1. BTE 450: Biotech Project
AHMED ABDULLAH FAHIM
Student ID:11336004
November 2015

2. Isolation of Reactive Red 3BX and Yellow
4GL dye degrading bacteria from the
textile sludge.

3. WHAT ARE AZO DYES?
AZO dyes are a large class of very effective synthetic dyes
used for coloring a variety of consumer goods
(examples: foods, cosmetics, carpets, clothes,
leather and textiles).

4. EXAMPLE OF AZO DYES
Blue Br
Orange 2R
Reactive Red
Reactive violet

5. BOND IN AZO COMPOUNDS

6. Effects: Textile dyes on environment

7. Textile Dye: Pollution

8. Treatment processes
Treatments
1
• Physico-chemical treatments
Treatments
2
• Adsorption with solid adsorbents
Treatments
3
• Biological treatments

9. Are biological treatments efficient over
other treatments?
• Less time consuming.
• Cost effective.
• Less chance of creating secondary reactive products.
• Do not need any specialized equipment.
• Environment friendly because it can lead to
complete mineralization of organic pollutants.

10. THIS PROJECT SCENARIOS
• In this project two commercially available
textile dyes were used to testify bacteria
whether they have the capability to degrade
these two dyes or not.
• Name of the two dyes used are
1. Yellow 4GL
2. Reactive Red 3BX

11. Methodology

12. Methodology
SAMPLING
• Soil collection :Industrial area of Bhalukha.
• Brought and processed in BRACU MNS laboratory.
TESTING
• Soil testing (Using yellow G2 dye): Checking the presence of dye degrading bacteria.
• After two weeks of incubation dye and soil containing media was diluted up to 10-5
dilution rate.
ANALYZING
• Continuous absorbance data : observe the de-colorization of the dyes.
• Presence confirmed: soil inoculated into the Yellow 4GL and Reactive Red 3BX dye
mixed with the SM broth media.

13. Processing
SAMPLING
• Spread plates made using diluted media : Observe the bacterial
colonies.
• 16 colonies : isolated by their morphological differences.
TESTING
• Single colonies : inoculated into two dyes to find out which one
has the capability to degrade dye.
• Finally two bacteria were identified.
ANALYZING
• To identify these colonies biochemical tests were done .
• 6 colonies out of 16 colonies found with better dye degrading
capacity, among which 3 colonies could degrade both dyes.

14. Processing

15. Figure: Bacterial Colonies

16. Biochemical tests :Applied
Catalase
Oxidase
Casein hydrolysis
Cimmon citrate
Nitrate reduction test
Starch hydrolysis test
MIU test ( motility indole
urease)
Methyl red test
In dole test
Vogasproskuras test.
TSI test
Aerobic growth test
Growth in 10% NaCl
Growth in 15% NaCl
Fructose test
Galactose test
Glucose test
Lactose test
Maltose test
Manitol test
Sucrose test
Treshalos test

17. Results
and
observation

18. Results and observation
Table: Chosen 6 colonies absorbance result in
Reactive Red dye is given bellow in 1%
concentration.
Colony name Wavelength (534nm)
control 0.529
Colony C 0.380
Colony D 0.237
Colony I 0.399
Colony K 0.391
Colony L 0.090
Colony M 0.143

19. Results and observation cont.
Colony name Absorbance (485 nm)
Yellow 4G control 0.065
Colony C 0.346
Colony D 0.360
Colony I 0.426
Table: And there 3colonies were found which have the
capability to degrade Yellow 4G dye as well as Re Active dye.

20. X axis is representing the absorbance values
Graph :De-colorization by 6 colonies in Reactive
Red dye
Control
Colony C
Colony D
Colony I
Colony K
Colony L
0.529
0.38
0.237
0.399
0.391
0.09
absorbed wavelength
Results and observation cont.

21. Figure: Degradation of Reactive Red dye

22. Figure: Degradation of Yellow 4GL dye

23. Results and observation cont.
Colony name shape Type
Colony C Small rod Gram positive
Colony D cocci Gram positive
Colony I cocci Gram positive
Colony K cocci Gram positive
Colony L cocci Gram positive
Colony M cocci Gram positive
Table: Grams staining

24. Results and observation cont.
Figure: Grams staining of colony C

25. Results and observation cont.
Test name Result
Gram staining Positive
Catalase Positive
Oxidase Negative
Nitrate reduction Positive
Citrate utilization Negative
Indole Positive
VogesProskauer test Negative
Casein hydrolysis Positive
Table: Biochemical test
results for Colony C

26. Results and observation cont.
Growth on 7% NaCl media Negative
Motility Positive
Growth on usual media Positive
Starch hydrolysis Negative
Acid production from Glucose Positive
Acid production from Fructose Positive
Acid production from Maltose Positive
Acid production from Mannitol Positive
Acid production from Trehalose Positive
Table: Biochemical test
results for Colony C

27. • By the help of these biochemical results, microscopic observation,
characteristics of the organism and software this colony C was found
as the Brevibacillus laterosporus

