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Saurashtra University Re – Accredited Grade ‘B’ by NAAC (CGPA 2.93)
Kothari, Charmy R., 2006, “Microbial degradation of Organopolutants”, thesis PhD, Saurashtra University
http://etheses.saurashtrauniversity.edu/id/801 Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given.
Saurashtra University Theses Service http://etheses.saurashtrauniversity.edu
I take pleasure in forwarding the thesis entitled “Microbial Degradation ofOrganopollutants” of Mrs. Kothari Charmy R for the acceptance of thedegree of Doctor of Philosophy in Microbiology. Thesis presented hereembodies a record of the results of original investigations carried out by her.
Date :Place : Rajkot
Forwarded through
Dr. B.R.M. Vyas Dr. S. P. SinghGuiding Teacher Professor & HeadDepartment of Biosciences Department of BiosciencesSaurashtra University, Saurashtra UniversityRajkot- 360 005, (INDIA) Rajkot- 360 005, (INDIA)
DECLARATION
I, Mrs. Kothari Charmy R., the undersigned hereby solemnly declare thatthe work presented in this thesis entitled “Microbial Degradation ofOrganopollutants” is original and independent. I declare further that this workhas not been submitted for any degree or diploma to any other Universities or
Summary of the Work and Concluding Remarks 105-110
Biodegradation of a Textile Dye Kemifix Red F6B by 88-104Pseudomonas aeruginosa CR-25 and Evaluation of Phytotoxicity
Isolation and Screening of Potential Dye Decolorizing Bacteria 16-42
General Introduction and Review of Literature 01-15
Influence of Nutritional Parameters on Decolorization of Dyesby Pseudomonas aeruginosa CR-25 60-73
Treatment of Mixtures of Dyes with Designer Bacterial 74-87Consortia and Evaluation of Toxicity on Ground nut
Influence of Cultural and Environmental Parameters on 43-59Decolorization of Dyes by Pseudomonas aeruginosa CR-25
CHAPTER -1
1
General Introduction and Review of Literature
INTRODUCTION
Environmental microbiology is the study of microorganisms which exist
in natural or/and artificial environment. It is a fascinating field of science and
the origin of this field rests in the observations of Antony van Leeuwenhoek.
During the last few decades we have begun learning how to harness microbial
biosynthetic and degradative activities. This harnessing, including the
intentional manipulation of microbial activities, constitutes the basis of
microbial biotechnology, whereby we direct the activity of microorganisms
within both natural and artificial environments for varieties of purposes. As
one example, we utilize microorganisms as tools to degrade both natural and
anthropogenic materials in wastewaters digesters, composters, landfills,
natural terrestrial environments and natural or artificial aquatic ecosystem.
There is a well known saying that “Everything touched by King Midas
turned to gold”. By a sort of inversion process, pretty well everything modern
men touch, including themselves, turns to a waste product sooner or later.
Wastes are usually discarded into water, with or without processing.
Presently, water is becoming a rare commodity, and the available water
sources are inadequate to meet the essential basic needs of man, which is
mainly due to increased industrialization of developing countries. Improper
disposal methods and inadequate control of toxic effluents from different
industries have led to the widespread contamination of surface as well as
ground water and have made the water resources polluted for usage1.
Industrial wastes consisting dyes and other pollutants are usually
discarded into water, with or without processing. When waste substances
reach such a concentration that they exert measurable effects upon
ecosystems then they are said to be pollutants. Thus, the problem of
environmental risk is caused by these xenobiotics.
Estimation of pollution created by these xenobiotics and recalcitrants
largely depends on the physical appearance of color, odour and turbidity.
Textile industries are country’s vital set ups. The major sources of colored
effluents are textile and dye stuff industries, particularly where the process of
bleaching, dyeing, printing and finishing in textile operations import huge
amounts of colored effluents. Apart from earning large amount of foreign
exchange, it catches the public attention from the standpoint of pollution.
Untreated effluents from dyestuff production and dyeing mills may be highly
colored and thus particularly objectionable and offensive if discharged into
open waters. Eventhough the dye concentration may be well below 1 ppm i.e.
lower than many other chemicals found in waters, the dye will be visible even
at such low concentrations2. The hazards associated with pollutants can be
reduced by conventional technologies that involve removal, alteration, or
isolation of the pollutants. These technologies are expensive, and in many
cases do not destroy the contaminating compounds but instead transfer them
from one environment or form to another.
