Ppt 27 08-2014

By:

ANWAR HUSSAIN PHULPOTO

(M. Phil. Scholar)

2

Department of Microbiology,

SALU, Khairpur.

Paint

• Liquid or mastic composition that converts to a solid film, when applied to a substrate

Paint

• Used to protect, to color, to provide texture and for the decoration

Uses

• Paint was made and used more than 35000 years ago

History

• Oil Based Paints• Water Based Paints

Types

3

Paints composition

• Allows the paint to be of correct consistency for application

Solvent

• Basic solid film former that remains after the solvent has been evaporated

Binder

• Gives paint its most important properties of colour

Pigment

• To improve production and storage properties

Additives

4

Environmental pollution

• Environmental pollution by oil-paints

effluents is of greater concern

nowadays due to its widespread

applications.

• Improperly used paint, accidental

spills, leakage & breakage can poison

people and animals in an environment.

• Environmental Protection Agency

(EPA) is working with state, local, and

tribal governments to reduce toxic

releases of 187 pollutants to the

environment and most of them are

present in paints

5

Hazards of oil-paints pollution

Oil-Paint hazards

Cancer

Organ and

CNS*Damage

Increased mortality

rate

Irritation and

Allergic Reactions

Disturbance of

Ecosystem

Global warming

Ozone layer depletion

Smog formation

6CNS* =Central Nervous System

Biodegradation and bioremediation

• Biodegradation: A process by which

microbial organisms transform or alter the

structure of chemicals introduced into the

environment

• Bioremediation: Microorganisms or their

enzymes to return the environment altered

by contaminants to its original condition

• Detoxification and Mineralization

• Safe

• Cheap

• Environmental friendly and

• Effective means of hazardous waste

removal

7

Aims & Objectives

To isolate, screen and identify oil paint-degrading bacteria from paints

warehouses of Khairpur and Sukkur cities.

To investigate the biodegradation potential of screened bacterial isolates.

To perform toxicological studies before & after biodegradation

experiments in order to assure safe and environment friendly process.

To suggest recommendations in order to get rid from oil paints

environmental pollution and hazards.

8

Methodology overview

• Paint contaminated soil samples were collected from warehouses of KHP and SUK

Samples collection

• Isolation of bacteria was done by soil enrichment technique

Isolation of oil paints degrading bacteria

• Identification through morphological, biochemical followed by molecular characterization

Identification of selected bacteria

• Experiments were carried out at shake flask level & evaluated using UV-Vis Spectrophotometer & FTIR

Biodegradation of oil paints

• Antimicrobial assays (antibacterial and antifungal)

• Brine-shrimps Cytotoxicity Assay

• Phytotoxicity assay using wild reddish seeds

Toxicity Analysis

• Lignin Peroxidases

• Laccases

Enzyme Assay

• DPPH free radical scavenging activity

Anti-oxidant Activity

10

Samples collection

The seven paints polluted soil samples, from seven warehouses

(one from each) were collected and processed according to the

method of Omar* et al. (2013).

Figure: Collection of paints polluted soil samples from Khairpur and Sukkur paints

warehouses

11

* Omar, O. C., Kirsteen, O. P., David, M. O., Anthony, S and Annete, O. W., 2013. Molecular clustering of microbial flora and

bacterial degradation of textile dyes by isolates from contaminated soils. Elixir. Bio. Tech: 54:12452-12459.

Media for isolation of oil-paints degrading bacteria

For screening & isolation

Mineral salt medium (MSM) agar

• Carbon source (oil paint) at

concentration 100 to 300 ppm (part per

million) w/v (Weight by Volume).

For colonial characteristics

Nutrient agar

Ingredient Conc. (g/L)

MgSO4, 7H2O, 10

CaCl2, 7H2O, 0.2

KH2PO4 13.6

(NH4)2, SO4 2.4

FeSO4, 7H2O 0.2

Na2HPO4, 12H2O 15

Agar 12

Ingredient Conc. (g/L)

Lab-Lemco powder 1.0

Yeast extract 2.0

Peptone 5.0

Sodium chloride 5.0

Agar 15.0

Table: Typical composition of Nutrient agar*

Table: Composition of MSM agar*

*purchased from Oxoid (Thermo Scientific, UK)12

* Yonetani, et al., 2004

Culture conditions

(MSM)

Inoculum(0.1ml)

Carbon source (Oil

paint)

Temperature (37 °C)

Agitation (160 rpm)

Incubation time (14

days)

Culture conditions for isolation of oil-paints degrading

bacteria

*-1 g paints-contaminated soil into 30 ml sterile distilled water, agitated at 160 rpm for 10 min and used for inoculation (0.1 ml clear

solution).

– Mineral Salts Medium (MSM)

– Inoculum (0.1mL of soil suspension*)

– Carbon source [Oil paint= White-32

(AlbaTM synthtic enamel)] at 100, 200,

& 300 **ppm conc.

– Growth OD*** were taken at 600 nm

using spectrophotometer at alternative

days up to 14 days including zero day

– Best growth ODs samples were then

used for the isolation of oil-paint

degraders.

