final report

“Biodegradation Of Congo Red By Aspergillus Niger & Pseudomonas Aeruginosa”

1.1 Bioremediation:

Bioremediation is a pollution control technology that uses biological systems to catalyze

the degradation or transformation of various toxic chemicals to less harmful forms. The general

approaches to bioremediation are to enhance natural biodegradation by native organisms

(intrinsic bioremediation), to carry out environmental modification by applying nutrients or

aeration (biostimulation) or through addition of microorganisms (bioaugmentation). Unlike

conventional technologies, bioremediation can be carried out on-site. Bioremediation is limited

in the number of toxic materials it can handle (Hart, 1996), but where applicable, it is cost-

effective(Atlas&Unterman,1997).

Biodegradation, mineralization, bioremediation, biodeterioration, biotransformation,

bioaccumulation and biosorption are some terms with minor differences but often overlappingly

used. Biodegradation is the general term used for all biologically mediated breaks down of

chemical compounds and complete biodegradation leads to mineralization. Biotransformation is

a step in the biochemical pathway that leads to the conversion of a molecule (precursor) into a

product. A series of such steps are required for a biochemical pathway. In environmental terms,

it is importance whether the product is less harmful or not (Bennett & Faison, 1997).

Biodeterioration refers usually to the breakdown of economically useful compounds but often the

term has been used to refer to the degradation of normally resistant substances such as metals,

plastics, drugs, cosmetics, painting, sculpture, wood products and equipment (Rose, 1981).

Bioremediation refers to the use of biological systems to degrade toxic compounds in the

environment. Bioaccumulation or biosorption is the accumulation of the toxic compounds inside

the cell without any degradation of the toxic molecule. This method can be effective in aquatic

environments where the organisms can be removed after being loaded with the toxic substance.

The fungi are unique among microorganisms in that they secrete a variety of extracellular

enzymes. The decomposition of lignocelluloses is rated as the most important degradative event

Dept of Biotechnology, BITM, Bellary.


in the carbon cycle of earth (Bennett & Faison, 1997). Enormous literature exists on the role of

fungi in the carbon and nitrogen cycles of nature (Frankland et al., 1982; Cooke & Rayner, 1984;

Carroll & Wicklow, 1992). The role of fungi in the degradation of complex carbon compounds

such as starch, cellulose, pectin, lignin, lignocelluloses, insulin, xylan, araban etc., is well

known. Trichoderma reesei is known to possess the complete set of enzymes required to

breakdown cellulose to glucose. Degradation of lignocelluloses is the characteristic of several

basidiomycetous fungi.

1.2 Biotransformation of Toxic Wastes to Harmless Products:

The rapid expansion and increasing sophistication of the chemical industries in the past

century and particularly over the last thirty years has meant that there has been an increasing

amount and complexity of toxic waste effluents. At the same time, fortunately, regulatory

authorities have been paying more attention to problems of contamination of the environment.

Industrial companies are therefore becoming increasingly aware of the political, social,

environmental and regulatory pressures to prevent escape of effluents into the environment. The

occurrence of major incidents (such as the Exxon Valdez oil spill, the Union-Carbide (Dow)

Bhopal disaster, large-scale contamination of the Rhine River, the progressive deterioration of

the aquatic habitats and conifer forests in the Northeastern US, Canada, and parts of Europe, or

the release of radioactive material in the Chernobyl accident, etc.) and the subsequent massive

publicity due to the resulting environmental problems has highlighted the potential for imminent

and long-term disasters in the public's conscience.

Even though policies and environmental efforts should continue to be directed towards

applying pressure to industry to reduce toxic waste production, biotechnology presents

opportunities to detoxify industrial effluents. Bacteria can be altered to produce certain enzymes

that metabolize industrial waste components that are toxic to other life, and also new pathways

can be designed for the biodegradation of various wastes. Since waste management itself is a

well-established industry, genetics and enzymology can be simply "bolted-on" to existing

engineering expertise.



Examination of effluents from the chemical and petrochemical industries shows that such

effluents typically contain either one or a limited range of major toxic components. In some

cases other considerations (such as aesthetic ones) can be important for removal of certain

components (such as dyes). This means that in general one industry may apply one or a few

genetically modified bacterial strains to get rid of its major toxic waste. However, it may be

important to contain the "waste-eating" bacteria within the manufacturing plant, and not release

these with the waste water. In such cases, filter installations will have to be built to separate the

bacteria from the effluent.

Of course, the bioprocesses for treating toxic effluents must compete with existing

methods in terms of efficiency and economy. However, the biotechnological solution to the

problem requires only moderate capital investment, a low energy input, are environmentally safe,

do not generate waste, and are self-sustaining. Biotechnological methods of toxic waste

treatment are likely to play an increasingly key role both as a displacement for existing disposal

methods and for the detoxification of novel xenobiotic compounds. On the other hand, however,

it is important to limit the generation of both hazardous and non-hazardous waste as much as

possible, and utilize recycling methods wherever possible.

Over the last few years, a number of companies have been established already to develop

and commercialize biodegradation technologies. Existence of such companies now has become

economically justifiable, because of burgeoning costs of traditional treatment technologies,

increasing public resistance to such traditional technologies, accompanied by increasingly

stringent regulatory requirements. The interest of commercial businesses in utilizing micro-

organisms to detoxify effluents, soils, etc. is reflected in "bioremediation" having become a

common buzzword in waste management. Companies specializing in bioremediation will need to

develop a viable integration of microbiology and systems engineering. As an example of a

bioremediation company, Envirogen (NJ) has developed recombinant PCB (polychlorinated

biphenyl)-degrading microorganisms with improved stability and survivability in mixed

populations of soil organisms.

The same company also has developed a naturally occurring bacterium that degrades

trichloroethylene (TCE) in the presence of toluene, a toxic organic solvent killing many other



microorganisms. A large number of similar companies can be found using a web search engine

and an appropriate keyword (such as "bioremediation").