Bioremediation
Bioremediation is the use of biological systems for the reduction of
pollution from air, aquatic or terrestrial systems. Microorganisms and plants
are the biological systems which are generally used for this purpose.
Biodegradation with microorganisms is the most frequently occurring
bioremediation option. Microorganisms can break down most compounds for
their growth and/or energy needs. These biodegradation processes may or
may not need air. In some cases, metabolic pathways which organisms
normally use for growth and energy supply may also be used to break down
pollutant molecules. In these cases, known as metabolism, the
microorganisms do not benefit directly, but researchers have taken
advantage of this phenomenon and use it for bioremediation. Complete
degradation, often termed mineralization, ultimately yields water and either
carbon dioxide or methane3, 4. Incomplete biodegradation will yield breakdown
products which may or may not be less toxic than the original pollutant.
Thus, bioremediation addresses the limitations of these conventional
techniques by bringing about the actual destruction of many organic
contaminants at reduced cost. As a result, over the last two decades,
bioremediation has grown from a virtually unknown technology to a
technology that is considered for the cleanup of a wide range of contaminants.
What are dyes?
Dyes have been used since ancient times for coloring and printing
fabrics. Most of these dyes were derived from plant or animal sources by long
and elaborate processes. Even the application of dyes was an elaborate art
involving a high degree of chemical skill. In these periods only the kings and
noblemen could afford colored fabrics. A dyed dress was a symbol of riches.
Indigo plant yielded a beautiful blue dye. Most of the dyes made in nineteenth
century were derived from the aromatic intermediate chemicals isolated from
coal tar distillation. The dyes are colored because they absorb light in the
visible range. The visible range of the spectrum consists of electromagnetic
radiation in the range of 400 to 800 nm.
In the first theory about color it was postulated that when aromatic
molecule combines with color forming groups called chromophores, the
resultant is called a chromogen. Chromogen, when it contains one or more
groups called auxochromes which intensify colors and improve its affinity for
the fiber, is referred to as a dye. Some chromophores are azo (-N=N-), nitroso
(-NO), nitro (-NO2), double bonded carbon (-C=C-) and keto or quinoid system
(-C=O-) etc. Some auxochromes are -NH2, -NCH3, -N(CH3)2 which generally
form cations and -SO3H, -OH and -COOH form anions. Azobenzene is a
chromogen and is colored but is useless as a dye as no auxochrome is
present while azobenzene with an auxochrome yields a dye.
Important pollutants in textile effluent are mainly recalcitrant organics,
color, toxicants and inhibitory compounds, surfactants, chlorinated
compounds, and salts. Dye is the most difficult constituent of the textile
wastewater to treat. Azo dyes are the class of dyes most widely used
industrially5 having a world market share of 60-70%. Reactive azo dyes are
becoming more popular in the textile industry; they are mainly used for cotton
dyeing. However, reactive dyes hydrolyze easily, resulting in a high portion of
unfixed (or hydrolyzed) reactive dyes, which have to be washed off during the
dyeing process. As much as 50% of the initial dye load is present in the dye
bath effluent6.
The wastewaters characteristics from a dye house are highly variable
from day to day depending on the type of dye, the type of fabric and the
concentration of the agents added. Treatment of such wastewaters is
therefore, essential but difficult. The discharge of dye house wastewater into
the environment is aesthetically disturbing, impedes light penetration,
damages the quality of the receiving stream and may be toxic to treatment
processes, to food chain organisms and to aquatic life.
The degradation of dyes in the environment by microorganisms is likely
to be slow 7, which means that it is possible for high levels of dye to persist,
and potentially accumulate. Furthermore, any degradation that does occur
may produce smaller molecules equally unfamiliar to the environment, such
as amines, and which may also be toxic. There is no universal method for the
removal of color from dye waste8, the alternative depend upon the type of dye
wastewater. As the characteristic of dye wastewaters are variable, many
different physical, chemical and biological treatment methods have been
employed for its treatment.
The physical and chemical techniques were numerous including anion-
exchange resin9, floatation10, electro-floatation11, electrochemical destruction12, irradiation13, ozonation14, adsorption15 and the use of activated carbon16.
Some of physical and chemical treatment techniques are effective for color
removal but use more energy and chemicals than biological processes. They
also concentrate the pollution into solid or liquid side streams requiring
additional treatment or disposal. In recent years, bioremedial approaches
have focused on some microorganisms that degrade and adsorb dyes in
wastewaters. A wide variety of microorganisms capable of decolorizing a wide
range of dyes include bacteria, fungi and yeast.
