The document summarizes a study on the biodegradation of reactive blue dye using freshwater microalgae. The objectives were to investigate the effects of dye concentration, temperature, pH, and incubation time on biodegradation. Maximum decolorization of 82.6% was achieved at 50mg/l dye concentration, 30°C temperature, and pH 8 over 20 days. Green microalgae like Chlorella and Scenedesmus were effective at biodegrading the dye under optimal conditions. FTIR analysis indicated the dye's chromophore structure was destroyed into smaller molecules. The study demonstrates that microalgae have potential for cost-effective treatment of dye-contaminated wastewater.

1. The 5th
International Conference on the Advancement of
Science and Technology (ICAST-2017)
Biodegradations of Reactive Blue-Dye Using Fresh
Water Microalgae
By Mr. Tadele A and Dr. Abraham M

2. Faculty of Chemical and Food Engineering
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Introduction
Dye contaminated wastewater is becoming serious problem new a days.
Presently a wide range of physical-chemical treatment methods is
available to decolorize dye-contaminated effluents.
But alternative processes based on biotechnological principles are
attracting increasing interest since they can avoid consumption of high
quantities of additional chemicals, energy and due to environmentally
friendly
So, the major advantage of biological decolourization is the partial or
complete mineralization with the actions of microorganism.

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Why microalgae?

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Objectives
The aims of this research is to investigate the
bioremediations of reactive blue dye using fresh water
microalgae. Under this study:
 Effect of dye concentration,
effect of temperature ,
effect PH,
incubation time for the biodegradation ability of the species
were determined.

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Motivation of the study
Problems concerning wastewater treatment stem from the
rise in Industrialization and the practice of discharging
untreated and poorly treated wastewater.
The current treatment technologies lack sustainability
Diverse metabolic pathways, Versatility of microalgae
species.
The ability to adapt to a variety of environmental conditions.
Also it is advantageous in terms of pathogenic to humans
than bacteria and fungi

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Advantages of microalgae remediation over
conventional wastewater treatment.
• Cost effective
• Low energy requirement
• Reductions in sludge formation
• GHG emission reduction
• Production of useful algal biomass

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Methodology
Experimental set up
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Photo bioreactor inside the
incubation chamber
Schematic diagram
representations

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Principles of azo dye Biodegradation with
microalgae (Jinqi et al., 1992)
1

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1ICAST/ 19-20 May 2017 8
Dye Decolorizing study
The primary step for this study is the determination of
maximum wave length of the dye(λ). Thus dye solution with a
range of wavelength were scanned (200-800nm) .
Then , 50 mg/L to 500 mg/L azo dye solution in Erlenmeyer
flasks with 10% Pre-grown cultures as inoculums were
prepared.
Treated dye was drawn with in five day interval and
centrifuged at 3,500 rpm for 10 minutes.

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Cont…
1
The supernatants were done in triplicate at maximum
absorbance (λmax).
The color removal efficiency achieved was determined
by monitoring the decrease in absorbance at the λ max of
the dye.
Percentage decolorization was calculated as shown by
(APHA, 1995).

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Effect of dye concentration
The substrate concentration have an effect on the
degradation ability of microalgae.
so, determining a series of concentration is important to
ensure the optimum decolorization capacity of the species.
Concentration of dye solution (50, 100,200, 300,400 ,500
mg/L) were used.
This is based on the assumptions of effluent concentration
( mostly ranged from 30 mg/l-450 mg/l).

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Effect of temperature
The effect of temperature were investigated by incubating the reaction
volumes at 20◦C, 30◦C, 40 ◦C ,50 ◦C and 60 ◦C.
Because the algal species can survive a range of temperatures.
Effect of PH
Medium with dye were prepared at pH 4, 5, 6, 7, 8 and 10, where the
dye concentration and temperature kept constant.
Different algae Species can survival on the water body in acidity and
basicity media.

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FTIR Analysis
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Control dye and treated samples were mixed with KBr (0.02 g) with
sample at a final weight of 0.4 g).
The samples were then ground, desorbed and pressed to obtain IR
transparent pellets.
The absorbance of FT-IR spectra of the samples were examined using
an FT-IR Spectrum Perkin–Elmer spectrometer.
The spectra were collected within a scanning range of 450-4000 cm-1
.
Calibration for background signal were scanned with a control sample of
pure KBr.

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Microalgae Identification
1ICAST/ 19-20 May 2017 13
The specific characteristics found
for algal strains under light
microscopy were assessed and
compared with Wehr and Sheath
(2002) to find the respective algal
genera.
The Predominant consortium of
microalgae species :Scenedesmus
sp., Chlorella sp., Synedra sp.,
Achnanthidium sp. were identified.
chlorella sp. dominantly grown but Synedra sp. and Achnanthidium sp.
was found in small amount.
This revealed that, mostly the fresh water ,where the sample taken,
microalgae species are chlorophyta phylum (green microalgae, specially
chlorella ).
Result and discussion

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Effect of dye concentration
1ICAST/ 19-20 May 2017 14
Zeroday50mg/l
20th
day50mg/lAfter
centrifugation
The graph depicts percent decolorization increases with contact time and
decreases with dye concentration. This is due to the toxicity of the dye to the
species and/or inadequate biomass concentration for the uptake of higher
concentrations of dye, resulting in the inhibition of enzyme systems gradually.

