ABSTRACT

In recent times river pollution is a serious and growing difficulty in most developing
countries. Industrial effluents and sewage entering the water bodies are one of the major
causes of environmental toxicity, which endangers aquatic biota and deteriorates water
quality.
Heavy metals and dyes are primarily one of the toxic pollutants that are released by textile
industries into water bodies. The chemical degradation of heavy metals and dyes is expensive
with the production of toxic by-products in certain chemical reduction processes. The
potential to biologically degrade the heavy metals and dyes by using biotic and abiotic
components like bacterial stains and some natural adsorbents has emerged as a potential nonpolluting and effective treatment method to eliminate toxic by-products. Biological methods
for the removal of heavy metals and dyes from textile industrial waste may provide an
attractive alternative to the physicochemical process.
In the present study we examined the industrial effluent wastes, soil and plant sample by
Atomic absorption spectroscopy to determine the presence of heavy metals like lead and iron
then through conventional and non-conventional microbes in submerged fermentation
cultures and then with the filter made of natural adsorbents coconut husk and sawdust. The
characterisation of major physical parameters initial concentration of dye and heavy metals
was

monitored

and

analysed

for

optimum

conditions.

The

atomic

absorption

spectrophotometric analysis was done to plot the standard calibration curve and then used to
determine percentage reduction as a function of the above mentioned physical parameters.
The disappearance of characteristic peaks was indicative of the degradation of dye and heavy
metal under aerobic conditions by Pseudomonas diminuta and Bacillus megaterium and
further with the filter made of natural adsorbents was known to partially degrade.
The determination of optimum physical parameters (Monitoring of natural attenuation) of
biodegradation of heavy metal and dye is therefore indicative of potential of the microbes to
utilise different sources of carbon for their functionality. The determination of optimum
conditions for bacterial strain growth in iron and lead broth and mythyl red broth can be used
for detection of degradation of heavy metals and dye from waste water. Thus the study
defines the application of utilisation of synthetic dyes and heavy metal as products for
medical detection also, while fostering waste water effluent treatment characteristics.
Keywords: Pseudomonas diminuta, Bacillus megaterium, Atomic absorption spectroscopy,
Coconut husk and sawdust, mythyl red (azo dye).

CHAPTER 1
INTRODUCTION
The Textile industry or apparel industry is principally apprehensive with the production of
fabric, yarn and the ensuing manufacture or plan of clothes and their circulation. The
unprocessed material may be natural, or it may be synthetic utilizing yield from the chemical
industry. The procedure of inserting color to textile/fabric products is called as Dying. In a
special solution include dyes and particular chemical material basically dye is formed. After
dyeing, dye particles have non breakable chemical bond with molecules of fibre. The two key
factors in dyeing are Time controller and temperature maintenance. It is found that the
effluent of textile industry is drained in water bodies directly without any treatment because
of this many aquatic lives are losing their existence from nature [1].
Textile chemicals have been shown the potential to not only act as allergens but also toxic
and even mutagenic or carcinogenic compounds, posing risks to the environment [2]. Effluent
discharge into water bodies containing dyes and heavy metals is both carcinogenic and
mutagenic, thus implicating its toxicity to aquatic life and humans [3]. Various microbial
decolorization systems for treating dyes and heavy metals by sequential anaerobic aerobic
system have been used [4]. In textile industries, the coloration process exudes large
percentage of the synthetic dyes which do not bind and are thus lost to the waste stream [5].
Figures approximate 10-15% dyes are released during dyeing process, thus imparting colour
and other toxic components. The effluent from textile industries thus carries a large number
of dyes and other additives which are added during the colouring process [6].

1.1 Bioremediation by micro-organisms

The process of treatment of biodegradable chemical compounds, which are potential sources
of environmental pollution by in-situ and ex-situ methods, is termed as bioremediation. Some
examples of bioremediation related in-situ technologies are
Phytoremediation,
Bioventing
Bioleaching

Bioaugmentation
Rhizofiltration, and
Bio-stimulation

Biodegradation mechanisms in a microbial cell

The application of bioremediation as a biotechnological process involving microorganisms

has emerged as an important field in microbiology and use of beneficial microbes, because of
its increasing potential of solving the dangers of many pollutants through biodegradation.
Microorganisms can be considered excellent pollutant removal tools in soil, water, and
sediments, mostly due to their advantage over other chemical and physical bioremediation
procedures on account of their inefficiency in treating stable aromatic compounds [7].