REVIEW OF LITERATURE
The use of microbes to detoxify or degrade pollutants is called
bioremediation. The great diversity of microorganisms provides vast genetic
resources for solutions to cleaning up the environment. Bioremediation, by
using the diversity and liability of microbes, enables more efficient and specific
degradation of contaminants. It is becoming a promising alternative to replace
or supplement present treatment processes for dye removal. There are
various reports of microbial degradation of textile and laboratory textile dyes
by fungi, actinomycetes, yeast, algae and bacteria.
Fungal biodegradation
White-rot fungi are those organisms that are able to degrade lignin, the
most complex structural polymer found in woody plants17. The most widely
studied white-rot fungus, in regards to xenobiotic degradation, is
Phanerochaete chrysosporium. This fungus is capable of degrading dioxins,
polychlorinated biphenyls (PCBs) and other chloro-organics18, 19. Other white-
rot fungi also decolorize dyes, e.g. Coriolus versicolor20, Trametes versicolor21, 22,
Pleurotus ostreatus23 and Coriolopsis polysona24.
Meanwhile, there are various other fungi, such as Umbelopsis isabellina
and Penicillium geastrivous25, Aspergillus foetidus and Rhizopus oryzae26 which
can also decolorize and/or adsorb dyes. Fungal decolorization is a promising
alternative to replace or supplement present treatment processes
Yeast biodegradation
Few reports are available on the application of yeast for decolorization
of textile effluents. The ability of Kluyveromyces marxianus IMB3 to decolorize
Remazol Black-B was investigated and 98% colour removal was achieved at
37oC27. A number of simple azo dyes were degraded in liquid aerated batch
cultures by a strain of the yeast Candida zeylanoides, the extent of colour
removal ranged from 44 to 90%, after 7 days28.
Microbial biosorption
The uptake or accumulation of chemicals by microbial mass has been
termed biosorption29, 30. Bacteria and fungi have been used for the purpose of
decolorizing dye-containing effluents. The decolorization of dyes with different
molecular structures by Cunninghamella elegans was evaluated using several
media31. The removal of Reactive Blue 19 was reported which was up to 60
and 91% in 10 and 80 min respectively14. Depending on the dye types and the
species of microorganisms used, different binding rates and capacities were
observed. It can be said that certain dyes have a particular affinity for binding
with specific microbial species. The use of biomass has its advantages,
especially if the dye containing effluent is very toxic. Biomass adsorption is
effective when conditions are not always favorable for the growth and
maintenance of the microbial population. Adsorption by biomass occurs by ion
exchange.
Bacterial degradation of textile dyes
The wastewaters from textile industry contain various dyes. The
bacteria should exhibit decolorizing ability for a wide range of dyes. Isolating
such microorganisms proved to be a difficult task. To gain a widespread
reception, efforts to isolate bacterial cultures capable of degrading azo dyes
started in the 1970s with reports of a Bacillus subtilis32 followed by numerous
bacteria: Pseudomonas spp. were isolated from an anaerobic-aerobic dyeing
house wastewater treatment facility as the most active azo-dye degraders33, 24.
Chang et al.34 used the extracellular metabolites of a dye-decolorizing strain,
Escherichia coli strain NO3, as a biostimulator to entice E. coli strain NO3 into
a beneficial mode of metabolism for an economically feasible decolorization.
Technical process was designed to decolorize textile wastewaters by sulfate
reducing bacteria35.
The ability of bacteria to metabolize azo dyes has been investigated by
a number of research groups. Under aerobic conditions azo dyes are not
readily metabolized, the intermediates formed by the degradative steps
resulted in disruption of metabolic pathways and the dyes were not actually
mineralized. Under anaerobic conditions, such as anoxic sediments, soluble,
cytoplasmic reductases, known as azo reductases, reportedly produce
colorless aromatic amines which can be toxic, mutagenic, and possibly
carcinogenic36.
Azo dyes and intermediates
Azo dyes contain at least one nitrogen-nitrogen (-N=N-) double bond,
however many different structures are possible37. Mono-azo dyes have only
one -N=N- double bond, while di-azo and tri-azo dyes contain two and three -
N=N- double bonds respectively. The azo groups are generally connected to
benzene and naphthalene rings, but can also be attached to aromatic
heterocycles or enolizable aliphatic groups37. These side groups are
necessary for imparting the color of the dye, with many different shades and
intensities being possible38.