13

**ppm= Part per million

***- OD= Optical Density

Identification of bacterial isolates

Cu

ltu

ral

Ch

ara

cter

isti

cs

• Colony:

• Size

• Shape

• Color

• Margins

• Texture

Mic

rosc

op

y

• Gram’s staining

• Capsule staining

• Spore staining

• Flagella staining (Motility test)

Bio

chem

ical

Tes

ts

• Table 1 (see Below in next slide)

14

Biochemical tests

TSI = Triple Sugar Iron assay Sugar Fermentation: LAC = lactose SUC = sucrose

MAN = mannoseGLU = glucose

IMViC : Indole (IND) test Methyl red (MR) test Voges Proskauer (VP) test

Citrate utilization (CIT) test

NR = Nitrate reduction OXD = Oxidase test CAT = Catalase test UT = Urease test

Table. 1

15

• The molecular identification of the bacterial isolates was carried out by 16S

rRNA sequence homology.

• The bacterial isolates as pure glycerol stocks were sent for commercial

amplification and sequencing of 16S rRNA at Macrogen Inc., Seol, Korea.

• The amplified nucleotide sequences were obtained and interpreted for

similarity index or sequence homology at NCBI GenBank database using

Basic Local Alignment Search Tool (BLAST) against available reference

nucleotide library.

• The evolutionary distances were computed by neigbour-joining (NJ) and

maximum-composite likelihood (MCL) methods.

• The phylogenetic trees were re-constructed with closely related bacteria

using Molecular Evolutionary Genetic Analysis (MEGA) version 6.0.

Molecular characterization of selected bacteria

16

Inoculum preparation for biodegradation

Pure Culture of selected isolates

grown in nutrient broth

Pure culture grown was

centrifuged for 10M at 4000rpm

Cell pellet was collected and

washed three times in normal

saline

Finally it was added into MSM

broth, that then serve as inocula

for all the experiments

preformed

Pure culture in Nutrient

broth

Centrifuged at

4000rpm for 10M

Collected cell pellet Pellet suspended into

MSM broth 18

Media and culture conditions for biodegradation

The inocula suspended in MSM was inoculated

(0.1ml) into MSM supplemented with oil paint

(300ppm)

The biodegradation experiment was run with and

without glucose.

The 5 mM Glucose was added (v/v) to MSM.

The experiment was preformed in triplicates with

all positive & negative controls

The cultural conditions have been already

discussed in slide 13.

19

Analysis of oil-paint biodegradation

• For analysis, the samples were drawn from each shake flask experiment afterevery 2nd day of incubation starting from zero day.

• Growth ODs were regularly monitored using spectrophotometer at 600 nm.

Samples Collection & Growth estimation

• Samples were analyzed using spectrophotometer (UV-Vis Scanning) at 285nm and FTIR spectrometry for biodegradation.

UV- Vis Scanning spectrometry and FTIR Analysis

FTIRShake flask experiment O. D by spectrophotometer 20

Estimation of oil-paint biodegradation using

UV-Vis spectrophotometer

Standard curve preparation

Paint standard at different conc. (i.e. 100, 200, 300,

and 400 ppm) was dissolved in petroleum ether and

its absorbance was recorded at 285 nm against a

blank of petroleum ether by using UV-VIS

spectrophotometer.

The absorbance was then plotted against conc. in a

graph in MS-Excel and a linear regression value (R2)

was calculated. An R2 value of 0.998 corresponds to

linear correlation between conc. vs absorbance and

was used to quantify the oil-paint conc. in unknown

test samples of the biodegradation experiments.

Quantification of the oil-paint

y = 0.008x - 0.016

R² = 0.998

Calculations

x = y+ 0.016 / 0.008

y = 0.0089x - 0.016

R² = 0.9984

0

0.5

1

1.5

2

2.5

3

0 100 200 300 400

Ab

sorb

an

ce (

28

5n

m)

Paint conc. (ppm)

21

Fourier transform infra-red spectroscopy

Fourier transform infrared (FTIR) spectra were

collected with a bench-top Spectrum™ 65 FT-IR

spectrometer equipped with Universal diamond

ATR (Attenuated Total Reflectance) accessory

(PerkinElmer Inc., USA)

Spectra were recorded in the ranges from 4000

cm-1 to 600 cm-1 with a speed of 16 scans per

scanning.FTIR

22

• Toxicity to

– Microorganisms (antibacterial & antifungal)

– Animal models (cytotoxic activity against

brine shrimps larvae)

– Plants (phytotoxic activity against wild reddish

seeds)

Toxicological studies

24

– Agar well diffusion method

– Control drugs Cefotexime (200 μg/well) [antibacterial]

Cloterimazole (200μg/well) [antifungal]

– Bacterial strains:• Micrococcus luteus ATCC* 10240

• Salmonella typhi ATCC 14028

• Pseudomonas aeruginosa ATCC 9027

– Fungal strains:• Aspergillus niger FCBP** 0198

• Aspergillus fumigatus FCBP 0066

• Aspergillus flavus FCBP 0064

Toxicity to microorganisms (Antibacterial & Antifungal Assay)

25

ATCC* = American type culture collection

FCBP** = Fungal cultural bank of Pakistan

Toxicity to eukaryotic animal model(Brine shrimp cytotoxicity assay):

• Cytotoxic activity against Brine shrimp

(Artemia salina) larvae at temp 37oC,

incubation time 24 to 36 hours By using

method of Jamil, et al., 2012.

• LD*50 estimation of each sample was

estimated before and after incubation with

respective bacterial inocula using

Statistical software Table Curve® (version

12.4)

26LD*= lethal Dose

Toxicity to Plants (Phytotoxicity assay using wild reddish seeds):

Phytotoxicity analysis was carried out according to Haq, et al.

