Final

A study on degradation of Congo red-an azo dye by Bacillus species
INTRODUCTION
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 through addition of (biostimulation) or 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.
Dept. of Biotechnology, S.R.N.M.N.College of Applied Sciences, Shimoga Page 1
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 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.
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, largescale 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 "wasteeating" 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 (hopefully), 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 (ranging from Love Canal to the ENSCO incinerator plans in Mobile AZ years ago), accompanied by increasingly stringent regulatory requirements. The interest of commercial businesses in utilizing microorganisms to detoxify effluents, soils, etc. is reflected in "bioremediation" having become a common buzzword in waste management. Companies specializing in bioremediation (or, as it was known several years ago, in biodegradation technologies) 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.
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. The 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 comply 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 (SO42-), 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. 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 insecticides.
Azo dyes
What are Azo Dyes? Azo dyes are a group of synthetic organic compounds that contain a nitrogennitrogen 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. What are Azo Dyes used for? 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. Are Azo Dyes Harmful? 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. Which Azo Dyes are Banned? 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 coloration under the EU Directive 2002/61/EC. 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, 4diaminodiphenylmethane 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
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. Remediation of Azo dyes: 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 - transitions in the UVvisible (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.
REVIEW OF LITERATURE Historical Perspective: In the past decade the United States has spent billions of dollars trying to clean up contaminated ground water and soils, the legacy of an era in which industry grew faster than knowledge about safe chemical disposal. Despite the large financial investment, ground water cleanup efforts are falling short of public expectations. Recent studies have revealed that, while conventional cleanup technologies have prevented the contamination problem from spreading, in most cases they are incapable of restoring the water to meet health-based standards in a reasonable time frame. Soil cleanups have been more successful in meeting regulatory standards. However, conventional soil cleanup methods may transfer contaminants to the air, posing risks that are not always acceptable to residents near the contaminated site. The limitations of conventional ground water cleanup technologies and the hazards of conventional soil cleanup methods have spurred investigations into in situ bioremediation, which uses microorganisms to destroy or immobilize contaminants in place. Conventional methods for ground water cleanup rely on pumping water to the surface and treating it there. Such pump-and-treat methods are slow; they require the withdrawal of large volumes of water to flush contaminants from aquifer solids, and they may leave behind reservoirs of contaminants that are lighter or denser than water and/or have low solubilities. These systems consist of a series of wells used to pump water to the surface and a surface treatment facility used to clean the extracted water. This method can control contaminant migration, and, if recovery wells are located in the heart of the contaminant plume, it can remove contaminant mass. However, recent studies have shown that because many common contaminants become trapped in the subsurface, completely flushing them out may require the pumping of extremely large volumes of water over very long time periods. The surface treatment methods that are part of
pump-and-treat systems use processes that transfer the contaminant to another medium (the air or the land) instead of destroying it. Few people realize that In Situ bioremediation is not a new technology. The use of biooxidation for environmental purposes has been practiced for many years. In fact organisms have been on earth for some 3 billion years. The thick GrenvilleLimestones suggest the presence of marine organisms of some sort existed in the Archeozoic Era. Thus, the organisms have had a very long time to evolve, adapt and disperse. This long period likely also led to excellent survival strategies. Extended to micro biology that suggests that biodegrative traits found in one soil or groundwater would be found in most other soils or groundwater around the world today. A novel bacterial strain capable of decolorizing reactive textile dye Red BLI is isolated from the soil sample collected from contaminated sites of textile industry from Solapur, India. The bacterial isolate was identified as Pseudomonas sp. SUK1 on the basis of 16S rDNA analysis. The Pseudomonas sp. SUK1 decolorized Red BLI (50mgl (-1)) 99.28% within 1h under static anoxic condition at pH range from 6.5 to 7.0 and 30 degrees C. This strain has ability to decolorize various reactive textile dyes. UV-Vis spectroscopy, FTIR and TLC analysis of samples before and after dye decolorization in culture medium confirmed decolorization of Red BLI. A significant increase in the activities of aminopyrine N-demethylase and NADH-DCIP reductase in cells obtained after decolorization indicates involvement of these enzymes in the decolorization process. Phytotoxicity testing with the seeds of Sorghum vulgare and Phaseolus mungo, showed more sensitivity towards the dye, while the products obtained after dye decolorization does not have any inhibitory effects (Kalyani et al., 2007). This work focused on the development of a practical biosorbent for the decolorization of textile effluents. The fermentation waste, Corynebacterium glutamicum biomass, when decarboxylated and immobilized in polysulfone matrix performed well in decolorization of simulated reactive dye bath effluent comprised of four different reactive dyes and other auxiliary chemicals. The regeneration of polysulfone-immobilized C. glutamicum was successful with the aid of 0.01M NaOH as the eluant, which enabled the biosorbent to maintain consistent decolorization
