jalgaon was used as the

parent source of inoculums in the present study. For

enrichment of the culture, the heterogeneous population was

first grown aerobically in a medium. The composition of the

synthetic medium used in the present study was as follows:

0.5% peptone, 0.3%beef/yeast extract, 1.5% agar, 0.5% NaCl

with distilled water. The culture was gradually exposed to

increasing concentrations of Reactive Red Dye in order to

acclimatize the microbial culture. This acclimatized

microbial culture was used for all experiments.



4.4 Preparation of Dye solution (synthetic effluent):

Reactive Red Dye was used in the present study is acidic

and soluble in water. A stock solution of 1000 ppm was

initially prepared and the solutions of the desired

concentrations for various experiments were obtained by

successive dilution method.

4.5 Batch experiments: The experiments was performed in batch mode in the bio-

reactor (Zenith)having 2 liters capacity which is available in our department.A working experiment was followed as follows:4.5.1 Experiment 1- At Constant PH 7 and temperature 300 C

4.5.2 Experiment 2- At constant pH 7 and temperature 350 C

4.5.3 Experiment 3- At Constant pH 7.5 and temperature 300 C

4.5.4 Experiment 4- At Constant pH 7.5 and temperature 350 C

In each experiment a working volume of 1500 ml was of

effluent employed throughout the study. The glucose media (1%)

and dye of concentration 50 ppm was added to the flasks. The

flasks were incubated with acclimatized inoculum. After adding

glucose media, inoculum and required concentration of dye, the

flasks were kept in an orbital shaker at 250 rpm and at

required temperature. The pH of the solution was adjusted to

required level with 1 N hydrochloric acid or sodium hydroxide.

In addition, control flasks containing only dye and media and

without inoculum were also kept to see the abiotic

decolourization, if any.

4.6 Analytical Methods:



At different time intervals, the samples was withdrawn

from the flasks and centrifuged at 4500 to 5000 rpm for 15

minutes to precipitate suspended biomass. The concentration of

dye in the supernatant was determined by reading absorbance at

400 nm. This absorbance was compared with standard curve

plotted using different concentrations of the dye. The

measurement of absorbance and centrifugation was done by using

Spectronic 20 D spectrophotometer and centrifuge,

respectively.

After this samples were titrated with Ferrous Ammonium

Sulfate using Ferroin as an indicator for estimation of COD

reduction. COD of initial dye concentration of 50 ppm was also

determined along with the degraded sample after experiment.

The COD of degraded samples was compared with the control

sample (of 50 ppm) for determining the COD reduction in

degraded samples at different process parameters. The

determination of COD reduction was done by COD digester.

Plate 1: Bioreactor Plate2: LCD of Bioreactor showing temp., pH



Plate 3: Mixed culture on petri plate Plate4: Mixed culture on petri plate

Plate 5: Result of Batch (expt.) no. 1, 2, 3, 4 Plate6: Result of Batch (expt.) no. 2

5. RESULTS AND DISCUSSIONS

Observation Table 1- Effect of pH and Temperature on dye concentration:-

Sr.

No.

pH Temp.

(0c)

Batc

h

Initial

Dye Conc.

(ppm)

Final Dye

Conc.

(ppm)

Removal %

of Dye

1 7 30 1 50 30 40School of Environmental and Earth Sciences, NMU, Jalgaon Page 20


2 7 35 2 50 10.5 793 7.5 30 3 50 18.5 634 7.5 35 4 50 16 68

Observation Table 2- Effect of pH and Temperature on COD Reduction:-

Sr.

No.

pH Temp.

(0c)

Batc

h

Initial

COD (mg/l)

Final COD

(mg/l)

Reductio

n % of

COD

1 7 30 1 13800 9522 312 7 35 2 13800 5106 633 7.5 30 3 13800 8556 384 7.5 35 4 13800 7590 45

5.1 Effect of pH (7 and 7.5) on decolourization of dye:



30 350

102030405060708090

40

79

Decolourization of dye at pH 7 at temp. 30°C and 35°C with time (24hrs)

pH 7

% De

colo

uriz

atio

n

TEMPERATURE (oC)

Fig. 1: Effect of pH (7) on decolourization of dye

30 3560616263646566676869

63

68

Decolourization of dye at pH 7.5 at temp. 30°C and 35°C with time (24hrs)

pH 7.5

Fig. 2: Effect of pH (7.5) on decolourization of dye

Effect of pH (7 and 7.5) on decolourization with time of

Reactive Red dye at 50 ppm of initial dye concentration with

inoculum is shown in figure 1 & 2. The figures clearly show

that the percentage removal of dye decreased with increase in

pH from 7-7.5 irrespective of temperature. The maximum removal



(79%) of dye was found at pH 7 after 24 hours of aerobic

digestion. Further increase in pH beyond 7 resulted in

decreased percentage removal of dye. The optimum pH was found

to be 7 for maximum removal of dye. The pH has a major effect

on the efficiency of dye decolourization, and the optimal pH

for color removal is often between 6.0 and 10.0 for most of

the dyes [19]. The pH tolerance of decolourizing bacteria is

quite important because reactive red dyes bind by addition or

substitution mechanisms under alkaline conditions and at high

temperatures.