Microorganisms have also been successfully applied during the removal of the Exxon

Valdez oil spill. A number of microorganisms can utilize oil as a source of food, and many of

them produce potent surface-active compounds that can emulsify oil in water and facilitate the

removal of the oil. Unlike chemical surfactants, the microbial emulsifier is non-toxic and

biodegradable. Also, "fertilizers" have been utilized to increase the growth rate of the indigenous

population of bacteria that are able to degrade oil.

Use of microbes for bioremediation is not limited to detoxification of organic

compounds. In many cases, selected microbes can also reduce the toxic cations of heavy metals

(such as selenium) to the much less toxic and much less soluble elemental form. Thus,

bioremediation of surface water with significant contamination by heavy metals can now be

attempted.

1.3 Role of Microbes in Bioremediation:

The goal in bioremediation is to stimulate microorganisms with nutrients and other

chemicals that will enable them to destroy the contaminants. The bioremediation systems in

operation today reply on microorganisms native to the contaminated sites, encouraging them to

work by supplying them with the optimum levels of nutrients and other chemicals essential for

their metabolism. Thus, today's bioremediation systems are limited by the capabilities of the

native microbes. However, researchers are currently investigating ways to augment contaminated

sites with nonnative microbes-including genetically engineered microorganisms-especially suited

to degrading the contaminants of concern at particular sites. It is possible that this process,

known as bioaugmentation, could expand the range of possibilities for future bioremediation

systems.

Regardless of whether the microbes are native or newly introduced to the site, an

understanding of how they destroy contaminants is critical to understanding bioremediation. This



types of microbial processes that will be employed in the cleanup dictate what nutritional

supplements the bioremediation system must supply. Furthermore, the byproducts of microbial

processes can provide indicators that the bioremediation is successful.

Microorganisms gain energy by catalyzing energy-producing chemical reactions that

involve breaking chemical reactions that involve breaking chemical bonds and transferring

electrons away from the contaminant. The type of chemical reaction is called an oxidation-

reduction reaction: the organic contaminant is oxidized, the technical term for losing electrons;

correspondingly, the chemical that gains the electrons is reduced. The contaminant is called the

electron donor, while the electron recipient is called the electron acceptor. The energy gained

from these electron transfers is then "invested", along with some electrons and carbon from the

contaminant, to produce more cells. These two materials the electron donor and acceptor are

essential for cell growth and are commonly called the primary substrates.

Many microorganisms, like humans, use molecular oxygen (O2) as the electron acceptor.

The process of destroying organic compounds with the aid of O2 is called aerobic respiration. In

aerobic respiration, microbes use 02 to oxidize part of the carbon in the contaminants to carbon

dioxide (CO2), with the rest of the carbon used to produce new cell mass. In the process the O2

gets reduced, producing water. Thus, the major byproducts of aerobic respiration are carbon

dioxide, water, and an increased population of microorganisms.

Many microorganisms can exist without oxygen, using a process called anaerobic

respiration. In anaerobic respiration, nitrate (NO3-), sulfate (SO4

2-), metals such as iron (Fe3+) and

manganese (Mn4+), or even CO2 can play the role of oxygen, accepting electrons from the

degraded contaminant. Thus, anaerobic respiration uses inorganic chemicals as electron

acceptors. In addition to new cell matter, the byproducts of anaerobic respiration may include

nitrogen gas (N2), hydrogen sulfide (H2S), reduced forms of metals, and methane (CH4),

depending on the electron acceptor.

Petroleum hydrocarbons and their derivatives are naturally occurring chemicals that

humans have exploited for a wide range of purposes, from fueling engines to manufacturing



chemicals. The representative types of petroleum hydrocarbons and derivatives are gasoline,

fuel, oil, polycyclic aromatic hydrocarbons (PAHs), creosote, ethers, alcohols, ketones, and

esters, dyes. Each of these chemicals has a broad range of industrial applications. For example,

PHAs are released when crude oil is refined and from the manufacture of petroleum products

such as plastics. Creosote is used in wood preservatives. Ethers, esters, and ketones are

components of chemicals ranging from perfumes, to anesthetics, to paints and lacquers, to

insecticide &dyes in textile industry.

Pseudomomas Aeruginosa:

Scientific classification

Kingdom : Bacteria

Phylum : Proteobacteria

Class : Gamma Proteobacteria

Order : Pseudomondales

Family : Pseudomonadaceae

Genus : Pseudomonas

Species : Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common bacterium which can cause disease in animals and

humans. It is found environments. It uses a wide range of organic material for food; in animals,

the versatility enables the organism to infect in soil, water, and most man-made environments

throughout the world. It thrives not only in normal atmospheres, but also with little oxygen, and

has thus colonized many naturals and artificial damaged tissues or people with reduced

immunity.



Identification

It is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. An

opportunistic human pathogen, P.aeruginosa is also an opportunistic pathogen of plants.

P.aeruginosa is the type species of the genus Pseudomonas (Migula 1984).

P.aeruginosa secretes a variety of pigments, including pyocyanin (blue –green),

fluorescein (yellow-green and fluorescent, now also known as pyoverdin), and pyorubin (red-

brown), King, Ward, and Raney developed Pseudomonas Agar P (aka King A media) for

enhancing pyocyanin and pyorubin production and Pseudomonas Agar F (aka King B media) for

enhancing fluorescein production.

P.aeruginosa is often preliminarily identified by its pearlescent appearance and grape-like

or tortilla-like odour in vitro. Definitive clinical identification of P.aeruginosa often includes

identifying ghe production of pyocyanin and fluorescein, as well as its ability to grow at

42.C.P.aeruginosa is capable of growth in diesel and jet fuel, where it is known as a

hydrocarbon-utilizing microorganism (or “HUM bug”), causing microbial corrosion. It creates

dark gellish mats sometimes improperly called “algae” because of their appearance.

