isolation and selection of thermal tolerant toluene degrading bacteria

The aim of this research is to isolate and select thermal-tolerant bacterial strains which are able to degrade Toluene.. Isolated bacteria which were able to use Toluene as a sole carbo

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS IN

BIOTECHNOLOGY

ISOLATION AND SELECTION OF THERMAL

-TOLERANT TOLUENE DEGRADING

APPROVAL

SUPPERVISOR STUDENT

Dr.NGUYEN HUU HIEP VO NGOC THAO NGUYEN

Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE

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Abstract

Nowadays, the development of industry offers people a better life However, industrialization without caring about the environment may lead to negative effects on the Earth as well as human health Therefore, the contamination of Toluene, an organic solvent which is a common ingredient in painting and leathering industry, has caught many attentions of scientists The aim of this research is to isolate and select thermal-tolerant bacterial strains which are able to degrade Toluene Soil samples from four Toluene-contaminated sites in Can Tho city were incubated with Toluene in both gas and liquid phase to increase bacterial mass MSB (mineral salt basal) medium was used for the isolation of these bacteria Isolated bacteria which were able

to use Toluene as a sole carbon source and synthesize enzyme amylase, cellulase, lipase and protease were selected to identify

at molecular level Moreover, drop count method was used to examine the ability of degrade Toluene of bacteria From four soil sources, 09 bacterial strains were isolated Most isolated bacteria could produce enzyme amylase, cellulase, lipase and protease T14, T38 and T50 strains were selected for 16S rRNA analysis and on the basis of 16S rRNA, they were assigned to Bacillus licheniformis, Bacillus subtilis, Acinetobacter sp, respectively T38 (Bacillus subtilis) could use Toluene as a sole carbon source at 1% and 1.5%

Key words: Bacteria, degradation, isolation, organic solvents,

Toluene contamination

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CONTENTS

ABSTRACT……… i

CONTENTS ……… ……… …… ii

1 INTRODUCTION 1

2 MATERIALS AND METHODS 2

2.1 Materials 2

2.2 Methods 2

2.2.1 Isolation of bacteria 2

2.2.2 Study of the ability to degrade Toluene 2

2.2.3 Study of the ability to produce some enzymes 3

3 RESULTS AND DISCUSSIONS 4

3.1 Result of isolation of thermal-tolerant Toluene degrading bacteria 4

3.2 Result of study of the ability to degrade Toluene 5

3.3 Result of the ability to produce enzymes of isolated strains 7

3.4 Result of identification of selected bacterial strains at molecular level 12

3.5 Result of experiment proving ability to degrade Toluene of selected bacterial strains 17

4 CONCLUSIONS AND SUGGESTIONS 19

4.1 Conclusions 19

4.2 Suggestions 19

REFERENCES 20

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1 INTRODUCTION

The development of industry offers human being a better life However, it cannot be denied that industrializing without caring about protecting the environment may bring serious environmental hazards Especially, toxic chemical like Toluene,

an organic solvent discarded from manufacture processes, could directly affect to human health as well as ecosystem (Lau, 2011) Among many traditional methods used to treat organic contamination, bioremediation appears as an outstanding method because it does not cost much and eco-friendly Therefore, scientists around the world have been interested in figuring out bacteria that have ability to degrade organic solvents Vietnam with an abundant bacterial population will be a promising site for isolating potential Toluene degrading bacterial strains

Objectives

To isolate and select thermal-tolerant Toluene-degrading bacteria

Chemicals and equipments in Microbiology laboratory

2.2 Methods

2.2.1 Isolation of bacteria

Collected samples from 04 different sites were incubated with Toluene in both liquid and gas phases in 2 weeks Then the samples were diluted into various concentrations and spread on sterilized MSB agar medium Then they were incubated at 48oC with Toluene in gas phase When colonies formed, colonies with different characteristics were subcultured until achieving single colonies with identical shape and size Colonies were then examined under microscope to determine the purity of bacteria Isolated bacteria and their colonies were observed their shape, size and Gram characteristics

2.2.2 Study of the ability to degrade Toluene

Isolated bacteria, in this experiment, were studied in order

to generally estimate their capability to utilize Toluene as a

2.2.3 Study of the ability to produce some enzymes

*Enzyme protease

Principle: 5 µl of bacteria nourished in Luria Bertani was dripped on MSB agar containing skim milk (2%) The diameter of Halo around the colony was measured to determine the ability to produce enzyme protease

*Enzyme lipase

Bacteria were cultured in MSB liquid media containing 2%

of oil to observe the ability to produce enzyme lipase The parameter used in this experiment was optical density of bacteria

at the wavelength of 600 nm

*Enzyme amylase

Similar to enzyme protease, halo diameter in MSB agar media containing 2% starch was used as a parameter to examine the ability to produce enzyme amylase

*Enzyme cellulase

MSB agar media containing CMC (2%) was used to determine the ability to produce cellulase of bacteria After dying with Iodine, the diameter of halo around colony would show the ability of bacteria to produce enzyme cellulase