(2013).

Distilled water was used as negative control

2-4dichloroacetic acid was used as control

Experiment was performed in triplicates.

The percent seed germination inhibition was calculated by

using equation

where; A= represents the total number of seeds germinated in control and

B= represents the total number of seed germinated in sample.

27

DPPH free radical scavenging activity (anti-oxidant

activity) using method Clarke et al. (2013)

The test sample (50 µl) from stock solution was added to respective well of

96 wells plate and then DPPH solution was added in each well to make 200

µl final volume.

Mixture was incubated at room temperature for half an hour.

Change in color from violet to yellow was observed.

Oxidation potential of samples was determined the color changing.

Absorbance of reaction mixture was measured at 517 nm on microplate

reader.

Figure : 96 well plate showing antioxidant activity of the cell-free supernatants

Ascorbic acid and DMSO were used

as positive and negative controls,

respectively.

Given formula was used for

calculation of scavenging activity of

sample.

28

• 2-6dimethoxyphenol (DMP) was used as substrate to calculate

the Laccase activity in sodium tartarate buffer.

• Activity was measured for cell free supernatant in reaction

mixture.

• The reaction mixture was incubated for 30 min and Absorbance

was measured at 469nm.

*DMP= 2-6dimethoxyphenol

Laccase Assay

No. Reagent Volume/Quantity

1 Sodium Malonate 400µl

2 DMP* 100µl

3 Distilled Water 700µl

4 Sodium Tartrate Buffer 500µl

5 Sample (Enzymes) 300µl

Total Volume 2000µl

Laccase enzyme assay

Table: Typical composition of the reaction mixture for laccase assay

30

Enzyme assay for Lignin Peroxidase

• Veratryl alcohol used as substrate for LiP* assay and activity was

measured by the rate of oxidation.

• Absorbance was measured at 310 nm after each 15 sec

Lignin peroxidase

No. Reagent Quantity/Volume

1 Sodium Tartrate Buffer 200µl

2 Veratryl Alkohol 200µl

3 Distilled Water 400µl

4 H2O2 100µl

5 Sample (Enzymes) 100µl

Total Volume 1000µl

*Lip Assay = Lignin peroxidase

Table: Typical composition of the reaction mixture for LiP assay

31

33

Phase 01:Isolation and Identification of oil-paint degrading

bacteria from paint contaminated soil

Enrichment of soil samples in MSM containing 100-300 ppm (w/v)

oil paint for the isolation of oil-paint degrading bacteria

NOTE:

SSSA = Sukkur Soil Sample A

SSSB = Sukkur Soil Sample B

34

NOTE:

KSSA = Khairpur Soil Sample A

KSSB = Khairpur Soil Sample B

Enrichment of soil samples…. Continued…

35

Enrichment of soil samples…. Continued…

NOTE:

SSSC = Sukkur Soil Sample C

KSSC = Khairpur Soil Sample C

KSSD = Khairpur Soil Sample D 36

Cultural characteristics

Paint Sample Isolate MSM AgarNutrient

AgarCultural Characteristics

200 ppm SSSA ACircular, Flat, Entire or

undulate, Smooth surface,

Opaque, white or creamy

300 ppm

KSSA B Circular, Flat, Entire or


Opaque, white or creamy

KSSC C Irregular, Glistening, low

convex, undulate and

translucent

KSSD D Circular, Flat, Entire or


Opaque, white or creamy37

38

Sub

-cult

ure

d o

n n

utr

ient

agar

Morphological characteristics

Bacterial Morphological Characteristics

Oil Paint Sample Isolate Microscopy

Gram's

Staining

Shape CS HDT SS

200ppm SSSA A + Bacilli _ + +

300ppm

KSSA B + Bacilli _ + +

KSSC C + Bacilli _ + +

KSSD D + Bacilli _ + +

NOTE:

CS = Capsule staining;

HDT = Hanging drop technique;

SS = Spore staining 39

B. subtilis LG4 gb|KF607095.1|

B. tequilensis LR3F3P gb|KF623287.1|

Isolate A

B. subtilis SBT-014 gb|KF601955.1|

B. subtilis VKK-3OL gb|JX852576.1|

B. subtilis ZLY gb|JX402129.1|

B. subtilis SMY gb|EU271854.1|

B. subtilis BCRC 17443 gb|EF423603.1|

B. subtilis ATCC 21331 dbj|AB018487.1|

B. pumilus ATCC 7061 ref|NR 043242.1|

B. cereus ATCC 4342 emb|AJ577288.1|

Brevibacillus parabrevis SR2729 gb|KF952775.1|

P. aeruginosa ATCC 23993 gb|FJ652615.1|

P. aeruginosa ATCC 25011 gb|FJ652613.1|100

98

99

98

99

0.02

Test Result

Gram reaction +

Motility +

Sporulation +

Capsulation -

Starch hydrolysis -

Lipid hydrolysis -

Casein hydrolysis -

Catalase +

Oxidase +

Indole _

Citrate utilization +

Lactose fermentation

+

Identified as: B. Subtilis

Fig. Phylogenetic tree of B. subtilis (Isolate, A) showing evolutionary relationshipwith other closely related taxa

Table. Microscopic & Biochemical characterization of B. subtilis, (isolate, A).