Although classified as an aerobic organisam, P.aeruginosa is considered by many as a

facultative anaerobe, as it is well adapted to proliferate in conditions of partical or total oxygen

depletion. This organism can achieve anaerobic growth with nitrate as a terminal electron

acceptor, and, in its absence, it is also able to ferment arginine by substrate-level

phosphorylation. Adaptation to microaerobic or anaerobic encironments is essential for certain

lifestyles of P.aeruginosa, for example, during lung infection in cystic fibrosis patents, where

thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.

Pathogenesis

An opportunistic pathogen of immune compromised individuals, P.aeruginosa typically

infects the pulmonary tract, urinary tract, burns, wounds, and also causes other blood infections.

It is the most common cause of infections of burn injuries and of the external ear (otitis externa),

and is the most frequent colonizer of medical devices (e.g., catheters), Pseudomonas can, in rare

circumstances, cause community-acquired pneumonias, as well as ventilator-associated



pneumonias, being one of the most common agents isolated in several studies. Pyocyanin is a

virulence factor of the bacteria and has been known to cause death in C.elegans by oxidative

stress.

Toxins

P.aeruginosa uses the virulence factor exotoxin A to ADP-ribosylate eukaryotic

elongation factor 2 in the host cell, much as the diphtheria toxin does. Without elongation factor

2, eukaryotic cells cannot synthesize proteins and nerose.

Prevention

Medical-grade honey may reduce colonization of many pathogens including

Pseudomonas aeruginosa. Probiotic prophylaxis may prevent colonization and delay onset of

pseudomonas infection in an ICU setting. Immunoprophylaxis against pseudomonas is being

investigated.

Aspergillus niger:

Scientific classification

Domain : Eukaryota

Kingdom : Fungi

Phylum : Ascomycota

Subphylum : Pezizomycotina

Class : Eurotiomycetes

Order : Eurotiales

Family : Trichocomaceae

Genus : Aspergillus

Species : Niger

Aspergillus niger is a fungus and one of the most common species of the genus

Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as

grapes, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is



commonly reported from indoor environments, where its black colonies can be confused with

those of Stachybotrys (species of which have also been called “black mold”).

Taxonomy

A.niger is included in Aspergillus subgenus Circumdati, section Nigri. The section Nigri

includes 15 related black-spored species that may be confused with A.niger, including

A.tubingensis , A.foetidus, A.carbonarius, and A.awamori. A number of morphologically similar

species were recently described by Samson et al.

Pathogenicity

A.niger causes black mold of onions. Infection of onion seedlings by a A.niger can

become systemic, manifesting only when conditions are conducive. A.niger causes a common

postharvest disease of onions, in which the black conidia can be observed between the scales of

the bulb. The fungus also causes disease in peanuts and in grapes.

Human and animal disease

A.niger is less likely to cuase human disease than some other Aspergillus species, but if

large amounts of spores are inhaled, a serious lung disease, aspergillosis can occur. Aspergillosis

is particularly frequent among horticultural workers who inhale peat dust, which can be rich in

Aspergillus spores. Less commonly, it has been found on the walls of ancient Egyptain tombs

and can be inhaled when the area is disturbed. A.niger is one of the most common causes of

otomycosis (fungal ear infections), which can cause pain, temporary hearing loss and, in severe

cases, damage to the ear canal and tympanic membrane.

Industrial uses

A.niger is cultured for the industrial production of many substances. Various strains of

A.niger are used in the industrial preparation of citric acid (E330) and gluconic acid (E574) and

have been assessed as acceptable for daily intake by the World Health Organisation. A.niger

fementation is “generally regarded as safe” (GRAS) by the United States Food and Drug

Administration. Many useful enzymes are produced using industrial fermentation of A.niger



glucoamylase is used in the production if high fructose corn syrup.and pectinases are used in

cider and wine clarification.

1.4 Azo dyes

Azo dyes are a group of synthetic organic compounds that contain a nitrogen-nitrogen

double bond (the azo group) as the key functional group that links to two aromatic ring systems.

Together these groups are principally responsible for the colour. Under aerobic

conditions (in the presence of oxygen) azo dyes are resistant to degradation, but under anaerobic

conditions (in the absence of oxygen) they can be reduced to certain aromatic amines called

Arylamines. This can be a problem because some aromatic amines have been found to be

carcinogenic.

The breakdown can happen in several ways, either chemically, usually through ‘reductive

cleavage’, or by bacteria. This process can occur in the body where intestinal bacteria, liver cells

and skin surface micro flora cause the breakdown. Exposure to light or high temperatures can

also cause some azo dyes to be broken down to aryl amines.

1.4.1 Uses of Azo Dyes

Azo dyes have many uses, although they are mainly used in dyeing textile fibres,

particularly cotton but also silk, wool, viscose and synthetic fibres. Azo dyes are usually red,

orange or yellow, and make up about half the dyes produced globally.

Certain colour shades for textiles cannot be produced without the use of azo dyes. They

are considered to be easy to use, to provide clear, strong colours and to be relatively cheap. At

present there are around 3,000 azo dyes in use worldwide and they account for 65% of the

commercial dyes. “Azo pigments”, which are designed to be insoluble (unlike dyes, where

solubility is needed), are extensively used in inks and paints.



1.4.2 Impact of Azo Dyes

All chemicals are potentially harmful when there is a significant amount of exposure to

them. The majorities of azo dyes is water-soluble and are therefore easy for the body to absorb.

This can take place through inhalation or ingestion of dust and to a lesser extent via skin contact.

For this reason it is best to minimize exposure to dyes when working with them.

There is concern that the arylamines that may be released from azo dyes can be absorbed

by the skin and accumulate in the body. A small number of the amines that contain a benzene

ring (aromatic amines) are classified as being carcinogenic or potentially carcinogenic to

humans. However, there are only a few commercial azo dyes that have been shown to release

these amines and most reputable manufacturers have removed these from their ranges. Some

arylamines are also judged capable of producing allergies on skin contact, irritating the eyes, and

being toxic by inhalation and if swallowed.