28. Results and observation cont.
Table: Cocci shape bacteria’s biochemical results
Test names Colony K Colony M Colony L Colony D
Aerobic growth positive positive positive positive
Growth on 10% NaCl
agar
Negative Negative Negative Negative
Growth on 15% NaCl
agar
Negative Negative Negative Negative
Nitrate reduction Negative Negative Negative
Urease Negative Negative Negative Negative
Catalase positive positive positive positive
Oxidase Negative Negative Negative Negative

29. Results and observation cont.
Sugar production
from
Colony K Colony M Colony L Colony D
Glucose Negative Negative positive Negative
Fructose positive Negative positive positive
Galactose positive positive positive positive
Maltose positive positive positive Negative
Mannitol positive Negative positive Positive
Lactose positive positive positive Positive
Sucrose Negative positive positive Positive
Thehalose positive positive Negative Positive
Table: Cocci shape bacteria’s biochemical results

30. • By the help of these biochemical results, microscopic observation,
characteristics of the organism and software this colony L was found
as Staphylococcus nepalensis

31. Results and observation cont.
Figure: Gram staining photo for Colony L which is
as Staphylococcus nepalensis

32. Again after identification of two bacteria
Brevibacillus laterosporus and Staphylococcus
nepalensis, again these two were tested into
higher concentration of Yellow 4GL and Reactive
Red dye. And the concentration was 5%.
Results and observation cont.

33. Results and observation cont.
Reactive Red Distilled water Control (534 nm) Colony C (assumed
Brevibacillus
laterosporus)
(534 nm)
0 hour later 0 3.537 3.541
24 hours later 0 3.537 0.598
48 hours later 0 3.541 0.388
72 hours later 0 3.539 0.326
Table: Degradation by Brevibacillus laterosporus in
Reactive Red dye

34. Results and observation cont.
Y axis is representing absorbance values and X axis is representing time period
Chart presenting the decolorization of Reactive Red dye by
Brevibacillus laterosporus
colony C
control0.2
0.7
1.2
1.7
2.2
2.7
3.2
3.7
Day 0
24 hours later
48 hours later
72 hours later
colony C
control

35. Results and observation cont.
0
0.5
1
1.5
2
2.5
3
3.5
4
0 hour later 24 hours later 48 hours later 72 hours later
Distilled water Control Colony C (assumed Brevibacillus laterosporus) Expon. (Colony C (assumed Brevibacillus laterosporus))
Y axis is representing absorbance values and X axis is representing time period
Chart presenting the decolorization of Reactive Red dye
by Brevibacillus laterosporus

36. Results and observation cont.
Decolorization percentage of Brevibacillus laterosporus :
Initial OD = 3.541nm
Final OD = 0.326 nm
Dye concentration = 5%
Decolorization% = { (Initial OD – Final OD) / Initial OD }*100
= { ( 3.541 – 0.326 ) / 3.541 } *100
= 90.8 %

37. Results and observation cont.
Figure: Decolorization of dye

38. Results and observation cont.
Staphylococcus nepalensis was unable to break the
5% concentration of Reactive Red and yellow 4G
dye. But it showed an excellent result in 1%
concentrated Reactive Red dye

39. Results and observation cont.
Decolorization percentage of assumed Staphylococcus nepalensis:
• Initial OD = 0.592 nm
• Final OD = 0.090 nm
• Dye concentration = 1%
Decolorization% = {(Initial OD – Final OD) / Initial OD} * 100
= {(0.592 – 0.090) / 0.592} *100
= 85% approximately.

40. Results and observation cont.
0.05
0.15
0.25
0.35
0.45
0.55
control Colony L (assumed
Staphylococcus
nepalensis)
De-colorization of Colony L
De-colorization of
Colony L
Here Y axis is representing Absorbance values
Staphylococcus nepalensis’s de-
colorization graph in 1% concentrated
Reactive Red dye

41. Discussion

42. Discussion
• Brevibacillus laterosporus and Staphylococcus nepalensis both have
dye degrading capacity. Between them Brevibacillus laterosporus is
more efficient. It can degrade higher concentration of dye. It
showed better result in Reactive Red 3BX dye. It takes only one
week to de-color the Reactive Red 3BX dye. And also showed
significant result in the de-colorization of Yellow 4GL dye.
• But Staphylococcus nepalensis is little inefficient to decolor the
higher concentrations of dyes in short time

43. Future aspects in the World as well as in
Bangladesh
• As other existing treatment are more expensive than this biological
process so industries will be more interested to use it. And in
Bangladesh Industries owners are not much willing to emphasis on
the treatments of pollutants, so they will take this opportunity very
eagerly.
• This can lead to a great business for the biotechnologist and
microbiologist if they start to isolate those dye degrading responsible
genes from organisms and create genetically modified organisms to
degrade dyes.
• Apart from all these the fast and foremost duty is to save the planet
for the future generations.

44. Acknowledgement
• Special thanks goes to my motivation, Professor Dr. A. A. Ziauddin Ahmad and Professor
Naiyyum Choudhury.
• I am likewise appreciative to my Supervisor, Romana Siddique, and all the senior teachers
of the Department.particularly to share her significant time and information about this
thesis.
Department of Mathematics and Natural Sciences
BRAC University
Dhaka, Bangladesh

45. Thank you