Natural attenuation or bio attenuation is the reduction of contaminant concentrations in the

environment throughout biological processes (aerobic and anaerobic biodegradation, plant
and animal uptake), physical phenomena (advection, dispersion, dilution, diffusion,
volatilization, sorption/desorption), and chemical reactions (ion exchange, complexation,
abiotic transformation).
Components of natural attenuation are biodegradation, the change in form of compounds
carried out by living creatures such as microorganisms. Under the right conditions,
microorganisms can cause or assist chemical reactions that change the form of the
contaminants so that little or no health risk remains. Natural attenuation occurs at most
polluted sites.
The most important component of natural attenuation is biodegradation, the change in form
of compounds by microbial action. Under optimum conditions, microorganisms can cause or
assist chemical reactions that transform the chemical structure of complex organic and
aromatic compounds. Natural attenuation occur at most polluted sites. However, the right
conditions must exist underground to clean sites properly. Scientists monitor these conditions
to make sure natural attenuation is working. This is called monitored natural attenuation or
(MNA). So, Monitored natural attenuation is a technique used to monitor or test the progress
of natural attenuation processes that can degrade contaminants in soil and groundwater. It
may be used with other remediation processes as a finishing option or as the only remediation
process if the rate of contaminant degradation is fast enough to protect human health and the
environment.

1.2 Toxicological considerations:

Heavy metals are elements having atomic weights between 63.5 and 200.6, and a specific
gravity greater than 5.0. Most of the heavy metals are dangerous to health or to the
environment. Heavy metals in textile industrial wastewater include lead, chromium, iron,
mercury, uranium, selenium, zinc, arsenic, cadmium, silver, gold, and nickel. The main
hazards to human health from heavy metals are associated with disclosure to lead, cadmium,
iron, chromium, mercury and arsenic. These metals have been extensively studied and their
effects on human health regularly reviewed by international bodies such as the WHO. Acute
heavy metal intoxications may damage central nervous function, the cardiovascular and
gastrointestinal (GI) systems, lungs, kidneys, liver, endocrine glands, and bones. Chronic
heavy metal exposure has been implicated in several degenerative diseases of these same
systems and may increase the risk of some cancers.
Non-biodegradable contaminants pose a serious health and environmental hazard and
removal of these wastes cannot be achieved using secondary methods. Hence,
tertiary/advanced wastewater treatment methods such as ion exchange, precipitation,
membrane separation, electrolysis and adsorption can be used to remove these recalcitrant
wastes.
So far, a number of efficient methods have been developed for heavy metal removal.
There are various methods for the removal of heavy metals such as chemical precipitation,
ion exchange, reverse osmosis, electrodialysis, ultrafiltration, nanofiltration, coagulation,
flocculation, floatation, etc. Generally, these processes are efficient in removing the bulk of
metals from solution at high or moderate concentrations. However, chemical processes
produce a large amount of metallic sludge, making metal recovery difficult.
The sludge also needs further disposal. In addition, effluent after such treatment usually has
unacceptably high total dissolved solids. When applied to dilute metal waste or lower
concentrations of metal ions, these processes are either ineffective or not cost-effective and
require high level of expertise; hence they are not applied by many end-users. However, most
of these methods are costly and require high level of expertise; hence they are not applied by
many end-users. For these reasons, adsorption technology and microbial strains has gained a
wider application due to its inherent low cost, simplicity, versatility and robustness. Low cost
adsorbents derived from agricultural by-products and industrial solid wastes could be used to
remove recalcitrant wastes from synthetic wastewater. Conversion of these materials into
adsorbents for wastewater treatment would help to reduce the cost of waste disposal. The

adsorption of toxic waste from industrial wastewater using agricultural waste and industrial
by-products has been massively investigated. The technical feasibility of various low-cost
adsorbents for heavy metal removal from contaminated water has been reviewed. Instead of
using commercial activated carbon, researchers have worked on inexpensive materials, such
as cocnut husk and sawdust, and other adsorbents, which have high adsorption capacity and
are locally available.