1.4.3 Banned Azo Dyes

There are approximately 3000 azo dyes on the market. Those azo dyes, which can break

down under reductive conditions to release carcinogenic arylamines, are prohibited by EU

Directive (2002) from being used in consumer goods which are considered to have regular skin

contact. The Directive led to numerous misleading and false statements, along the lines that “azo

dyes can no longer be used after September 2003” - this is incorrect. What this actually means is

that from September 2003 all EU countries were required to prohibit the manufacture and sale of

those defined consumer goods, which on chemical analysis are found to contain the listed

aromatic amines originating from a small number of azo dyes.

Articles coloured with all other azo dyes will be able to be manufactured and sold

without restriction. Since most coloured textile and leather articles are treated with azo dyes and

pigments, it is an important basic point to realise that only a very few azo dyes will be affected.

It has been estimated that less than 4 % of known azo dye structures would release any of the

prohibited amines. Additionally, over the past few years all reputable dye manufacturers have

stopped manufacturing such azo dyes and test institutes report the vast majority of samples tested

today do comply with the EU Directive.



Currently 22 aromatic amines are considered to be harmful and are banned in textile

industry

4-aminodiphenyl

Benzidine

4-chloro-o-toluidine

2-naphthylamine

4-amino-2’,3-dimethylazobenzene

2-amino-4-nitrotolue

4-chloroaniline

2,4-diaminoanisole

4,4’-diaminodiphenylmethane

3, 3’-dichlorobenzidine

3, 3’-dimethoxybenzidine

3, 3’-dimethylbenzidine

3, 3’-dimethyl-4, 4’diaminodiphenylmethane

4-cresidine

4, 4’-methylene-bis-(2-chloroaniline)

4, 4’-oxydianiline

4, 4’-thiodianiline

2-aminotoluene

2, 4-diaminotoluene



2, 4, 5-trimethylaniline

2-methoxyaniline

4-aminoazobenzene

1.4.4 Congo Red

It is a synthetic dye, a derivative of benzidine and naphthionic acid. It is used for

differential staining of elastic fibers for microscopic examination. Amyloid is stained a light

orange-red with Congo red and exhibits apple green birefringence under polarized light.

Amyloid in cats stains poorly. Congo red undergoes a change in hue with acidity and thus can

be used as an indicator of pH, turning red in the presence of alkalis (bases) and blue when

exposed to acids. It is a brownish-red powder, C32H22N6Na2O6S2, used in medicine and as a dye,

indicator, and biological stain. It is an acid dye used as an indicator in testing for free

hydrochloric acid in gastric contents, as a laboratory aid in the diagnosis of amyloidosis, and as a

histologic stain for amyloid.

Structural formula

Molecular formula: C32H22N6Na2O6S2


http://www.answers.com/topic/amyloidosis

http://www.answers.com/topic/gastric

http://www.answers.com/topic/hydrochloric-acid

http://www.answers.com/topic/dye


1.5 Remediation of congo red:

Azo dyes are used in a wide variety of products and can be found in the effluent of most

sewage treatment facilities. Substantial quantities of these dyes have been deposited in the

environment, particularly in streams and rivers. Azo dyes were shown to affect microbial

activities and microbial population sizes in the sediments and in the water columns of aquatic

habitats. Only a few aerobic bacteria have been found to reduce azo dyes under aerobic

conditions, and little is known about the process. A substantial number of anaerobic bacteria

capable of azo dye reduction have been reported. The enzyme responsible for azo dye reduction

has been partially purified, and characterization of the enzyme is proceeding. The nematode

Ascaris lumbricoides and the cestode Momezia expansa have been reported to reduce azo dyes

anaerobically. Recently the fungus Phanerochaete chrysosponum was reported to mineralize azo

dyes via a peroxidation-mediated pathway.

The chemical structure of dyes is comprised of a conjugated system of double bonds and

aromatic rings. The major classes of dyes have antroquinoid, indigoid, and azo aromatic

structures. All of these structures allow strong π-π transitions in the UV-visible (UV-Vis) area,

with high extinction coefficients that allow us to consider these structures dye chromophores. Of

all of these structures, the azo aromatic one is the most widespread dye class in the industry.

The main drawback of this class of dyes is that they are not easily degraded by aerobic

bacteria, and with the action of anaerobic or micro aerobic reductive bacteria, they can form toxic

and/or mutagenic compounds such as aromatic amines. There is a great environmental concern

about the fate of these dyes, with special emphasis on reactive dyeing of cellulosic fibers, where

large amounts of unbound dye are discharged in the effluent.

Congo red dye is one of important azo dyes. Its colored substances have complex

chemical structures and high molecular weights. The chemical structure is the sodium salt of

benzidinediazo-bis-1-naphtylamine-4-sulfonic acid. It is highly soluble in water and persistent in

the environment, once discharged into a natural environment. Thus, the study on Congo red is

interesting not only for being possible pollutants of industrial effluents but also because it is a

good model of complex pollutants.



Paper 1: “Microbial degradation of azo dyes” work was done by N.Puvaneswari ,J

Muthukrishnan ,P. Gunasekaran(Dept Of Genetics, Centre Of Excellence In Genomic Sciences,

School Of Biological Sciences Madurai Kamaraj University, Madurai 625 021, India).

Toxic effluents containing azo dyes are discharged from various industries and they

adversely affect water resources, soil fertility, aquatic organisms and ecosystem integrity. They

pose toxicity (lethal effect, genotoxicity, mutagenicity and carcinogenicity) to aquatic

organisms (fish, algae, bacteria, etc.) as well as animals. They are not readily degradable under

natural conditions and are typically not removed from waste water by conventional waste water

treatment systems. Benzidine based dyes have long been recognized as a human urinary bladder

carcinogen and tumorigenic in a variety of laboratory animals. Several microorganisms have

been found to decolorize, transform and even to completely mineralize azo dyes. A mixed

culture of two Pseudomonas strains efficiently degraded mixture of 3-chlorobenzoate (3-CBA)

and phenol/cresols. Azoreductases of different microorganisms are useful for the development

of biodegradation systems as they catalyze reductive cleavage of azo groups (-N=N-) under

mild conditions. In this review, toxic impacts of dyeing factory effluents on plants, fishes, and

environment, and plausible bioremediation strategies for removal of azo dyes have been

discussed.