Eighty to ninety five percent of all reactive dyes are based on the azo
chromogen37, 39. Reactive dyes are colored compounds that contain one or
two functional groups capable of forming covalent bonds with the active sites
in fibers. A carbon or phosphorous atom of the dye molecule will bond to
hydroxyl groups in cellulose, amino, thiol, and hydroxyl groups in wool, or
amino groups in polyamides37, 40. Most fiber-reactive azo dyes are used for
dyeing cellulosic materials, such as cotton, and are a major source of dye
wastes in textile effluents. Between 20-50% of the reactive dye used by the
textile industry is lost in exhaust and wash water41. Fiber-reactive azo dyes
exhibit a high wet-fastness, due to their ability to covalently bond to
substrates. However, dyes that hydrolyze in solution prior to bonding to a
substrate are often lost in the washing processes42.
Much of the work undertaken in dye degradation has involved the
decolorization of azo dyes. An aerobic azo dye degradation by several
bacterial strains capable of using the dye as the sole source of carbon and
nitrogen has been reported43. Anthraquinone-based dyes are highly resistant
to degradation due to their fused aromatic structures. Some anthraquinone
dyes undergo decolorization and degradation by Bacillus subtilis44. Walker and
Weatherly45 reported degradation of an acid anthraquinone dye by three
strains of Pseudomonas and Bacillus.
Physico-chemical methods are applied for the treatment of many textile
dye effluents achieving high dye removal efficiencies46. On the other hand, in
recent years there is a tendency to use biological treatment systems to treat
dye-bearing wastewaters47. The recalcitrant nature of azo dyes, together with
their toxicity to microorganisms, makes aerobic treatment difficult. On the
other hand, a wide range of azo dyes is decolorized anaerobically48, 49, 50, 51.
Under anaerobic conditions, azo dyes are readily cleaved via a four-electron
reduction at the azo linkage generating aromatic amines. In addition, it is
known that methanogenic and acetogenic bacteria in anaerobic microbial
consortium contain unique reduced enzyme cofactors, such as F430 and
Vitamin B12 that could also potentially reduce azo bonds52, 53. These steps
remove color of the dye, however they do not completely mineralize the
aromatic amines generated in the anaerobic environment53, 55, 56, with a few
exceptions52, 57. Unfortunately, the aromatic amines cannot be regarded as
environmentally safe end products. On the other hand, it is known that most of
the aromatic amines can be biodegraded under aerobic conditions50, 58, 59.
Although, in recent studies dealing with anaerobic treatment of textile
wastewater several high rate anaerobic reactors such as up flow anaerobic
sludge blanket reactors (UASB) and anaerobic baffled reactors (ABR) were
used.
Mixed bacterial cultures from a wide variety of habitats have also been
shown to decolorize the diazolinked chromophores of dye molecules60, 61
demonstrated decolorization of mixture of dyes by anaerobic bacteria using
free growing cells or in the form of biofilms on various support materials.
Toxicity considerations: The problem of carcinogenic azo colorants
Colorants (dyes and pigments) are important industrial chemicals.
Following the technological nomenclature, pigments are colorants that are
insoluble in the application medium whereas dyes are applied in soluble form.
The question of systemic bioavailability, upon inhalation and skin contact, is of
particular importance for azo colorants based on carcinogenic aromatic
amines62.
In the past azo colorants based on benzidine, 3, 3’-dichlorobenzidine,
3,3’-dimethylbenzidine (o-toludine) and 3’-dimethoxybenzidine (o-dianisidine)
have been synthesized in large amounts and numbers, especially in
Germany.
Metabolism and bio-activation of azo colorants
The ecotoxicity of the textile dyes and their possible hazard to human
health was frequently reported63, 64, 65, 66, 67. As reported by Chung et al.68, the
azo dyes extensively used in textile, printing, leather, paper making, drug and
food industries, following oral exposure, these dyes might be metabolized to
aromatic amines. Many of these aromatic amines are mutagenic as indicated
by AMES Salmonella/microsomal assay system.
Azo colorants are biologically active through their metabolites.
Azoreduction of these compounds occur in vivo69, 70, 71 by an enzyme-mediated
reaction. Azo-reductases are found in mammalian tissues, particularly in
liver72, 73, 74, 75 in gut bacteria76, 77, 78, 79, 80 and in skin bacteria such as
Staphylococcus aureus81. Extensive azo reduction was also described in one of
40 investigated isolates of Pseudomonas fluorescence82. The result of this azo-
reduction is the release of the (carcinogenic) aromatic amine from the
colorant74.