CHAPTER 2
LITERATURE REVIEW
2.1 Types of Biological treatment:
2.1.1 Anaerobic biological treatment
Anaerobic biological treatment process is methods that make use of the anaerobic bacteria
putrefy organic matter in anaerobic conditions. This method was first used for sludge
digestion. In recent years it was steadily used in high concentration and low concentration
organic wastewater treatment. In textile industry, there are many types of high concentration
organic wastewater, such as wool washing sewage, textile printing and dyeing wastewater
etc., which the organic matter content of it is as high as 1000 mg/L or more, the anaerobic
wastewater treatment process can achieve good results. The anaerobic aerobic treatment
process is usually adopted in actual project that is using anaerobic treatment to treat high
concentration wastewater, and using aerobic treatment to treat low concentration wastewater.
An anaerobic pre-treatment step could be a cheap alternative compared to aerobic systems as
expensive aeration is omitted and problems with bulking sludge are avoided. Currently, the
hydrolysis acidification process is the main anaerobic treatment process, which can increases
the biodegradability of the sewage to facilitate the following biological treatment process.
The hydrolysis acidification process is the first two stages of the anaerobic treatment.
Through making use of the anaerobic bacteria and facultative bacteria, the macromolecule,
heterocyclic organic matter and other difficult biodegradable organic matter would be
decomposed into small molecular organic matter, thereby enhancing the biodegradability of
the wastewater and destructing the heavy metal and coloured groups of dye molecules
remove from wastewater. More importantly, due to the molecular structure of the organic
matter and coloured material or the chromophore has been changed by the anaerobic bacteria,
its easy to decompose and decolour under the aerobic conditions, which improve the
decolourization effect of the sewage. Operating data shows that the pH value of the effluent
from hydrolysis tank usually decrease 1.5 units. The organic acid which is produced in
hydrolysis can effectively neutralize some of the alkalinity in wastewater, which can make
the pH value of sewage drop to about 8 to provide a good neutral environment for following
aerobic treatment. Currently, the anaerobic digestion process is an essential measure in the
biological treatment of textile dyeing wastewater.

.
.1.2 Aerobic biological treatment
According to the oxygen requirements of the different bacteria, the bacteria can be divided
into aerobic bacteria, anaerobic bacteria and facultative bacteria. Aerobic biological treatment
can purify the water with the help of aerobic bacteria and facultative bacteria in the aerobic
environment. Aerobic biological treatment can be divided into two major categories:
activated sludge process and bio film process.
Activated sludge is a kind of floc which is mainly comprised of many microorganisms, which
has strong decomposition and adsorption of the organics, so it is called activated sludge.
The wastewater can be clarified and purified after the separation of activated sludge.
Activated sludge process is based on the activated sludge whose main structure is the aeration
tank. Presently, oxidation ditch process and sequencing batch reactor activated sludge process
are in use amongst activated sludge process.
The bio film process is a kind of biological treatment that making the numerous
microorganisms to attach to some fixed object surface, while letting the wastewater flow on
its surface to purify it by contact. The main types of the bio film process are biological
contact oxidation, rotating biological contactors and biological fluidized bed.
Various factors like concentration of pollutants, dyestuff concentration, initial pH and
temperature of the effluent affect the decolourisation process. Although the aerobic treatment
is suitable for some dyes, most of them are recalcitrant to biological breakdown or are
nontransformable under aerobic conditions.