Paper 2: “Effect of carbon and nitrogen source amendment on synthetic dyes decolourizing

efficiency of white-rot fungus, Phanerochaete chrysosporium”

Decolourization activity of Phanerochaete chrysosporium for three synthetic dyes viz.,

congo red, malachite green and crystal violet and impact of additional carbon and nitrogen

supply on decolourization capacity of fungus were investigated. Maximum decolourizing

capacity was observed up to 15 ppm. Addition of urea as nitrogen source and glucose as carbon

source significantly enhanced decolourizing capacity (up to 87%) of fungus. In all the cases, both

color and COD were reduced more in non-sterilized treatments as compared to sterilized ones.

Significant reductions in COD content of dye solutions (79- 84%) were recorded by fungus



supplied with additional carbon and nitrogen. A highly significant correlation (r = 0.78, p<0.001)

between color and COD of dye solutions was recorded. Thus, a readily available carbon and

nitrogen source is imperative to enhance the bioremediation activity of this fungus which has

been the most suitable for synthetic dyes and textile industry wastewater treatment.

Deepak Pant*1, 2, Anoop Singh1, 2, Yamini Satyawali1, 2 and R. K. Gupta 1Department of

Environmental Sciences, College of Basic Sciences and Huminities, G.B. Pant University of

Agriculture and Technology, Pantnagar - 263 145, India

2The Energy and Resources Institute (TERI), Darbari Seth Block, India Habitat Centre, Lodhi

Road, New Delhi - 110 003, India

Paper 3: Decolourization & detoxification of congo red and textile industry effluent by an

isolated bacterium pseudomonas sp.SU-EBT

The biodegradation of Reactive Red (RR) textile azo dye by the bacterial isolates

Enterobacter cloacae, Pseudomonas spp. and Bacillus spp was investigated. A small bench scale

suspended bed bioreactor was used to study the capacity of the three bacterial isolates to

decolorize the dye solutions supplemented in five successive additions trial. The degradation of

azo dyes is usually judged by the formation of aromatic amines. All bacterial isolates used under

anoxic conditions were found to produce aromatic amines. The evidence of biodegradation of

RR textile azo dye was tested using three bioassay methods. Six strains related to soil biofertility

were grown in spent media obtained from RR biodegradation. The growth of each isolate on

biodegradation products was as high as the same growth on specific media of either. This is an

evidence for removal of toxicity in biodegradation products. The effect of RR dye

biodegradation products on wheat and berseem clover seed germination was investigated. The

biodegradation of RR dye removed the dye phytotoxic effects. The COD decreased from 2048 to

599 ppm under aerobic conditions. The increased bacterial growth indicated the breakdown of

the organics.The continued decreases in COD value indicated steady biodegradation of the

anoxic biodegradation products under aerobic conditions. The study shows the potential for

using this approach for biodegradation of toxic textile azo dyes by potent bacterial strains in the

sequential anoxic/aerobic bioremediation system.



Amar A. Telke Swati M. Joshi Sheetal U. Jadhav

Dhawal P. Tamboli Sanjay P. Govindwar.

Paper 4: Evidence Of Biodegradation Of Reactive Red Textile Azo Dye In

Anoxic/Aerobic Bioremediation System

By O. S. Barakat, O. M. Darwesh*, M. Z. Sedik , H. Moawad* and W. M. Abd El-Rahim*

Microbiology Department, Faculty of Agriculture, Cairo University and

*Agricultural Microbiology Department, National Research Center, Giza, Egypt

Among 44 yeast strains tested for ability to degrade textile dyes, two novel strains,

Pseudozyma rugulosa Y-48 and Candida krusei G-1, were selected and identified because they

exhibited excellent color removal from various dyes. P. rugulosa Y-48 and C. krusei G-1 could

remove the reactive azo dye, Reactive Brilliant Red K-2BP 200mgL, giving up to 99%

decolorization in 24 h. P.rugulosa Y-48 could also remove 22–98% of seven other dyes 50mgL

and C. krusei G-1 62–94%. Of the seven dyes, all azo dyes were most easily decolorized by these

yeasts. Further analysis showed that decolorization of the dyes tested proceeded primarily by

biodegradation. Screening and identification of yeasts for decolorizing synthetic dyes in

industrial wastewater Zhisheng Yu_, Xianghua Wen State Key Joint Laboratory of Environment

Simulation & Pollution Control, Department of Environmental Science and Engineering,

Tsinghua University, Beijing 100084

Paper 5: Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by

an isolated bacterium Pseudomonas aeruginosa strain BCH

Jyoti P. Jadhav1 , Swapnil S. Phugare1, Rhishikesh S. Dhanve1 and Shekhar B. Jadhav1

(1) Department of Biochemistry, Shivaji University, Kolhapur, 416 004, India



A newly isolated novel bacterium from sediments contaminated with dyestuff was

identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The

bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C)

and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7.

This strain was capable of decolorizing Direct Orange 39; 50 mg l−1 within 45 ± 5 min, with

93.06% decolorization, while maximally it could decolorize 1.5 g l−1 of dye within 48 h with

60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed

to confirm the biodegradation of dye and formation of new metabolites. Induction in the

activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating

the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies

with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded

metabolites.



3.1 Materials required:

All the glass wares, media, equipment used were sterilized according to the microbiological

techniques before carrying out the study.