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1ICAST/ 19-20 May 2017 15
Sample137.Sample
Sample138.Sample
Name Description
190 750200 300 400 500 600 700
3.7
-0.1
-0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
nm
A
50mg/l UV-Vis Spectra of before and after degraded
dye: a) Control b) Dye with algae
In addition to biodegradation, a biosorption might also play an important
role in the decolorizations of dye. For dead/inactive cells, the mechanism is
biosorption. The chemical functional groups (carboxyl, phosphate, amine
and hydroxyl) present on the cell wall plays vital role in biosorption (van
der Wal et al, 1997).
It can be assumed that the aromatic ring structure was destructed
or the color intensity of the dye was decreased by the action of
microalgal species.

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Effect of Temperature
1ICAST/ 19-20 May 2017 16
Among different temperature tested,
300
C was found to be an optimum on
dye decolorization (78% for 20 days).
It is noted that the percentage
decolorization of dye increased with an
increase in temperature from 200
C to 30
0
C but decreased with further increase in
temperature up to 60 0
C.
This can be attributed due to the
decline in microalgal activity that led to
the deactivation of enzyme and
eventually the loss of cell viability.

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Effect of PH
1ICAST/ 19-20 May 2017 17
 82.6% of dye decolorization
was found at pH 8.
 A rapid increase in decolorization
was observed as the pH increased
from 4 to 5 and 6 to 8 for the 15
and 20 day.
However, a relative decrease in
decolorization was found when pH
increases from 9 to 10.
Neutral range of pH would be
more favorable for decolorization of
the azo dyes
Most likely pH affects the
enzymatic activity involved in
decolorization of dye in addition to
cellular growth of algae.

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Global Effluent standard Guidelines (DOE)
Most o f the time the color intensity can be determined in terms
of BOD, TDS values. Because effluent color have direct response
for BOD, TDS.
DOE: Department of Environment

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FTIR analysis Before dye degradation
peaks were detected at 2078
cm-1
and the conjugated
aromatic rings or amide
functional groups at 1648 cm-
1
.
Dye spectrum before
biodegradation showed two
prominent peaks and shows
one peak after degradation and
also existing peaks showed
peaks shifts.
 It was found that the
presence of new peaks at 3477
cm-1 attributes to –N-H-
stretch vibration.

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Cont…
1ICAST/ 19-20 May 2017 20
The results revealed that the large conjugated chromophore
structure of dye was destroyed yielding smaller organic molecule.
The obtained variations in IR spectra of the dyes could be
attributed to the cleavage of azo linkage of the dye and the
subsequent formation of aromatic amines.
Additionally, the differences in spectral intensity and the
occurrence of stretched vibration of algae treated samples also
manifest possible biosorption besides the algal degradation
activities.

22. Faculty of Chemical and Food Engineering
Conclusion
1ICAST/ 19-20 May 2017 21
It can be deduced that, Microalgae have bioremedation
ability of dye contaminated waste.
Mostly the green microalgae, of common fresh water algae,
can be used to treat the azo dye in different concentrations.
 In this study maximum of 82.6% of dye decolorization was
obtained for time course studies within 20 days at,[50mg/l] dye
concentration, 300
C and PH 8 .

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Cont…
1ICAST/ 19-20 May 2017 22
Biodegradation activity are significantly suppressed at high
temperature, the basic and acidic PH range and at high dye
concentration.
This is due to the loss of cell viability or deactivation of the enzymes
responsible for Biodegradation.
It can be conclude that microalgae undoubtedly have the potential to
rapidly, efficiently and effectively biodegrade dyes having moderate
concentrations at environmental temperature and neutral PH range.
Moreover, the biosorption process could be adopted as a cost effective and
efficient approach for decolorization of effluents and it may be an alternative to
more costly materials.

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Recommendation and Future work
After degradation, the algal residue can be used for bioenergy.
Utilize the microalgae for wastewater treatment is environmentally
friendly.
The nature of the metabolic intermediates formed should be done .
Further study should be conducted for the maximum percent
decolorization with different external nitrogen and phosphorus
source.
This information will increase knowledge concerning the
mechanisms employed by microalgae for biodegrading dyes.

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Acknowledgement
Faculty of Chemical and Food Engineering
Dr. Abraham Mebrat
Dr. Bizuayehu S from biology Department
Prof. Mulugeta Kibret from biology Department
Dr. Atikilt Abebe Chemistry department
Friends

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Thank you