Molecular identification

Gene Bank submission

Nucleotide sequence were

submitted in NCBI Gene Bank

USA & accession number

(KJ872852) was received

Identification of isolate, A

40

Test Result

Gram reaction +

Motility +

Sporulation +

Capsulation -

Starch hydrolysis -

Lipid hydrolysis -

Casein hydrolysis -

Catalase +

Oxidase +

Indol -



+

Identified as: B. subtilis

Table. Microscopic & Biochemical characterization of B. subtilis (isolate, B)

Isolate B

B. subtilis BAB-2437 gb|KC443082.1|

B. subtilis VITLWS2 gb|KF929418.1|

B. subtilis BC18 gb|KF636528.1|

B. subtilis NM1C5 gb|EU221331.1|



Bacillus cereus ATCC 14579 |NR 074540.1|

B. thuringiensis IAM 12077 |NR 043403.1|

B. weihenstephanensis DSM 11821 |NR 024697.1|

B. mycoides 273 |NR 036880.1|


96

96

100

99

99

99

0.01


Fig. Phylogenetic tree of B. subtilis (Isolate, B) showing evolutionary relationshipwith other closely related taxa






Identification of isolate, B

41

Isolate C

Brevibacillus parabrevis M3 dbj|AB215101.1|

Brevibacillus parabrevis HDYM-18 gb|EF428244.2|

Brevibacillus parabrevis NBRC 12334 dbj|AB680274.1|

Brevibacillus formosus SBZ2-10 gb|HQ236045.1|

Brevibacillus agri M1-5 dbj|AB039334.1|

Brevibacillus limnophilus DSM 6472 dbj|AB112717.1|

Brevibacillus agri 7-2 emb|FN397521.1|

Brevibacillus panacihumi DCY35 gb|EU383033.1|

Paenibacillus sonchi X19-5 gb|DQ358736.1|

Paenibacillus sp. 172 gb|AF273740.1|

100

99

62

47

50

99

0.01

Table. Microscopic & Biochemical characterization of Brevibacillus. parabrevis (isolate, C)

Test Result

Gram reaction +

Motility +

Sporulation +

Capsulation +

Starch hydrolysis _

Lipid hydrolysis _

Casein hydrolysis _

Catalase +

Oxidase +

Indol _


Lactose fermentation +

Identified as:Brevibacillusparabrevis


Fig. Phylogenetic tree of Brevibacillus parabrevis (Isolate, C) showingevolutionary relationship with other closely related taxa






Identification of isolate, C

42

Isolate D

B. subtilis BAB-2437 gb|KC443082.1|

B. subtilis BCRC 14716 gb|EF423595.1|

B. subtilis BC18 gb|KF636528.1|



B. cereus XX2010 |JX993816.1|

B. cereus RIVM BC00068 |AJ577283.1|

Bacillus cereus ATCC 14579 |NR 074540.1|

B. thuringiensis IAM 12077 |NR 043403.1|

Brevibacillus panacihumi DCY35 gb|EU383033.1|


Brevibacillus limnophilus DSM 6472 dbj|AB112717.1|

Paenibacillus sonchi X19-5 gb|DQ358736.1|

Paenibacillus sp. 172 gb|AF273740.1|

61

100

100

76

76

100

100

100

100

94

0.01

Table. Microscopic & Biochemical characterization of B. subtilis (isolate, D)

Test Result

Gram reaction +

Motility +

Sporulation +

Capsulation -

Starch hydrolysis -

Lipid hydrolysis -

Casein hydrolysis -

Catalase +

Oxidase +

Indol _



+

Identified as: B. sublitis


Fig. Phylogenetic tree of B. subtilis (Isolate, D) showing evolutionary relationship withother closely related taxa






Identification of isolate, D

43

Cont…

44

Four bacterial isolates were identified and nucleotide

sequence of the amplified 16S rRNA gene were submitted

to NCBI GenBank and Accession No. received against each

sequence:

Bacillus subtilis strain NAP1 gb│KJ872852│


Brevibacillus parabrevis strain NAP3 gb│KJ872853│


Phase 02:

Biodegradation of oil paint (300ppm, v/v) by the

selected isolates in MSM supplemented with and

without glucose

45

Enrichment of pure culture of selected isolates for biodegradation

in MSM containing 300ppm (v/v) oil paint with & without glucose

A=Isolate + MSM (300ppm) + Glucose

B= Isolate + MSM (300ppm) + Glucose;

C=Isolate + MSM (300ppm) + Glucose46

NOTE:

D=Isolate + MSM (300ppm);

CONSORTIUM= ABCD Isolates+ MSM+ paint (300ppm)

47

Enrichment of selected isolates…… continued

Biodegradation of Oil Paint: Potential Of Bacterial Isolates For Oil-paint Degradation

51

Biodegradation of oil paint (300ppm, w/v) by Isolate, A

52

Isolate A, FTIR spectra

A-0A-14AG-0AG-14

53

Biodegradation of oil paint (300ppm, v/v) by Isolate, B

54

B-0B-14BG-0BG-14

Isolate B, FTIR spectra

55

Biodegradation of oil paint (300ppm, v/v) by Isolate, C

56

C-0C-14CG-0CG-14

Isolate C, FTIR spectra

57

Biodegradation of oil paint (300ppm, v/v) by Isolate, D

58

D-0D-14DG-0DG-14

Isolate D, FTIR spectra

59

Biodegradation of oil paint (300ppm, v/v) by using

bacterial consortium

60

Con-0Con-14ConG-0ConG-14

Consortium, FTIR spectra

61

TOXICITY ANALYSIS OF THE OIL-PAINT: BEFORE & AFTER MICROBIAL TREATMENT

62

Toxicity to microorganisms (Antibacterial & Antifungal Assay)

