Ahmed Abdullah Fahim thesis presentation

BTE 450: Biotech ProjectAHMED ABDULLAH FAHIM

Student ID:11336004 November 2015

Isolation of Reactive Red 3BX and Yellow 4GL dye degrading

bacteria from the textile sludge.

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).

EXAMPLE OF AZO DYES

Blue Br

Yellow 2G

Yellow 4G

Orange 2R

Reactive Red

Reactive violet

BOND IN AZO COMPOUNDS

Effects: Textile dyes on environment

Textile dyes can i. absorb and reflect sunlight from entering

water which can hinder photosynthesis.

ii. change the pH of soil and water.

iii. create bladder cancer, splenic sarcomas and nuclear anomalies in animals.

Textile Dye: Pollution

Treatment processes

Treatments 1

• Physico-chemical treatments

Treatments 2

• Adsorption with solid adsorbents

Treatments 3

• Biological treatments

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.

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

Methodology

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.

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.

Processing

Figure: Bacterial Colonies

Biochemical tests :AppliedCatalaseOxidaseCasein hydrolysisCimmon citrateNitrate reduction testStarch hydrolysis testMIU test ( motility indole urease)Methyl red testIn dole testVogasproskuras test.TSI test

Aerobic growth testGrowth in 10% NaClGrowth in 15% NaClFructose testGalactose testGlucose testLactose testMaltose testManitol testSucrose testTreshalos test

Results and

observation

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

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.

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

0.380000000000001

0.237

0.399000000000001

0.391000000000001

0.0900000000000001

absorbed wavelength

Results and observation cont.

Figure: Degradation of Reactive Red dye

Figure: Degradation of Yellow 4GL dye

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


Figure: Grams staining of colony C

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

Results and observation cont.Growth on 7% NaCl media Negative

Motility PositiveGrowth on usual media Positive

Starch hydrolysis NegativeAcid 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

• By the help of these biochemical results, microscopic observation,

characteristics of the organism and software this colony C was found

as the Brevibacillus laterosporus


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

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

• By the help of these biochemical results, microscopic observation,

characteristics of the organism and software this colony L was found

as Staphylococcus nepalensis


Figure: Gram staining photo for Colony L which isas Staphylococcus nepalensis

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.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


Y axis is representing absorbance values and X axis is representing time period

Chart presenting the decolorization of Reactive Red dye by Brevibacillus laterosporus

Day 0

24 hours later

48 hours later

72 hours later

0.2

0.7

1.2

1.7

2.2

2.7

3.2

3.7

colony C

control

colony C

control


0 hour later 24 hours later 48 hours later 72 hours later0

0.5

1

1.5

2

2.5

3

3.5

4

Distilled water Control Colony C (assumed Brevibacillus laterosporus) Exponential (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

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


Figure: Decolorization of dye


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

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.


0.05

0.15

0.25

0.35

0.45

0.55

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

Discussion

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

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.

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 SciencesBRAC University

Dhaka, Bangladesh

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Ahmed Abdullah Fahim thesis presentation