All chemicals were of highest purity and of analytical grade. Congo red

was obtained from Vasa Scientific Chemicals, Bangalore. Peptone, yeast

extract, beef extract, potato dextrose agar, nutrient agar, nutrient broth and

potato dextrose broth of LR grade were obtained from Hi-media Laboratory.

Acetate buffer, phosphate buffer and Tris HCL were prepared.

3.2 Collection of Microorganisms

For the study of various parameters such as optimum temperature, pH, incubation time

and degradation capability, we have collected the pure cultures of Pseudomonas aeruginosa and

aspergillus niger from Department of Microbiology, Vijayanagara Institute of Medical Sciences

(VIMS) Bellary.

Pseudomonas aeruginosa

FIG 1: PURE CULTURE OF P.Aeruginosa



Aspergilllus niger

FIG 2: PURE CULTURE OF A.Niger

3.2 Methods:

Preparation of media:

100ml of Nutrient broth and nutrient agar was prepared by dissolving 1.3gms of nutrient

broth and 2.8gm of nutrient agar which contains; peptic digest of animal tissue(5 gm/lt),sodium

chloride(5 gm/lt),beef extract(1.5 gm/lt), yeast extract(1.5 gm/lt) and 15 gm/lt of agar was added

while preparing the nutrient agar in 100ml of double distilled water by maintaining pH 7.4±0.2

under aseptic conditions. A loop full of pure culture (p.aeruginosa)was inoculated in the prepared

media to obtain the sub culture under sterile conditions and was kept for incubation at 37c for 24

hours. Whereas for sub culturing of a.niger 2.4gm of potato dextrose broth and 4.1gms of potato

dextrose agar; which contains potatoes infusion(200 gm/lt), dextrose(20 gm/lt) and 15gm/lt of

agar was added while preparing potato dextrose agar dissolved in 100 ml of double distilled

water by maintaining pH of 5.1±0.2. For maintainence of pure cultures, sub culturing was done

every week.



Preparation of congo red solution:

0.5% of congo red was prepared by adding 0.05 gms of congo red in 10 ml of distilled water.

FIG 3: CONGO RED DYE AND SOLUTION

3.2.1 pH optimization:

For the optimization of pH, buffers of different pH were prepared ranging from 4-9.

Buffers of pH 4 and 5 were prepared using acetate buffer and ph 6, 7 and 8 were prepared by

using phosphate buffer and tris HCl buffer was used to prepare pH 9 buffer. Subcultures of

Pseudomonas aeruginosa and aspergillus niger were prepared. 6 test tubes were taken for each

organism for pH ranging from 4-9 1ml of buffer and 1ml of 0.5% congo red were added to all the

test tubes. To measure the OD, blank solutions for pH ranging from 4-9 were prepared i.e., for pH



4 the blank was prepared by using 1ml of pH 4 buffer along with 1ml of 0.5% congo red dye,

likewise the blanks were prepared for the rest of the pH from 4-9.

3.2.2 Optimization of temperature:

For the optimization of temperature, take three test tubes for each organism and add 1ml

of buffer of Ph 4 and 1ml 0f 0.5 % congo red for all the test tubes. Then add 5ml of subcultured

organism to all test tubes i.e., P.Aureginosa with 3 test tubes and Aspergillus niger with other 3

test tubes. Incubate the test tubes for 24hrs at three different temperatures, first test tube at 37ºc,

second at 45ºc and third at 55ºc. After incubation all the test tubes will be centrifuged at

10,000rpm for 3 minutes and OD is measured at 497nm by preparing blank(1ml of Ph 4 buffer

and 1ml of dye).

3.2.3 Effect of incubation:

The specific time period for degradation of dye with the organisms was done by the

following procedure; test tubes with 1ml of pH 4 buffer, 1ml of 0.5% congo red and 5ml of

cultured organism were incubated at different time periods i.e., 1st test tube for 24 hours, 2nd test

tube at 48 hours , 3rd for 72 hours and 4th for 96 hours and were analysed for the degradation. The

24 hours incubated test tube was centrifuged and the OD was noted at 497nm. This process was

repeated for 48 hours, 72 hours and 96 hours test tubes and the OD was noted.

3.2.4 Study of media optimization:

Four different media were prepared i.e., one is free with beef extract, next media free

with peptone, other is free with sodium chloride and the last media is free with yeast extract.

Adjust the pH of all the media and sterilize. Four test tubes for each organism were taken and



5ml of the different media was added to four different test tubes of both the organisms and

inoculated with the organisms for all the test tubes. These test tubes were incubated for optimum

temperature pH and incubation time and the OD was measured at 497nm to find the maximum

degradation.

3.2.5 Effect of UV exposure:

Nutrient agar and potato dextrose agar for P.aeruginosa and A.niger were prepared and

sterilized. The petriplates were prepared and allowed for solidification under aseptic conditions.

Then 200µl of the culture was poured to the petriplates by pour plate technique and incubated for

24 hours then exposed the petriplates under UV light for 10 min. Comparison of the maximum

degradation capacity of the UV unexposed organism along with the UV exposed organism was

done by the following procedure.

Six test tubes for each organism were taken and then the respective broth media of 5ml

was added to all the test tubes. Then three test tubes were inoculated with the UV unexposed

organism and the other three with the UV exposed organism and incubated for 24 hours (same

process with other organism).The OD was measured at 497nm and analyzed for the maximum

degradation capacity of the UV unexposed organism and UV exposed organism.



4.1 Results:

The media and chemicals were prepared under the strict laboratory procedures and sterile

conditions as per chapter 3 and annexure. These were used for optimizing the parameters. The

media and chemicals prepared under such conditions gave 100% results without any

contamination.

4.1.1 Optimization of pH:

Buffers of different pH ranging from 4-9 and sub cultures of P.aureginosa and A.niger were

prepared. Six test tubes for each organism were taken. Add 1ml of the buffer, 1ml of 0.5%

congored and 5ml of sub cultured organisms for all the test tubes. All these test tubes were

incubated for 24 hours and OD was noted at 497nm to find the maximum degradation at a

particular pH. From the readings obtained by measuring the OD at 497nm, maximum

degradation for the organisms was found at pH 4 which was depicted from graph shown above.