Cefotexime (200 μg/well) [antibacterial]

Cloterimazole (200μg/well) [antifungal] 63

Toxicity to eukaryotic animal model (Brine shrimp cytotoxicity assay):

LD50 estimation of each sample before incubation with respective bacterial inocula

0

25

50

75

100

LD

50

(μL/

mL)

Sample

*NS (P<0.05)

64

Cytotoxicity assay

Figure: Cytotoxic activity of control and treated paint samples by each bacterial isolate

before and after incubation in MSM broth supplemented without (A) and with (B)

glucose as additional carbon source. 65

Experiment:

Toxicity to Plants (Phytotoxicity assay using wild reddish seeds):

Root Length (mm)

66

Phytotoxicity assay

Shoot Length (mm)

67

Phytotoxicity assay

68

DPPH* free radical scavenging activity (anti-oxidant

activity)

0

5

10

15

20

25

30

35

40co

ntr

ol A B C D

Co

nso

rtiu

m A B C D

Co

nso

rtiu

m

con

tro

l A B C D

Co

nso

rtiu

m A B C D

Co

nso

rtiu

m

0 day 14 days 0 day 14 days

without Glucose With Glucose

% D

PP

H S

cave

ngi

ng

( 4

00

µl/

mL)

Sample & Incubation time (days)

*NS (P<0.05)

**S (P<0.01) **S (P<0.01)

*S (P<0.05) *S (P<0.05) **S (P<0.01)

69DPPH*= diphenyl picryl hydrazine

Enzymes Assay

70

Enzymes Assay

71

Conclusion

The most predominant bacterial isolate in all the soil samples was Bacillus subtilis (A, B, D).

A novel oil-paint degrading bacterial strain of Brevibacillus parabrevis (C) was isolated and

characterized from the paint contaminated soil sample of Khairpur paint warehouse.

Among four bacterial isolates and their consortia, the maximum oil-paint % removal was achieved with

bacterial consortia (94%, 81% followed by Brevibacillus parabrevis (C) (83%, 73%) with and without

glucose, respectively.

The isolate C (Bravibacillus parabrevis) and consortium showed significant removal of toxicity

followed by the B. subtilis (B), B. subtilis (D), and B. subtilis (A) with glucose among the all

treatments.

All the isolates have strong potential for the enzymes and bioactive metabolites production. The highest

Laccase and LiP production threshholds were recorded for the isolate C, followed by consortium,

isolate B, Isolate A and isolate D, respectively.

The isolate A as well as bacterial consortium, revealed the release of some cytotoxic metabolites in the

absence of glucose, while in the presence of glucose there were no production of such cytotoxic

metabolites.

72

Future Prospects

The isolated bacterial strains could be explored for the biodegradation & bioremediation of various

other environmental pollutants

Future optimization studies are needed for complete mineralization of the oil-paints used.

Genes involved in biodegradation could be studied in detail.

Mechanism of oil-paint biodegradation and metabolites could be studied in detail.

The isolates could be a good source of antibacterial as well as antifungal agents, and should be

studied in more detail for the production of antimicrobial metabolites.

Enzyme profiling of the isolates for different enzymes could be done in details, as the isolates

showed an excellent potential for some certain enzymes

Strains improvement could be accomplished in order to achieve desired results for biodegradation.

73

I am thankful to Almighty Allah

All Teaching & Non Teaching staff (Dept. Microbiology, SALU, Khairpur).

Director Research & Staff (Hi Tech. Laboratory, SALU, Khairpur)

Ms. Nazia Khatoon (Dept. Microbiology, QAU, Islamabad)

Mr. Abdul Rehman Phul (Dept. Pharmacy, QAU, Islamabad)

All friends, laboratory fellows (seniors and colleagues)

All family members

Acknowledgements

74

76

Research images

77

Research images

78

Research images

Paint

conc.

(v/v)

Sample Description Optical Density at 600 nm

incubation time (days)

0d 2d 4d 6d 8d 10d 12d 14d

100ppm

SSSA PC 0.096±0.01 0.099±0.02 0.106±0.04 0.112±0.06 0.118±0.07 0.121±0.05 0.125±0.06 0.127±0.02

Test 0.201±0.02 0.256±0.01 0.335±0.07 0.375±0.06 0.548±0.01 0.479±0.16 0.329±0.15 0.238±0.01

SSSB PC 0.085±0.03 0.093±0.04 0.101±0.05 0.106±0.05 0.109±0.04 0.113±0.03 0.116±0.02 0.119±0.01

Test 0.148±0.05 0.309±0.02 0.329±0.02 0.339±0.01 0.479±0.08 0.421±0.11 0.323±0.01 0.251±0.04

SSSC PC 0.099±0.03 0.106±0.04 0.111±0.06 0.114±0.06 0.118±0.06 0.123±0.02 0.123±0.02 0.127±0.01

Test 0.165±0.07 0.425±0.03 0.423±0.02 0.535±0.02 0.585±0.09 0.525±0.01 0.375±0.07 0.283±0.05

KSSA PC 0.089±0.02 0.098±0.02 0.107±0.01 0.112±0.01 0.117±0.02 0.121±0.03 0.125±0.04 0.129±0.05

Test 0.135±0.04 0.328±0.05 0.395±0.02 0.435±0.02 0.485±0.01 0.483±0.11 0.277±0.14 0.181±0.01