efficiency for up to 25 cycles. An up-flow packed column loaded with polysulfoneimmobilized biomass performed well in the continuous treatment of Remazol effluent. Samples collected after 14hours of column operation revealed almost zero colour and TOC (Vijayaraghavan, 2008). Under nitrogen-limiting, secondary metabolic conditions, the white rot basidiomycete Phanerochaete chrysosporium extensively mineralized the specifically 14C-ring-labeled azo dyes 4-phenylazophenol, 4-phenylazo-2methoxyphenol, Disperse Yellow 3 [2-(4'-acetamidophenylazo)-4methylphenol], 4-phenylazoaniline, N,N-dimethyl-4-phenylazoaniline, Disperse Orange 3 [4-(4'-nitrophenylazo)-aniline], and Solvent Yellow 14 (1-phenylazo2-naphthol). Twelve days after addition to cultures, the dyes had been mineralized 23.1 to 48.1%. Aromatic rings with substituents such as hydroxyl, amino, acetamido, or nitro functions were mineralized to a greater extent than unsubstituted rings. Most of the dyes were degraded extensively only under nitrogen-limiting, ligninolytic conditions. However, 4-phenylazo-[U-14C] phenol and 4-phenylazo-[U-14C] 2-methoxyphenol were mineralized to a lesser extent under nitrogen-sufficient, nonligninolytic conditions as well. These results suggest that P. chrysosporium has potential applications for the cleanup of textile mill effluents and for the bioremediation of dye-contamin Degradation of Azo Dyes by Trametes villosa Laccase over Long Periods of Oxidative Conditions. Trametes villosa laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 hours of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by
13
C-nuclear magnetic
resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in
effluents, limiting the application of laccases as bioremediation agents (Zille et al., 2005).
Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC50) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with blank using water. However, when the effluents were decolorized with immobilized lactase, they could be used for dyeing and acceptable color differences (DE) below 1.1 were measured for most dyes (Abdulla et al., 2000).
OBJECTIVES
Screening of organisms for the effective degradation of Congo red. Optimization of cultural parameters for the effective degradation. Determination of effect of mutations on degradation of Congo red.
pH METER
The negative logarithm of hydrogen ion concentration of any solution is known as its pH and indicates the amount of hydrogen ion available for reaction.
pH meter is an instrument which uses an ion selective electron (ISE) that ideally responds to only one specific ion, such as the H+ concentration of the solution, to measure Ph of the solution.
The electrode produces the current, proportional to the concentration of the H+, which is converted into pH reading and displaced on the meter. Based on the type of display, PH meters are of two types: Digital and Analog.
pH meter consists of: H+ selective membrane. Internal reference electrode. External reference electrode. Meter with control electronics and display.
Working Design and Principle: Earlier pH meters used to have two separate electrodes; glass and reference. Now a days single or combination electrodes are being used. The internal reference electrode is filed with a saturated KCl and a 0.1M HCl solution. The cathode terminus of the reference probe is also present within this electrode. The inner tube is the reference end. It does not come in contact with the solution, thus KCl remains unchanged. The anode terminus is wrapped around the outside of the inner tube. Reference solution is present in both the inner as well as outer tube. The outer tube is in contact with the test solutions by means of a porous plug, which acts as salt bridge. It contains the solution of KCl, which mixes with outer solution. The round thin glass bulb at the bottom of a pH electrode is the measuring part of the pH meter. When ions move diffused from one region of activity of another, there is a free energy change, which is measured by pH meter. Measurement of pH: pH of solution is measured as follows:
Remove electrodes from standardized solution. Rinse electrodes (and blot dry). Place electrodes in an unknown solution. Turn function selector from the stand by to pH. Read pH of the unknown solution. Return function selector to stand by.
CENTRIFUGE
A centrifuge is used to separate a heterogonous mixture of solid and liquid by spinning it. It is a device used to separate two or more substances of different density by using centrifugal force. After the successful centrifugation, the solid precipitate settles to the bottom of the tube and the clear solution called the centrifuge is obtained. It consists of a fixed base or frame and a rotating part in which the mixture is placed and then spins at high speed. 0.5 or 1.5ml disposable plastic or glass tubes are used to place the sample. Tubes are placed in the rotating part in holders so arranged that when the rotator motion begins, the test tube swing into a slanted or a horizontal position with the open ends towards the axis of rotation the heavier the solid part of solution settles in the bottom of the tube and the lighter liquid part forms layer on the tube.
Types of Centrifuge