Therefore pH 4 was maintained for all the other parameters.

P.Aeruginosa



FIG 4: PH OPTIMIZATION (pH 4 - pH 9)

Table 6 : PH Optimization for P.Aeruginosa

Sl no

Buffer

(ml)

Dye

( ml)

Subculture of P.aeruginosa

(ml)

Incubation

For 24 hrs

At 37oC

OD at

497nm

1 pH 4 1 1 5 0.06

2 pH 5 1 1 5 0.04

3 pH 6 1 1 5 0.02

4 pH 7 1 1 5 0.04

5 pH 8 1 1 5 0.01

6 pH 9 1 1 5 0.02

A.niger



FIG 5: PH OPTIMIZATION (4 -9)

Table 7: PH Optimization for A.Niger

Sl no

Buffer

(ml)

Dye

( ml)

Subculture of A.niger

(ml)

Incubation

For 24 hrs

At 37oC

OD at

497nm

1 pH 4 1 1 5 0.11

2 pH 5 1 1 5 0.01

3 pH 6 1 1 5 0.01

4 pH 7 1 1 5 0.02

5 pH 8 1 1 5 0.01

6 pH 9 1 1 5 0.08



4 5 6 7 8 90

0.02

0.04

0.06

0.08

0.1

0.12pH optimization

p.aeruginosaa.niger

pH

OD at497nm

4.1.2 Optimization of temperature:

Three test tubes for each organism were taken and 1ml of buffer of pH 4,1ml of 0.5% congored and 5ml of the subcultured organisms were added to all the test tubes. These test tubes were incubated for 24 hours at three different temperatures i.e., 370c, 450c and 550c and OD was noted at 497nm to analyse the maximum degradation at a particular temperature. From the readings obtained, maximum degradation was found at 450c for A.niger and 550c for P.aeruginosa which is shown in the graph. Therefore these temperatures were maintained for the respective organisms for the optimization of other parameters.

P.aeruginosa



FIG 6: OPTIMIZATION OF TEMPERATURE

Table 8: Optimization of temperature for P.Aeruginosa

Sl no

Temperature

(0C)

OD at

497nm

1 37 1.39

2 45 1.44

3 55 1.45

4 65 1.35

A.niger

FIG 7: OPTIMIZATION OF TEMPERATURE



Table 9: Optimization of temperature for A.Niger

Sl no

Temperature

(0C)

OD at

497nm

1 37 1.46

2 45 1.48

3 55 1.42

4 65 1.41

37 45 55 651.25

1.3

1.35

1.4

1.45

1.5 Temperature optimization

p.aeruginosaA.niger

Temperature

OD at 497nm

4.1.3 Effect of incubation time:

To know the specific time for the degradation of the dye, four test tubes were taken and 1ml of pH4 buffer, 1ml of 0.5% congored and 5ml of sub cultured organisms was added for all the test tubes. Incubate all these test tubes for different time periods i.e., at 24 hours,48 hours,72 hours and at 96 hours by maintaining specific temperatures for the particular organisms. From the readings and the graph, maximum degradation was found at 48 hours for A.niger and 96 hours for P.aeruginosa. Therefore these time periods were maintained to get the maximum degradation of the dye.



24 hours 48 hours

72 hours 96 hours

FIG 8: EFFECT OF INCUBATION

Table 10: Effect of incubation time for P.Aeruginosa

Sl no Time OD at 497 nm

1 24 0.06

2 48 0.05

3 72 0.02

4 96 1.06



Table 11: Effect of incubation time for A.Niger

Sl no Time OD at 497 nm

1 24 0.04

2 48 0.2

3 72 0.04

4 96 0.03

Graph:

24 48 72 960

0.2

0.4

0.6

0.8

1

1.2Incubation time

p.aeruginosa

a.niger

Time in hrs

OD at 497nm

4.1.4 Study of media optimization:

Four different media were prepared i.e., one is free with beef extract, next media free with peptone, other is free with sodium chloride and the last media is free with yeast extract. Adjust the pH of all the media and sterilize. Four test tubes for each organism were taken and 5ml of the different media was added to four different test tubes of both the organisms and inoculated with the organisms for all the test tubes. These test tubes were incubated for optimum temperature Ph and incubation time and the OD was measured at 497nm to find the maximum degradation. From



the readings obtained and the graph plotted the maximum degradation was found in the sodium chloride free media for A.niger and peptone free media and yeast free media for P.aeruginosa.

P.Aeruginosa

Yeast NaCl Peptone Beef

FIG 9: OPTIMIZATION OF MEDIA

Table 12: Optimization of media for P.Aeruginosa

Media OD at 497nm

Beef free 0.15

Peptone free 1.6

NaCl free 0.30

Yeast free 1.54

A.Niger



Yeast NaCl Beef Peptone

FIG 10: OPTIMIZATION OF MEDIA

Table 13: Optimization of media for A.Niger

Media OD at 497 nm

Beef free 0.03

Peptone free 0.04

NaCl free 0.15

Yeast free 0.06

beef free peptone free nacl free yeast free0

0.20.40.60.8

11.21.41.61.8

media optimization

p aeruginosaa.niger

MEDIA

OD at 497nm



4.1.5 Effect of UV exposure:

Nutrient agar and potato dextrose agar for P.aeruginosa and A.niger were prepared and

sterilized. The petriplates were prepared and allowed for solidification under aseptic conditions.

Then 200µl of the culture was poured to the petriplates by pour plate technique and incubated for

24 hours then exposed the petriplates under UV light for 10 min. Comparison of the maximum

degradation capacity of the UV unexposed organism along with the UV exposed organism was

done by the following procedure.