KSSB PC 0.088±0.02 0.093±0.02 0.950±0.04 0.099±0.04 0.104±0.06 0.108±0.06 0.114±0.01 0.121±0.01

Test 0.198±0.06 0.291±0.06 0.342±0.01 0.381±0.01 0.493±0.02 0.428±0.01 0.321±0.12 0.251±0.10

KSSC PC 0.091±0.01 0.097±0.11 0.103±0.03 0.107±0.05 0.112±0.02 0.115±0.06 0.122±0.07 0.122±0.01

Test 0.158±0.01 0.297±0.21 0.351±0.01 0.353±0.09 0.503±0.08 0.415±0.02 0.311±0.01 0.237±0.20

KSSD PC 0.103±0.04 0.108±0.05 0.115±0.31 0.121±0.10 0.123±0.20 0.125±0.02 0.126±0.10 0.128±0.01

Test 0.208±0.11 0.281±0.01 0.435±0.01 0.455±0.09 0.523±0.91 0.475±0.13 0.322±0.02 0.275±0.01

Control MSM 0.128±0.01 0.135±0.02 0.138±0.03 0.147±0.04 0.153±0.05 0.155±0.06 0.153±0.10 0.159±0.23

NC 0.091±0.01 0.092±0.01 0.098±0.03 0.105±0.03 0.099±0.07 0.108±0.06 0.115±0.06 0.113±0.05

NOTE: SSSA = Sukkur Soil Sample A; SSSB = Sukkur Soil Sample B; SSSC = Sukkur Soil Sample C; KSSA = Khairpur Soil Sample A; KSSB =

Khairpur Soil Sample B; KSSC = Khairpur Soil Sample C and KSSD = Khairpur Soil Sample D; PC = Positive Control; MSM = Mineral Salts

Medium; NC = Negative Control ; 0d = Zero day and so on;

Enrichment of soil samples in MSM containing 100ppm (w/v) oil paint for the

isolation of bacteria using shake flask method

79

Paint conc.

(v/v)

Sample Descriptio

n

Optical Density at 600 nm


0d 2d 4d 6d 8d 10d 12d 14d

200ppm

SSSA PC 0.096±0.01 0.099±0.02 0.106±0.04 0.112±0.06 0.118±0.07 0.121±0.05 0.125±0.06 0.127±0.02

Test 0.259±0.11 0.478±0.01 0.526±0.02 0.641±0.01 0.838±0.06 0.642±0.11 0.421±0.01 0.315±0.08 Selected

SSSB PC 0.085±0.03 0.930±0.04 0.101±0.05 0.106±0.05 0.109±0.04 0.113±0.03 0.116±0.02 0.119±0.01

Test 0.249±0.21 0.458±0.04 0.523±0.31 0.542±0.21 0.713±0.01 0.542±0.04 0.323±0.08 0.299±0.01

SSSC PC 0.099±0.03 0.106±0.04 0.111±0.06 0.114±0.06 0.118±0.06 0.123±0.02 0.123±0.02 0.127±0.01

Test 0.237±0.05 0.465±0.03 0.516±0.01 0.519±0.09 0.625±0.19 0.596±0.11 0.365±0.18 0.258±0.11

KSSA PC 0.089±0.02 0.98±0.02 0.107±0.01 0.112±0.01 0.117±0.02 0.121±0.03 0.125±0.04 0.129±0.05

Test 0.231±0.01 0.461±0.05 0.469±0.21 0.499±0.11 0.726±0.05 0.589±0.22 0.343±0.11 0.256±0.12

KSSB PC 0.088±0.02 0.930±0.02 0.95±0.04 0.099±0.04 0.104±0.06 0.108±0.06 0.114±0.01 0.121±0.01

Test 0.247±0.09 0.388±0.01 0.436±0.11 0.528±0.12 0.639±0.13 0.544±0.12 0.308±0.01 0.173±0.14

KSSC PC 0.91±0.011 0.970±0.11 0.103±0.03 0.107±0.05 0.112±0.02 0.115±0.06 0.122±0.07 0.122±0.01

Test 0.278±0.04 0.368±0.04 0.396±0.09 0.539±0.15 0.733±0.01 0.503±0.14 0.389±0.13 0.221±0.09

KSSD PC 0.103±0.04 0.108±0.05 0.115±0.03 0.121±0.11 0.123±0.21 0.125±0.02 0.126±0.10 0.128±0.01

Test 0.268±0.03 0.381±0.07 0.402±0.01 0.428±0.13 0.618±0.11 0.506±0.15 0.381±0.04 0.273±0.13

Control MSM 0.247±0.01 0.251±0.01 0.262±0.02 0.267±0.11 0.281±0.21 0.291±0.01 0.292±0.04 0.191±0.02

NC 0.091±0.01 0.092±0.01 0.098±0.03 0.105±0.03 0.099±0.07 0.108±0.06 0.115±0.06 0.113±0.05



80

Paint conc.