Low speed: Go up to about 5000 rpm. High speed: Go up to a 20000rpm. Air fuge: can reach 1, 00,000 rpm Micro centrifuge: operates between 10000 to 13000 rpm. Ultra centrifuge: operates at the speed of more than about 20000 rpm. Vacuum type ultra centrifuge. Centrifuge general purpose. High speed refrigerated research centrifuge. Programmable high speed research centrifuge.
Using a Centrifuge: Place test tube in the centrifuge holder balance with another test tube filled to the same level in the opposite holder close cover and turn knob. Centrifugation a minute are more. Note that you must turn off the centrifuge with the switch and wait for it to stop spinning, to effectively separate the precipitate and the solution. Precaution: Mechanical stress can result in rotor failure. Improper loading and balancing of rotors can cause the rotor to break loose while spinning. Prior to centrifuge operation, check the following: The total mass and balance. Distance or position of all counter weights. Secure all elements.
The centrifuge chamber should be clear of abstraction.
MICROPIPPETE
Pipettes are used for volumetric measurements and transfer of known quantity of solution from one container to another vessel. Micropipettes are precision instruments. There are two types: single and multi channel. They are provided with plastic disposable tips for dispersing liquid. Micropipette has 3 positions. Procedure: Attach the tip to the end of the micropipette shaft. Adjust the setting. Press and twist slightly to make sure it is air tight Hold the pipette vertically and press the plunger to the first stop. This displaces equal to the volume of setting. Now immerse the tip into liquid. Release the plunger back to the rest position. Wait a second for the liquid to be sucked up into the tip. The volume of the liquid in the tip is equal to the volume of the setting of the micropipette.
Place the tip at the angle (10-450) against the wall of the tube or flask or a micro well plate and once again press the plunger to the first stop.
After one second, press the plunger to the second stop to the expel all the liquid.
Remove the tip away and bring the plunger to the rest position.
Precautions: The grips should fit tightly on the end of the pipette. Pipette should always be kept in an upright position to prevent liquids from running inside the shaft of the pipette. Always leave the pipette on the pipette stand. The liquid drawn up should reach the expected level in the tip and there should be no air bubbles in the tip. Discard contaminated/blocked/broken tips.
HOT AIR OVEN
Doubled walled thermostatically controlled: Inner chamber made of aluminum or stainless steel. Outer body is made mild steel. Beaded heating elements are placed under the ribs at the bottom and sides. Temperature controlled by hydraulic thermostat from 100 C above ambient to 2400C. dial setting approximate basis. Correct reading as per l shape thermometer fitted on the front panel at the top. Sterilization Temperature: Either 1600C for 2hours or 1800C for 30 minutes. Uses: Sterilization of
Glass wares like syringes, Petri dishes, test tubes, flasks, pipettes. Surgical instruments like forceps, scalpel and scissors. Oily fluid which are impermeable to water such as oils & fats. Chemicals such as the powders which would clump or form into cake in presence of moisture.
Precautions: Substances to be sterilized should be absolutely dry. Articles like rubber goods, fabrics or any inflammable or volatile substances should not be put in the oven. The instrument should not be overloaded & adequate space must be left inside for the circulation of air. After disconnecting from electric line, oven should be allowed to cool for 2 hours before opening the dorsa to prevent cracking of glass
AUTO CLAVE