2.2 Chemical Method:

The chemical methods applied for colour removal includes

Coagulation or flocculation combined with floatation and filtration chemical

treatment of dye waste water with a coagulating/ flocculating agent is one of the
robust ways to remove colour. The process involves adding agents such as
Aluminium (Al3+), Calcium (Ca2+) or Ferric (Fe3+) ions to the waste water and
inducing flocculation. Sometimes a combination of reagents may be used to enhance
the efficiency of the process. Generally the process is economically feasible with
satisfactory removal of dyes. However, the main drawback of the process is that the
final product is a concentrated sludge whose disposition is difficult and secondly the
removal is pH dependent. This process is not suitable for the basic dyes.

Conventional oxidation methods it is a method by which waste water is treated by

using oxidising reagents. Generally two forms viz. Chemical oxidation and UV
assisted oxidation using chlorine, KMnO4, Fentons reagent are used. This is the most
commonly used method for dye removal, because of low quantities of reagents and
short reaction time. This process degrades the dyes partially or completely to low
molecular weight compounds. pH and catalysts play an important role in oxidation.
Chlorine is a good oxidising agent and used in the form of sodium or calcium
hypochlorite, water soluble dyes are readily decolourised but water insoluble dyes are
resistant to this process.

Fentons reagent, a solution of hydrogen peroxide and an iron catalyst is used to and
However this method is applicable within a narrow pH range of<3.5, involves sludge
generation and reaction time is long.

Ozonation it has been suggested that this can be used to remove completely the
colour and COD of waste water to an extent which is sufficient to allow water reuse.

Advanced Oxidation Processes (AOPs) these are the processes involving

simultaneous use of more than one oxidation processes, since sometimes a single
oxidation system is not sufficient for the total decomposition of dyes. This involves
the accelerated production of hydroxyl free radicals and includes techniques as

Fentons reagent oxidation, UV photolysis and sonolysis. They are capable of

degrading dyes at ambient temperature and pressure and may also have an advantage
over biological treatment for waste streams containing toxic or inhibitory
contaminants.

Photolysis involves the formation of hydroxyl radicals, which is formed when light
energy from a light source excites an electron from the valence band of the catalyst to
the conduction band. These hydroxyl radicals have high oxidising potential and
therefore can attack most organic structures.

Sonolysis is the use of ultrasonic waves for the decolourisation and degradation of
dyes. The mechanism proposed for the sonochemical processes is usually based on
the formation of short-lived radical species generated in violent cavitation events [17].

1.3 Need of Study:

The current study focuses on comparative analysis of 2 micro-organisms (bacterium), gram
positive Bacillus megaterium and gram negative Pseudomonas diminuta in the degradation of
azo dye and heavy metal like iron and lead by submerged culture fermentation. The use of
conventional and non-conventional microbes for waste water treatment has been debated
through numerous researches. The analysis of bond peaks and its appearance and
disappearance in untreated and treated samples can be utilised for detection of toxic
derivatives of incomplete or complete degradation of their structures. These carcinogenic and
mutagenic compounds are potential environmental pollutants entering the aquatic ecosystem
via effluent discharges. Thus the study aims to determine natural attenuation parameters of
bio-degradation of microbes in submerged culture fermentation (Flask culture). Also some of
the natural materials locally available in certain regions can be employed as low-cost
adsorbents due to their metal binding capacity. The film of natural adsorbents like coconut
husk and sawdust makes them particularly suitable for removing undesirable heavy metal
ions from industrial effluent waters. The adsorption
behavior of natural adsorbents was studied and the results shows that natural adsorbents can
also be used effectively for the removal of heavy metal from wastewater.
Besides, the adsorption behavior of formulated coconut husk and sawdust mixture for heavy
metals removal efficiency was studied as a substitute for activated carbon for wastewater
treatment.
The study therefore characterizes the physicochemical parameters involved in the process of
biodegradation and determines the optimum parameters for complete degradation or
incomplete degradation. The objective of this work is to study the biodegradation through
gram positive and gram negative bacteria and some low-cost adsorbents such as coconut
husk and sawdust with respect to Pb and Fe. The batch method was employed: parameters
such as time, and initial metal concentration, were studied.