5.2 Effect of Temperature (30°C and 35°C) on decolourization of dye:

7 7.5010203040506070

40

63

Decolourization of dye at temperature 30°C at pH 7 and 7.5 with time (24hrs)

TEMP. 30°C

pH

FIG.3- Effect of Temperature (30°C) on decolourization of dye



7 7.562646668707274767880 79

68

Decolourization of dye at temperature 35°C at pH 7 and 7.5 with time (24hrs)

TEMP. 35°C

FIG.4- Effect of Temperature (35°C) on decolourization of dye

Figure 3 and 4 shows decolourization of dye with time at

different temperatures (300 and 350C) at 50 ppm of initial dye

concentration. It is clear from the figures that percentage

removal of dye increased with an increase in temperature from

300 to 350C irrespective of pH. The maximum removal (79 %) of

dye was found at temperature 350C after 24 hours of aerobic

digestion. Decolourizing activity would significantly be

suppressed at 450C, this might be due to the loss of cell

viability or deactivation of the enzymes responsible for

decolourization at 450C [21].



5.3 Effect of pH (7 and 7.5) on COD reduction:

30 35010203040506070

31

63

Reduction % of COD at pH 7 at temp. 30°C and 35°C with time (24hrs)

pH 7

FIG.5- Effect of pH (7) on COD reduction

30 3534363840424446

38

45

Reduction % of COD at pH 7.5 at temp. 30°C and 35°C with time (24hrs)

pH 7.5

FIG.6- Effect of pH (7.5) on COD reduction



Effect of pH (7 and 7.5) on COD reduction with time of

Reactive Red dye at 50 ppm of initial dye concentration is

shown in figure 5 & 6. The figures clearly show that the

percentage reduction of COD decreased with increase in pH from

7-7.5 irrespective of temperature. The maximum reduction (63

%) of COD was found at pH 7 after 24 hours of aerobic

digestion. Further increase in pH beyond 7 resulted in

decreased percentage reduction of COD. The optimum pH was

found to be 7 for maximum reduction of COD.

5.4 Effect of Temperature (30°C and 35°C) on COD reduction:

7 7.50510152025303540

3138

Reduction % of COD at temperature 30°C at pH 7 and 7.5 with time (24hrs)

TEMP. 30°C

FIG.7- Effect of Temperature (30°C) on COD reduction



7 7.5010203040506070

63

45

Reduction % of COD at temperature 35°C at pH 7 and 7.5 with time (24hrs)

TEMP. 35°C

FIG.8- Effect of Temperature (35°C) on COD reduction

Figure 7 and 8 shows COD reduction with time at different

temperatures (300 and 350C) at 50 ppm of initial dye

concentration. It is clear from the figures that percentage

reduction of COD increased with an increase in temperature

from 300 to 350C irrespective of pH. The maximum reduction

(63%) of COD was found at temperature 350C after 24 hours of

aerobic digestion.

5.5 CPCB Standards for Textile industry effluent discharge:

Parameters CPCB Standard

pH 6-8.5

Temperature Shall not exceed 5oC abovethe ambient temperature of

the receiving body.BOD 100COD 250TSS 100TDS 2100



Sulphide 2Sulphate 1000Chloride 600Calcium 75

Magnesium 50All the values except pH are in mg/L

6. CONCLUSION

The present study reveals that enriched aerobic mixed

culture can be used successfully for decolourizing Reactive

Red dye. The culture exhibited maximum decolourization ability

at pH 7 and temperature 35° C .Hence pH 7 and temperature 35°C

were found to be optimum for decolourization. At 50 ppm

initial dye concentration a maximum of 79% removal of colour

was observed after 24 hour of aerobic digestion and, Enriched



aerobic mixed culture can be used successfully for reduction

of COD. The culture exhibited maximum COD reduction ability at

pH 7 and temperature 35° C .Hence pH 7 and temperature 35°C

were found to be optimum for reduction of COD. At 50 ppm

initial dye concentration a maximum of 63% of COD reduction

was observed after 24 hour of aerobic digestion. On the basis

of the results of the present study suitable strategy can be

developed for the treatment of waste water contaminated with

Reactive Red dye by using aerobic mixed microbial culture.



7. FUTURE WORK

Present research work can be extended by carrying out

following experiments by:

1. Changing food concentration

2. Extending retention time period for the degradation

(for eg. 48, 72, 96…)

3. Taking different inoculum concentration

4. Taking wide range of pH and temperature

5. Taking different initial dye concentration



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