Studies performed on exposed workers have demonstrated that the
azo-reduction of benzidine-based colorants occurs in man83, 84, 85, 86, 87. As early
as 1995 increased rates of bladder cancer were observed in workers involved
in dye manufacturing88. Since that time, many studies have been conducted
showing the toxic potential of azo dyes. An understanding of the toxicity
problem can be found in the works of89 as well as90. Both papers indicate that
the problem associated with azo dyes is created by the dye metabolites.
A plant seed germination/root elongation and plant genotoxicity
bioassays has been reported to evaluate the remediation of some of the
contaminated soils91. The same test was employed by many other
researchers92, 93, 94, 95, 96, 97 to assess the phytotoxicity of different chemicals.
The evaluation of biotoxicity of textile dyes using germination of seeds to test
the mutagenic effects of several textile dyes also has been reported98.
Understanding the dye structures and how they are degraded is crucial
to understand how toxic by-products are created. The three-part list of the
biological mechanisms thought to be responsible for carcinogenic activation of
azo dye compounds89. This list is based on an extensive review of the
literature regarding azo dye toxicity, and places each mechanism in order of
their frequency of citation. They postulated that (i) azo dyes may be toxic only
after reduction and cleavage of the azo linkage, producing aromatic amines,
(ii) azo dyes with structures containing free aromatic amine groups that can
be metabolically oxidized without azo reduction may cause toxicity and (iii)
azo dye toxic activation may occur following direct oxidation of the azo linkage
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4. Bhalodi, P.V., Falguni J., Kothari C.R. & Kothari, R.K. 2003. Isolation, identification andcharacterization of an indigenous bacterial population from textile effluents-abiodiversity approach. In proc of National Symposium on Environmental Biotechnologyand Biodiversity Conservation sponsored by UGC (DSA) programme Department ofBiosciences, Sardar Patel University, Vallabh Vidhyanagar. January 31-February 1,2003.
5. Kothari, C.R. & Kothari, R.K. 2005. Analysis of dye decolorizing potential amongbacterial population of textile effluents. In Proc. of 46th AMI annual conference on Micro-Biotech 2005. Department of Microbiology, Osmania University. December 8-10, 2005.
6. Kothari C.R., Kothari, R.K., & Vyas B.R.M. 2006. Sequential anaerobic-aerobictreatment of artificial textile effluents and its effects on seed germination. In Proc. ofUGC-DSA sponsored National conference on Bioresources: Utilization andConservation. Department of Biosciences, Saurashtra University, Rajkot. February 17-18, 2006.
7. Kothari, R.K., Kothari, C.R., & Pathak, S.J. 2006. Bioremediation of textile effluents-anindispensable need for better and cleaner tomorrow. In proc. of UGC-DSA sponsoredNational conference on Bioresources: Utilization and Conservation”. Department ofBiosciences, Saurashtra University, Rajkot. February 17-18, 2006.
8. Purohit, M., Kothari, R.K., Kothari, C.R., & Pathak, S.J. 2006. Biodegradation of textiledyes by a seven member bacterial consortium. In proc. of UGC-DSA sponsoredNational conference on Bioresources: Utilization and Conservation. Department ofBiosciences, Saurashtra University, Rajkot. February 17-18, 2006.
9. Maniar, E., Kothari, C.R., & Pathak, S.J. 2006. Isolation, identification andcharacterization of naphthalene utilizing bacteria. In Proc. of UGC-DSA sponsoredNational conference on “Bioresources: Utilization and Conservation. Department ofBiosciences, Saurashtra University, Rajkot. February 17-18, 2006.
STATE1. Kothari, R.K., Kothari C.R. & Jani N.N. 2004. Bioinformatics a novel tool for biological
sciences. In proc. of XVIII Gujarat science congress. Saurashtra University, Rajkot.March 13, 2004.
TRAININGS/WORKSHOPS
1. Fluorescence In Situ Hybridization (FISH) technique, organized by cell biologydivision, Gujarat cancer and research institute, Ahmedabad. September 10, 2003.
2. Workshop cum Hand on Experience Course in GCMS sponsored by DST, Govt. ofIndia, New Delhi, and organized by sophisticated instrumentation centre for appliedresearch & testing (SICART), Vallabh Vidhyanagar, New Delhi. March 5-7, 2006.