CHAPTER 3
METHODOLOGY AND DATA COLLECTION
4. Material required
Apparatus used:
Test tubes, Erlenmeyers flasks, Beakers, measuring cylinder,
Instrumentation:
Autoclave, Incubator and shaker, UV/VIS Spectrophotometer, pH meter,Oven, micropipette,
Laminar airflow, Centrifuge, Microwave, Refrigirator, AAS instrument (PERKIN ELMER A.
Analyst 200; Germany consisting of a hollow cathode lamp, slit width of 0.7 nm and an air
acetylene flame was used for this work. The samples were analyzed for two heavy metals
namely Pb & Fe.
Chemicals and media:
Chemical used: All chemicals and reagents of analytical grade were used for analysis.
Concentrated HNO3 was used for the digestion of the samples while corresponding metal
salts Pb and Fe were used as standards.
Dye: Mythyl red
Sampling
Sampling was done as per standard way to ensure representation of industrial effluent.
Samples were collected from various industrial units situated in the industrial area. Effluent
samples were mostly collected from drains coming out of the industrial site. Plastic cans of 2
litre capacity were used for sampling of effluent. Samples were collected from the effluent
release point or a little away from it (this was generally the drains coming out from the
industrial unit). Also one plant sample and soil sample were collected to determine the
presence of heavy metal. Samples were collected and preserved after nitric acid treatment.
Analysis of samples was done by flame atomic absorption spectroscopy at IIT BHU,
Varanasi.

Sample preparation
To ensure removal of organic impurities and prevent interference during analysis, each of 50
ml volume sample was digested using 10 ml conc. HNO3 in a 250-ml conical flask placed on
a fume cupboard. The samples were covered properly with aluminium foil to avoid spillage
and heated on a hot plate until the solution reduced to 10 ml. This was allowed to cool and
made up to mark with distilled water before filtering into a 50-ml standard flask, labelled and
ready for analysis. The blank constituted 5% HNO3.
Standard preparation
Standard solutions were taken for two metals Pb and Fe. Solution of 1000 ppm strength
served as the stock solution, subsequently lower concentrations of 0.25 ppm, 0.5 ppm, 1 ppm,
2 ppm, 5ppm, 10ppm, 25ppm were prepared from the stock by serial dilution.

Concentration(ppm)

Absorbance
Lead (Pb)

Iron (Fe)

0.25

0.009

0.003

0.5

0.027

0.011

0.025

0.029

0.06

0.080

0.152

0.179

10

0.215

0.308

25

0.225

0.250

Table 1

Preparation of stock culture of Bacillus megaterium:

1 ml of inoculums from the Seed culture was grown in the nutrient broth and incubated at 120
rpm, 30 0C, and pH 6.5 for 48 hours. It was then stored at 40C for further use in
decolourisation studies of Azo dye and degradation of heavy metal Pb and Fe. The first
decolourisation studies were conducted with Malachite green.

Preparation of Stock culture of Pseudomonas diminuta:

1ml of inoculums from seed culture was grown in nutrient broth and incubated at 150 rpm, 30
0C, and pH 6.5 for 48 hours. It was then stored at 40C for further use for degradation of azodyes and heavy metal Pb and Fe.

Fig
Procedure for determination of Decolourisation studies in Azo dye
The experimental biodegradation of Azo dye by Pseudomonas diminuta NCIM 2865 and
Bacillus megaterium NCIM 5374 was carried out to study the decolourisation potential of the
micro-organism. The absorbance values were noted for concentration of 0.005 g/L at 450 nm.
3% inoculums are inoculated into 120 ml of prepared Azo dye solution in a laminar hood
under aseptic conditions. 3.6 ml of P.diminuta and B.megaterium was inoculated in azo dye
solution and further analysis of the sample before degradation was done by UV-Visible
Spectrophotometer (UV-1800) Shimadzu, and then incubated at 280C, under mild shaking
conditions of 120 rpm and 400ul of each sample was withdrawn at intervals for absorbance
readings. The absorbance readings of the supernatant were observed after centrifugation
carried out of the degraded samples at 10,000 rpm for 10 minutes at 100C in Heraeus Biofuge
Stratos Centrifuge (Thermo Fischer Scientific).