(v/v)

Sample Description Optical Density at 600 nm


0d 2d 4d 6d 8d 10d 12d 14d

300ppm

SSSA PC 0.096±0.01 0.099±0.02 0.106±0.04 0.112±0.06 0.118±0.07 0.121±0.05 0.125±0.06 0.127±0.02

Test 0.341±0.03 0.437±0.01 0.531±0.11 0.652±0.01 0.738±0.02 0.674±0.02 0.415±0.11 0.308±0.12

SSSB PC 0.085±0.03 0.093±0.04 0.101±0.05 0.106±0.05 0.109±0.04 0.113±0.03 0.116±0.02 0.119±0.01

Test 0.333±0.01 0.537±0.08 0.592±0.12 0.751±0.02 0.859±0.08 0.665±0.21 0.436±0.13 0.288±0.11

SSSC PC 0.099±0.03 0.106±0.04 0.111±0.06 0.114±0.06 0.118±0.06 0.123±0.02 0.123±0.02 0.127±0.01

Test 0.322±0.09 0.537±0.9 0.562±0.13 0.645±0.03 0.833±0.09 0.635±0.11 0.515±0.02 0.298±0.17

KSSA PC 0.089±0.02 0.098±0.02 0.107±0.01 0.112±0.01 0.117±0.02 0.121±0.03 0.125±0.04 0.129±0.05

Test 0.345±0.04 0.635±0.06 0.728±0.09 0.843±0.09 0.949±0.16 0.736±0.15 0.526±0.07 0.293±0.12 Selected

KSSB PC 0.088±0.02 0.093±0.02 0.95±0.04 0.099±0.04 0.104±0.06 0.108±0.06 0.114±0.01 0.121±0.01

Test 0.331±0.02 0.435±0.07 0.623±0.30 0.724±0.50 0.849±0.13 0.731±0.05 0.442±0.15 0.321±0.10

KSSC PC 0.091±0.01 0.097±0.10 0.103±0.03 0.107±0.05 0.112±0.02 0.115±0.06 0.122±0.07 0.122±0.01

Test 0.352±0.05 0.532±0.03 0.646±0.06 0.793±0.03 0.895±0.11 0.627±0.01 0.541±0.21 0.305±0.10 Selected

KSSD PC 0.103±0.04 0.108±0.05 0.115±0.03 0.121±0.10 0.123±0.21 0.125±0.02 0.126±0.10 0.128±0.01

Test 0.362±0.08 0.532±0.04 0.545±0.01 0.623±0.31 0.998±0.21 0.671±0.11 0.449±0.11 0.313±0.12 Selected

Control MSM 0.301±0.01 0.331±0.10 0.338±0.20 0.342±0.02 0.341±0.02 0.351±0.02 0.351±0.02 0.353±0.02

NC 0.091±0.01 0.092±0.01 0.098±0.03 0.105±0.03 0.099±0.07 0.108±0.06 0.115±0.06 0.113±0.05



81

Positive Control = Isolate+ Base; A=Isolate + MSM (300ppm)

B= Isolate + MSM (300ppm); C=Isolate + MSM (300ppm)

D=Isolate + MSM (300ppm); ABCD+ Isolates+ MSM (300ppm)

PC = Paint Control (300ppm); BC = Base Control

ENRICHMENT OF PURE CULTURE OF SELECTED ISOLATES FOR BIODEGRADATION

IN MSM CONTAINING 300PPM (V/V) OIL PAINT WITHOUT GLUCOSE USING SHAKE

FLASK METHOD.

82

Description 2d 4d 6d 8d 10d 12d 14d

Positive Control, A 0.14±0.11 0.14±0.06 0.15±0.03 0.15±0.52 0.09±0.04 0.08±0.73 0.07±0.05

A 0.45±0.08 0.60±0.04 0.53±0.09 0.43±0.09 0.27±0.03 0.19±0.06 0.14±0.04

Positive Control, B 0.18±0.02 0.20±0.11 0.17±0.51 0.16±0.06 0.14±0.72 0.13±0.05 0.11±0.04

B 0.48±0.05 0.77±0.05 0.61±0.12 0.55±0.06 0.32±0.13 0.29±0.11 0.15±0.05

Positive Control, C 0.19±0.51 0.23±0.02 0.19±0.11 0.19±0.72 0.11±0.06 0.09±0.33 0.09±0.04

C 0.33±0.06 0.92±0.06 0.67±0.07 0.44±0.07 0.30±0.06 0.23±0.18 0.09±0.01

Positive Control, D 0.17±0.31 0.20±0.71 0.14±0.02 0.15±0.12 0.12±0.05 0.09±0.06 0.09±0.33

D 0.52±0.05 0.71±0.07 0.57±0.05 0.36±0.04 0.24±0.03 0.16±0.04 0.13±0.06

Positive Control Consortium 0.21±0.07 0.22±0.04 0.24±0.03 0.20±0.02 0.19±0.12 0.14±0.04 0.12±0.06

ABCD 0.66±0.11 0.94±0.06 0.98±0.11 0.78±0.11 0.65±0.05 0.30±0.05 0.25±0.04

PC 0.28±0.04 0.31±0.05 0.39±0.07 0.29±0.04 0.31±0.03 0.12±0.02 0.06±0.03

BC 0.14±0.05 0.14±0.04 0.14±0.03 0.14±0.42 0.11±0.03 0.08±0.13 0.08±0.02


Incubation Time (Days)