Autoclave is very widely used in the medicine and metallurgy. Autoclaving is a process of sterilization by saturated steam under high pressure above 1000C. Autoclave is doubled walled chambers made up of stainless steel or gunmetal with a supporting frame. The steam circulates within the jacket and is supplied under high pressure to close dinner chamber where goods are kept for sterilization. 1/5th part of cylinder is filled with water and materials to be sterilized are placed inside. The lid is closely securely discharge tap on it open. The heater is put on. Safety valve is adjusted to required pressure. For sometime after boiling of water inside the chamber the steam and air mixture is allowed to escape to the cylinder becomes air free. Then the discharge tap is closed. When desired pressure in the chamber rises to one chosen for autoclaving i.e., 15lbs pressure the sterilization period is maintained for 15-20 minutes. Sterilization Period: Autoclaving is usually done at a temperature of 1210C and pressure of 15 lbs for 15-20min. sterilization can also be done higher temperature at 1260C (20 lbs pressure) for 10 min. or 1330C (30lbs pressure) for 3 min. Principle of Autoclaving: Steam saturated at high pressure temperature is better sterilizing agent than dry heat. Saturated steam heats the articles to be sterilize rapidly latent heat which participates in bacterial killing. Steam contracts in volume & enhances penetration.
Uses: Autoclaving is used sterilize culture media, rubber goods, syringes, gowns, dressings etc and is the surest method of destruct ion of bacterial spores. Precautions:
The air must be escape from the chamber as temperature of air steam mixture at given pressure is lower than that pure steam.
Contents should be arranged loosely to ensure free circulation of steam inside the chamber.
MATERIALS AND METHODOLOGY All the chemicals used in this study were of analytical grade supplied from Himedia, SRL and Rankem, Mumbai. Screening of Organisms for Degradation: Nutrient agar plate of 25ml was prepared by dissolving ingredients like peptone (0.25g) NaCl (0.25g) , yeast extract (0.125g), 0.01% Congo red and 0.5 g Agar in 25ml of distilled water and autoclaved. After autoclaving the media was poured in to preautoclaved petridish and allowed for solidification. Totally 4 organisms were screened, viz., four Bacillus species (SCWS-5, SCWS-3, PAWN and CR) and Labactobacillus (KCL). The Bacillus species isolates were collected from the culture collection of BioGenics, Hubli, which were isolated from textile effluents of Chennai. The plate was incubated at 450C for 48 hours and the zone of Congo red degradation around the colonies was observed. The Bacillus species (SCWS-3) was found to have a larger zone around, so the culture was used for further studies.
Determination of optimum Congo red concentration for degradation: The nutrient broth tubes (5 ml) containing different concentrations of Congo red (0.01, 0.05, 0.1, 0.5 and 1 mg/ml) were prepared, autoclaved and inoculated with 0.1 ml of Bacillus species. An un-inoculated tube without Congo red was also kept along with a series of tubes with different concentrations of Congo red (as mentioned above) kept as control. The tubes were incubated at 450C for 24 hours. The cultures with inocula were centrifuged at 10,000 rpm for 3 min and the absorbance of the supernatant (dye left undegraded) was measured at 497 nm against media without inocula and Congo red. The absorbances of the control tubes were also measured and the percentage of degradation was measured according to the following formula.
pH optimization: Nutrient broth medium was prepared and it was taken in beaker then pH was adjusted from pH 7, 8 and 9 sterilized. After sterilization to all the test tubes 0.5ml Congo red solution was added then organism was inoculated and incubated at 45oC for 24hours. After incubation all the test tubes were centrifuged and absorbance was read at 497nm.
Optimization of Temperature: Nutrient broth medium was taken in 4 test tubes and sterilized. After sterilization, one tube was kept as blank and for other 3 tubes 0.01 mg/ml of Congo red solution was added and organism was inoculated. Then one tube was incubated at room temperature and other at 45oC and 55oC were incubated for 24hours. After incubation tubes were observed for degradation.
Effect of Incubation Time: Medium was prepared and taken in test tubes. The tubes were sterilized. After sterilization, to all tubes Congo red solution was added and organism was inoculated and kept for incubation at 45oC for 24, 48 and 72 hours and analyzed for the degradation at every 24 hours respectively.