Fig
UV-VIS Spectroscopy: Spectrum Scanning for Determination of Absorbance in dye
Spectrum Scan of azo dye solution containing P.diminuta and B.megaterium was done at 450
nm. In the UV-1800 Shimadzu spectrophotometer, the spectrum scanning of the 0.005g/L,
diluted by taking 2.5 ml of dye solution and 2.5ml of distilled water was done in
spectrophotometric cuvettes. Thus, the peak detection values for the scan in the range: 220700 nm showed the highest peak at 450 nm and was selected as the wavelength for
absorbance based decolourization studies of azo dye.

Time (hr)

Table

0
3
6
9
12
15
18
24

Absorbance
Bacillus megaterium
Pseudomonas diminuta
0.707
0.296
0.276
0.248
0.225
0.211
0.16
0.125

0.707
0.464
0.445
0.387
0.371
0.36
0.334
0.318

Procedure for determination of Percentage reduction in 1 ppm Solution of Pb and Fe

2% inoculums are inoculated into 150 ml of prepared 1 ppm solutions of Pb and Fe into
250ml Erlenmeyer flask containing 100ml nutrient broth (gL-1Peptone- 5, Beef extract-1,
Yeast extract-2, Nacl-5,pH-6.5) in a laminar hood under aseptic conditions. 3ml of
P.diminuta and B.megaterium was inoculated in 1 ppm solution of Pb and Fe and further
analysis of the sample before degradation was done by AAS instrument (PERKIN ELMER A.
Analyst 200) and then incubated at 300C, under mild shaking conditions of 120 rpm and 50
ml of each sample was withdrawn at intervals 6 hr, 12 hr and 24 hr for absorbance readings.
The absorbance readings of the supernatant were observed after centrifugation carried out of
the degraded samples at 10,000 rpm for 10 minutes at 100C in Heraeus Biofuge Stratos
Centrifuge (Thermo Fischer Scientific).

Fig
Atomic Absorption Spectroscopy: Spectrum Scanning for Determination of Absorbance
in heavy metal
Spectrum Scan of 1 ppm solution of Pb and Fe containing P.diminuta and B.megaterium was
done. In the AAS instrument (PERKIN ELMER A. Analyst 200; Germany consisting of a
hollow cathode lamp, slit width of 0.7 nm and an air acetylene flame), the spectrum scanning

of the 1 ppm solution of Pb and Fe was done to analyse the degradation of heavy metals by
gram-positive and gram-negative bacteria.

Lead (Pb)

Iron (Fe)

0.115

0.266

Table

Time (hr)

Absorbance
Lead (Pb)
B.megaterium

Iron (Fe)

P.diminuta

B.megaterium

P.diminuta

0.095

0.095

0.097

0.087

12

0.086

0.075

0.078

0.072

24

0.40

0.012

0.068

0.057

Table
Preparation of filter made of natural adsorbents
Wash saw dust and coconut husk with d/w 4-5 times and dry it at 100 C to 110 C.
Maintain their pH from 4 to 8 by digital pH meter. Weigh 3 gm of coconut husk and saw dust
and pack in syringe of 50 ml capacity to make a thin film which will work as a filter. Now
connect the syringes to conical flask. Further solutions will be passed from these filters to
adsorb the heavy metal present in the sample.

Time (hr)

Absorbance
Lead (Pb)
B.megaterium

P.diminuta

Iron (Fe)
B.megaterium

P.diminuta

0.068

0.044

0.082

0.080

12

0.046

0.022

0.072

0.060

24

0.02

0.008

0.058

0.052

Table

Fig

AAS Analysis of effluent water for

determination of Heavy metal

Preparation of seed culture of

B.megaterium and P.diminuta

Cultivation of 1 ppm Pb and Fe in

acclimatized medium

Inoculation of 2% seed culture in 1

ppm prepared nutrient broth

Characterization of process, reading at

6 hr, 12 hr, 24 hr

AAS Analysis of 50ml sample

Passing the remaining 100 ml solution

through filter of natural adsorbents

AAS Analysis
Fig. Overview of degradation process and analysis of toxic heavy metal by bacterial
cultures.