0d

0.14±0.02

0.28±0.01

0.12±0.01

0.26±0.02

0.21±0.31

0.26±0.03

0.28±0.07

0.14±0.05

0.16±0.51

0.26±0.02

0.21±0.03

0.26±0.02

Negative Controls

ENRICHMENT OF PURE CULTURE OF SELECTED ISOLATES FOR

BIODEGRADATION IN MSM CONTAINING 300PPM (V/V) OIL PAINT WITH

GLUCOSE (5MM) USING SHAKE FLASK METHOD

Positive Control = Isolate+ Base +Glucose; A=Isolate + MSM (300ppm) + Glucose

B= Isolate + MSM (300ppm) + Glucose; C=Isolate + MSM (300ppm) + Glucose

D=Isolate + MSM (300ppm) + Glucose; ABCD+ Isolates+ MSM + Glucose

PCG = Paint + Glucose Control; PC = Paint Control (300ppm)

BCG = Base + Glucose Control; BC = Base Control

83

Description 2d 4d 6d 8d 10d 12d 14d

Positive Control, A 0.23±0.02 0.24±0.06 0.24±0.31 0.23±0.04 0.22±0.14 0.23±0.14 0.22±0.06

A 0.71±0.07 1.32±0.61 0.82±0.06 0.74±0.14 0.58±0.06 0.39±0.03 0.33±0.01

Positive Control, B 0.24±0.14 0.24±0.32 0.14±0.06 0.15±0.13 0.25±0.03 0.25±0.01 0.23±0.14

B 0.75±0.06 0.95±0.13 1.42±0.12 0.92±0.33 0.71±0.01 0.5±0.04 0.35±0.06

Positive Control, C 0.22±0.05 0.23±0.06 0.24±0.04 0.24±0.03 0.24±0.01 0.22±0.33 0.21±0.05

C 0.66±0.06 0.87±0.13 1.23±0.02 0.95±0.31 0.7±0.15 0.55±0.11 0.43±0.01

Positive Control, D 0.21±0.06 0.21±0.14 0.23±0.03 0.23±0.01 0.21±0.04 0.21±0.05 0.21±0.03

D 0.82±0.05 1.43±0.4 1.43±0.53 0.91±0.11 0.82±0.14 0.61±0.02 0.43±0.01

Positive Control Consortium 0.24±0.04 0.27±0.03 0.27±0.01 0.25±0.14 0.24±0.14 0.23±0.04 0.23±0.05

ABCD 0.86±0.04 1.65±0.1 1.37±0.52 0.99±0.01 0.81±0.04 0.52±0.04 0.41±0.01

PCG 0.38±0.03 0.40±0.01 0.41±0.04 0.45±0.10 0.45±0.14 0.36±0.14 0.33±0.04

PC 0.28±0.01 0.31±0.03 0.31±0.01 0.29±0.31 0.29±0.13 0.12±0.03 0.27±0.14

BCG 0.16±0.04 0.16±0.06 0.15±0.03 0.15±0.11 0.10±0.04 0.11±0.01 0.23±0.01

BC 0.14±0.01 0.14±0.04 0.14±0.06 0.13±0.03 0.11±0.01 0.11±0.04 0.21±0.30

0.32±0.06

0.28±0.03

0.15±0.01

0.14±0.06

0.19±0.06

0.41±0.01

0.24±0.06

0.44±0.02

Negative Controls

0.42±0.02

0.23±0.31

0.43±0.02

0.21±0.14

0.42±0.03


Incubation Time (Days)

0d

0.22±0.14

Similarity of all the isolates

84

Similarity of all the isolates

85

y = 0.0025x + 0.0878R² = 0.9894

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 50 100 150

Ab

sorb

an

ce (

31

0n

m)

Enzyme units

Molecular characterization of selected bacteria

• Preparation of template DNA

Suspend pure colonies in 0.5

ml saline

Centrifugation 10,000 rpm, 10

M

Discard supernatant

Suspend pellete in 0.5 mlInsta Gene Matrix (Bio-

Rad, USA).

Incubate at 56°C, 30 M

Incubate at 100°C, 10 MCollect supernatant and use for PCR

Pure culture

86

Primers for amplification and sequencing of 16S rRNA

gene

Table 1 Primers of amplification and sequencing

Analysis : http://rdp.cme.msu.edu/html/

http://rdp.cme.msu.edu/

Reference : Nucleic Acids Research, Vol. 18, Supplement

87

Polymerase Chain Reaction (PCR)

Procedure and PCR conditions

10㎕ of template DNA was (v/v) added in 20㎕of PCR reaction solution

27F/1492R primers were used for bacteria, andthen 35 amplification cycles at 94℃ for 45 sec,55℃ for 60 sec, and 72℃ for 60 sec wereperformed

DNA fragments were amplified about 1,400 bp inthe case of bacteria

Included a positive control (E. coli genomic DNA)and a negative control in the PCR.

Purification of PCR products

Unincorporated PCR primers and dNTPs fromPCR products were removed by using MontagePCR Clean up kit (Millipore).

88

Sequencing

Sequencing procedure and conditions:

The pure amplified PCR products weresequenced by using two primers asdescribed (Table 1)

Sequencing were performed by using BigDye terminator cycle sequencing kit v.3.1(Applied Bio Systems, USA)

Sequencing products were resolved on anApplied Biosystems model 3730XLautomated DNA sequencing system(Applied BioSystems, USA) at theMacrogen, Inc., Seoul, Korea.

89

90

0

20

40

60

80

100

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GI

91

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% D

PP

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cave

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ng

( 4

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µl/

mL)

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RG

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MPFG

Ppt 27 08-2014

Education

Transcript of Ppt 27 08-2014