Six test tubes for each organism were taken and then the respective broth media of 5ml

was added to all the test tubes. Then three test tubes were inoculated with the UV unexposed

organism and the other three with the UV exposed organism and incubated for 24 hours (same

process with other organism).The OD was measured at 497nm and analyzed for the maximum

degradation capacity of the UV unexposed organism and UV exposed organism. From the

readings obtained and the graph plotted maximum degradation was found in UV exposed

organism compared to UV unexposed organism for P.aeruginosa, and for A.niger maximum

degradation was found in UV unexposed organism compared to that of the UV exposed

organism.

P.Aureginosa

UV Exposed Normal

FIG 11: COMPARING DEGRADATION CAPACITY OF NORMAL & UV EXPOSED

Table 14: Comparing degradation capacity of normal and UV exposed P.Aeruginosa

Sl no UV unexposed OD at 497 nm

UV exposed OD at 497 nm



1 0.02 0.04

2 0.02 0.03

3 0.02 0.04

A.Niger

UV Exposed Normal

FIG 12: COMPARING DEGRADATION CAPACITY OF NORMAL & UV EXPOSED

Table 15: Comparing degradation capacity of normal and UV exposed A.Niger

Sl no UV unexposed OD at 497 nm

UV exposed OD at 497 nm

1 0.08 0.04

2 0.06 0.05

3 0.06 0.05

4.2 Discussion:

Earlier works has been done on the biodegradation of congo red, azo carmine, direct orange dyes by organisms like bacillus sp, pseudomonas sp, citrobacter sp candidazeylanoides, etc



(1) Decolorization of direct orange 39(Orange TGLL) by an isolated bacterium Pseudomonas aeruginosa was done by Jyothi.P.Jadhav, Swapnil S. Phugare1, Rhishikesh S. Dhanve1

and Shekhar B. Jadhav

(2) Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium Pseudomona sp. SU-EBT Amar A. Telke Swati M. Joshi Sheetal U. Jadhav Dhawal P. Tamboli , Sanjay P. Govindwar

In the present study, we have taken two organisms namely P.aeruginosa and A.Niger for the degradation of the congo red dye which is generally used in the textile industry for coloring purpose.

We have taken few parameters like PH, temperature, incubation time, dye concentration, media optimization, effect of carbon and nitrogen sources, and effect of exposure to UV light for stabilizing the P.aeruginosa and A.Niger for effective degradation.

In our project, we first stabilized the PH by checking maximum degradation in range PH

4-9. After taking the OD it was concluded that PH 4 was optimum for both the organism.

Then in the temperature optimization, the maximum degradation was checked for different temperature like 370c, 450c, 550c. After the OD results, graphs were plotted and concluded that P.aeruginosa optimum temperature was450c and A.Niger was 550c.

For incubation time, the maximum degradation was checked by incubating for different time periods like 24hr, 48hr, 72hr, and 92hrs. After the OD results, graphs were plotted and concluded that P.aeruginosa incubation time was 96hrs and A.Niger was 48hrs.

Media optimization: The maximum dye degradation was checked by using the media lacking yeast or peptone or beef or NaCl subsequently. By the OD results, it was concluded that the nutrient media lacking peptone free was suited for P.aeruginosa and NaCl was suited for A.niger.

Further the work can be done on the following:

• Analysis of metabolites obtained after decolorization:After the degradation of the congo red dye by the micro organisms the

metabolites can be identified and studied.



• Analysis of the degradation pathway:For the degradation of the congo red dye the pathways so followed by the

organisms can be found and studied to further follow the appropriate pathway to obtain maximum degradation.

• The enzyme responsible for the degradationAspergillus sp and pseudomonas sp have many enzymes which are responsible

for the degradation of the dye at different pH, these enzymes can be identified and purified for degradation of the dyes.

• Effect of mutation due to UV exposure.After the exposure to UV light, confirmation of mutation can be done and the

mutated gene can be analysed to further improve the degradation capacity.

The industrial pollution has grown as one of the most potent challenges for the

environment. The removal of the pollutants from the nature has become one of the prime areas

of research. The bio based removal of pollutants has gained niche because of permanent, short

period and safe bioconversions. Various microorganisms have been reported to have such

capability and the present study is on microbial degradation of congo red, a prominently used azo

dye in textile industry. Congo red at high concentration is toxic to microorganisms, plants and

animals including human beings. Naturally pseudomonas occurring are found to be well suited to

congo red degradation process in which temperature, can be monitored and controlled easily.

The congo red degradability of the isolates can be maintained and used at large scale effluent

treatment. . The pseudomonas sp. isolate was found to have a better degradation capability than

the aspergillus niger.



Annexure :

Table 5: Preparation of buffers:

1. Acetate buffer:Solution A: 0.2 mol/lt of acetic acid(11.55ml/1000ml)Solution B: 0.2 mol/lt of sodium citrate(16.4gm/1000ml)

Mix A & B and make the volume to 100ml.

pH Solution A(in ml) Solution B(in ml)4 41 95 14.8 35.2

2. Phosphate buffer:

Solution A: 0.2mol/lt of monobasic sodium phosphate(NaH2PO4)(31.2gm/lt)



Solution B: 0.2mol/lt of dibasic sodium phosphate(Na2HPO4)(28.3gm/lt)

Mix A & B and make the volume to 100ml.

3.4.

3. Tris HCl:

Solution A: Amount of tris(0.2m) 24.2gm in 100mlSolution B: Volume of HCl(0.2m) 16.1ml in 1000ml

pH Solution A(ml) Solution B(ml) Dilute with distilled water and make volume to

200ml.9 50 5

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Websites:

http://en.wikipedia.org/wiki/Azo_compound http://en.wikipedia.org/wiki/Congo_red http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa http://en.wikipedia.org/wiki/Aspergillus_niger


http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa

http://en.wikipedia.org/wiki/Congo_red

http://en.wikipedia.org/wiki/Azo_compound