CHAPTER 4
RESULT AND DISCUSSION
Detection of heavy metal:
Result from Table no.1 is:

Fig
The standard curve of Lead (Pb) was plotted graphically in order to evaluate the
concentration of known absorbance of effluent sample, soil and plant sample. After
calculating the concentration in samples it has been found that there is no content of Lead is
present in soil and plant sample, whereas less than1 ppm concentration of Pb was present
in textile effluent water.

Result from Table no.1 is:

Fig.
The standard curve of Iron (Fe) was plotted graphically in order to evaluate the concentration
of known absorbance of effluent sample, soil and plant sample. After calculating the
concentration in samples it has been found that there is no content of Lead is present in soil
and plant sample, whereas 1 ppm concentration of Fe was present in textile effluent water.

Decolourization study in Azo dye:

Fig.
The results obtained for gram positive and gram negative bacteria indicating the higher
capacity decolourization of azo dye.
According to the graphical representation after Highest Reduction has been observed at 24
hr for both the bacterial strains.

By using above formulae Percentage Reduction can be calculated.

Maximum Percentage reduction at 24 hr for

B.megaterium = 82.319661%

P.diminuta = 55.021216%

Study of degradation of heavy metal through biotic and abiotic components:

Fig.
The above graph shows the degradation of Pb after treatment with gram positive and gram
negative bacteria and further after filteration through natural adsorbents higher amount of
reduction in Pb concentration has been found. Maximum Percentage reduction was found at
24 hr .
Maximum Percentage reduction after treatment with microbial strain:

B.megaterium = 65.217391%

P.diminuta =89.565217%

Maximum Percentage reduction after filtration throught the film of natural adsorbents:

B.megaterium =82.608696%

P.diminuta =93.043478%

Fig.
The above graph shows the degradation of Fe after treatment with gram positive and gram
negative bacteria and further after filteration through natural adsorbents higher amount of
reduction in Fe concentration has been found. Maximum Percentage reduction was found at
24 hr.
Maximum Percentage reduction after treatment with microbial strain:

B.megaterium = 74.43609%

P.diminuta =78.571429%

Maximum Percentage reduction after filtration throught the film of natural adsorbents:

B.megaterium =78.195489%

P.diminuta =80.451128%

CONCLUSIONS AND FUTURE DIRECTIONS

5.1 Summary of Results
The results obtained for azo dye provided comparative analysis of biodegradation of gram
positive and gram negative bacteria, which showed Bacillus megaterium to have a maximum
decolorisation of 82% within a time of 24 hours whereas Pseudomonas diminuta was able to
achieve a lower decolorisation of 55% under optimum conditions. The UV-Vis
spectrophotometric analysis of azo dye mythyle red showed the maximum absorbance of the
dye at 450 nm. Thus decolorisation potential as a function of important physical parameters
was determined.
The results obtained for heavy metal Lead (Pb) and Iron (Fe) also provided comparative
analysis of biodegradation of gram positive and gram negative bacteria, which showed
Bacillus megaterium to have a maximum Percentage reduction of 78% for Fe within a time of
24 hours whereas Pseudomonas diminuta was able to achieve a maximum Percentage
reduction of 89% for Pb after treatment with bacterial strain only under optimum conditions.
Further after treatment with natural adsorbents maximum percentage reduction of 82% for
Bacillus megaterium for Pb whereas Pseudomonas diminuta was able to achieve a maximum
Percentage reduction of 93% for Pb. The AAS analysis of heavy metals showed the
maximum absorbance. Thus percentage reduction as a function of important physical
parameters was determined.
Many textile industries specially treat dye with water itself but then also the toxic properties
cannot be removed as it required equipments. Survey defines that most of the small scale textile
industries do not have any acknowledgement regarding harmful effects of dye to them as well as
nature. These small scale industries do not effort to buy treatment units as they are very cost
effective. Large scale industries avoid using treatment units/plants because of high cost of
maintenance and electricity. Now the treatment has been done with using microbial strain s and
natural adsorbents - Saw dust & Coconut husk.