Effect of Carbon and Nitrogen Sources: Nutrient broth medium was prepared and was taken in test tubes. Then carbon source like Dextrose, Maltose, Lactose, Fructose and Sucrose, of 1% was added to respective tube and similarly nitrogen source like Peptone, Ammonium sulphate and Yeast extract were weighted and added to respective tube. All the test tubes were sterilized, to each test tube 0.01 mg/ml of Congo red was added and organism was inoculated. Tubes were kept for incubation at 45oC for 24 hours and analyzed for degradation.
RESULTS AND DISCUSSION The present study was aimed at studying the biodegradation of textile pollutant Congo red. Screening of organisms for degradation: Five organisms were screened for the degradation. One isolate belonging to Bacillus species (SCWS-5) showed the maximum degradation which was much better than other four organisms screened. So, the same isolate was further studied.
Analysis of degradation capability: The degradation capability of the culture was screened by growing it in presence of different concentrations of Congo red. The result indicated at 0.01 mg/ml concentration was more suitable for the culture to degrade more, and the same concentration of the dye was maintained in further studies.
Optimization of Temperature: The incubation temperature was screened for the effective degradation. The tubes were incubated at 3 different temperatures. At 450C more than 87.2% Congo red was degraded. The other temperatures were unsuitable as less degradation was noted.
Effect of Incubation time: The culture was screened for the effect of incubation time on degradation of Congo red. 86 % of degradation was found at 48 hours and complete degradation was observed by 72 hours.
Optimization of pH: The incubation temperature was screened for the effective degradation. The tubes were incubated at 3 different pH. At pH 7 more than 87.2% Congo red was degraded. The other pH was unsuitable as less degradation was noted.
Effect of Carbon sources: After the incubation carbon sources like Dextrose, Maltose, Lactose, Fructose and Sucrose. Fructose gave 88.63% degradation, indicating the best suitability. Dextrose showed 86.34% degradation, Maltose showed 71.50% degradation, Lactose showed 86.33% degradation and Sucrose showed 51.41% degradation.
Effect of Nitrogen sources: After the incubation nitrogen sources like Peptone, Yeast extract and Ammonium sulphate. Peptone gave 65.35% degradation, indicating the best suitability. Yeast extracts showed 36.22% degradation and also Ammonium sulphate showed 48.95% degradation.
SUMMARY 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. that other 4 bacteria screened. Peptone as nitrogen source. The Bacillus species isolate (SCWS-5) was found to have a better degradation capability The Bacillus species strain was found to work efficiently at pH 8, at temperature 450C, at 72 hours, with Fructose as carbon source,
CONCLUSION Congo red at high concentration is toxic to microorganisms, plants and animals including human beings. In spite of the physical and chemical methods in use, the microbial degradation of toxic xenobiotics is gaining much importance. Naturally occurring Bacillus species are found to be well suited to Congo red degradation process. Therefore, the Congo red degradability of the isolates can be maintained and used at large scale effluent treatment.
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A study on degradation of Congo red-an azo dye by Bacillus species

Bioremediation is a pollution control technology that uses biological systems

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

Biotransformation of Toxic Wastes to Harmless Products:

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A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

Role of Microbes in Bioremediation:

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

The centrifuge chamber should be clear of abstraction.

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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A study on degradation of Congo red-an azo dye by Bacillus species

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