Discussion and Future Prospects:

Submerged culture fermentation systems are used for onsite monitoring of waste water
effluents, by microbes. However in-vitro studies involving the characterization of optimum
parameters is essential efficient treatment of toxic unchanged chemical aromatic derivatives
formed from dye degradation. The use of sequential reactors involving better monitoring of
parameters as well as oxygen supply can be crucial in enhancing decolorisation levels. The
degradation is governed by aerobic as well as anaerobic mechanisms and the type of microorganism utiilised for the degradation. The implementation of microbes in bioremediation is
therefore governed by a multitude of parameters, an effective analysis of which is perennial
in the understanding of bio-degradation kinetics.
The future prospects of research in the field of biodegradation by microbial cultures and the
studies involving examination of the extracellular enzymes present in potential dye degrading
microbes. The kinetic studies of enzyme degradation can be a better indicator of the specific
decolorisation efficiencies. The ultimate future work and various other current researches are
targeted towards enzyme purifications and extraction for enzymatic degradation by the
microbes. The kinetics of enzyme degradation are also essential in elucidating the pathways
underlying degradation of complex aromatic compounds and their potential usage in
determination of rate limiting parameters of degradation.
With the advancement of genetic engineering, use of genetically engineered microbes for
targeted treatment of the wastes is proving to an essential research platform. However the use
of chemical processes of bleaching, redox reactions and photocatalytic reactions is still
prevalent due to upscaling restrictions on use of biological systems for handling large effluent
volumes. Thus the optimization of physical parameters and analysis of chemical structures is
essential in understanding how the upscaling restrictions can be overcome.

REFRENCES
1. Hildenbrand, S., Schmahl, F.W., Wodarz, R., Kimmel, R. and Dartsch, P.C. (1999); Azo dyes
and carcinogenic aromatic amines in cell cultures. Int. Arch. Occup. Environ. Health, 72, 5256.
2. Supaka N, Juntongjin K, Damronglerd S, Delia ML, Strehaiano N. Microbial decolorization of
reactive azodye in sequential anaerobic-aerobic system. Chemical Engineering Journal (2004);
99(2):16- 76.
3. Weber, E. J., Adams, R. L., (1995); Chemical and sediment mediated reduction of the azo dye
Disperse Blue 79. Environmental Science & Technology, 29, 1163-1170.
4. Wang, C., Yediler, A., Linert, D., Wang, Z., Kettrup, A., (2002); Toxicity evaluation of
reactive dye stuff, auxiliaries and selected effluents in textile finishing industry to luminescent
bacteria vibrio fisheri. Chemosphere, 46, 339-344.
5. Sandhya, S., Padmavathy, S., Swaminathan, K., Subrahmanyam, Y.V., Kaul, S.N., (2005);
Microaerophilic-aerobic sequential batch reactor for treatment of azo dyes containing simulated
wastewater. Process Biochem. 40 (2), 885890.
6. Deepali, (2011); Bioremediation of Chromium (VI) from textile industrys effluent and
contaminated soil using Pseudomonas putida. Iranica J. Energy. Environ. 2 (1), 2431.
7. Anliker, R. (1979); Proceedings, ecotoxicological assessment of dyes with particular reference
to ETAD's activities. JSDC 9, 317}326.
8. Rintamaki-Kinnunen P., Rahkonen M., Mykra H., Valtonen E.T. (2005); Treatment of
ichthyophthiriasis after malachite green. II. Earth ponds at salmonid farms. Diseases of Aquatic
Organisms, 66, 1520.
9. Saglam N., Ispir U., Yonar M.E. (2003); The effect of a therapeutic bath in malachite green on
some haematological parameters of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792).
Fresenius Environmental Bulletin, 12, 12071210.
10. Army US. Interim army policy on natural attenuation for environmental restoration:
Washington, DC, Department of the Army, Assistant Chief of Staff for Installation Management
(1995); DAIM-ED-R (200-1c).