2.2.5 Experiment proving the ability of bacteria to degrade Toluene

The experiment was carried out with 4 treatments, 3 replicates, completely randomized design

Four treatments consisted of MSB liquid media containing Toluene at 0%, 1%, 1.5%, 2% Bacteria were inoculated in each treatment and incubated at 45oC, shaking 120 rpm

Drop count method was used to determine bacterial density

in each treatment after 0, 2, 4, 6 days

3 RESULTS AND DISCUSSIONS

3.1 Result of isolation of thermal-tolerant Toluene degrading bacteria

From 04 soil samples, 09 bacterial strains which were capable of degrading Toluene were isolated Among these, there were 07 strains isolated from Tay Do leather factory and 02 strains isolated from Xang Thoi lake All bacteria had rod shape Colonies on MSB agar media were circular or irregular, entire and undulate edge with raised and flat elevation Colors of colonies were milky white except T4 with clear white The growth of these bacteria was quite slow (from 48-72 hours) Gram

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staining showed that all bacteria were Gram negative Matsumoto

et al (2002) also reported that genus Pseudomonas Gram

negative could degrade organic solvent

Figure 3 Characteristics of some bacterial colonies

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Figure 4 Gram staining of some bacteria

3.2 Result of study of the ability to degrade Toluene

Isolates were observed their growth in media with Toluene

as a sole carbon source After two days of incubation, almost all bacterial strains decreased their growth The reason for this might

be explained that the changing of environment made bacteria die

We found a different pattern among bacterial strains after four days Besides strains that had OD decrease such as T3, T4 strain, the other bacterial strains grew differently at different concentration This meant that the ability of bacteria to adapt to Toluene concentration was not the same among bacterial strains For example, T38 strain, at 1% of Toluene could increase gradually but at 2%, its growth decreased This might be because T38 strain was only able to use Toluene at low concentration If the concentration was high, it became toxin to the bacteria Previous results also reported that some bacteria which could degrade organic solvent but they could only survive when organic

belonging to genus Bacillus were able to use Toluene as a sole

carbon at 90% of Toluene The results showed that T14, T38 and T50 were promising strains in degrading Toluene

3.3 Result of the ability to produce enzymes of isolated strains

*Enzyme protease

Time OD(600nm)

Note:

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06 out of 09 strains were able to produce enzyme protease Among these T14, T9, T50 and T51 strains had significantly large diameter comparing to T3 and T34 strains T4, T12 and T38 strains did not produce enzyme protease (Table 8)

Table 8 Ability to produce enzyme protease of isolated bacterial strains

No Bacterial strain Halo diameter (cm)

(Note: F= 54.42, means followed by the same letters in the same column were

not significantly different (P<0.05))

(Note: F= 15.31 , means followed by the same letters in the same column were

not significantly different (P<0.05))

*Amylase

After dying with Iodine, all strains could form halo around the colony T4, T38 and T12 strains formed significantly larger halo than others T9 strain formed the smallest halo

(Note: F= 98.88, means followed by the same letters in the same column were

not significantly different (P<0.05)

The result of enzyme test on the ability to produce enzyme protease, lipase, amylase and cellulase showed that almost all bacterial strains could produce these enzymes These results were

in agreement with previous studies on organic solvent degrading bacteria (Ogino et al., 1995; Rahman et al., 2010; Trivedi et al., 2011; Pandey, 2012) Ogino et al (1995) reported that

Pseudomonas aeruginosa could synthesize protease which was

stable in organic solvents Moreover, the ability to produce enzyme lipase of organic solvent degrading bacteria,

Pseudomonas sp and Bacillus sp were also recorded (Rahman et al., 2010) Bacillus aquimaris could also produce enzyme

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cellulase stable in organic solvents (Trivedi et al., 2011) According to Pandey (2012), Bacillus agaradhaerens was able to synthesize enzyme α-amylase at 30% of organic solvent

In summary, the results from enzymes tests reflected that T14, T38 and T50 strains were promising strains in degrading Toluene and producing some enzymes

Figure 5 Halo created on starch and CMC medium by bacterial strains

rRNA sequence similarity with Bacillus licheniformis strain W11 and T38 strain had 89% similarity with Bacillus subtillus BSn5

Whereas, T50 strain showed 85% homology with the sequence of

Acinetobacter sp.G16(2009)

*16s rRNA gene sequence of T14 strain (995 nucleotides) TCGAGCGGACGACGGGAGCTTGCTCCCTTAGGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATCATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGAGCGGTGAAATGCG

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TAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCCTTCC

The sequence was identical to Bacillus licheniformis at

99% Strain T14 was a potential strain in degrading not only organic solvents but also crude oil (Das and Chandran, 2010; Reda and Ashraf, 2010) According to Das and Chandran (2010),

Bacillus licheniformis which was isolated from polluted stream

could degrade crude oil Moreover, Reda and Ashraf (2010) also

reported that Bacillus licheniformis was potential in degrading

Toluene

*16s rRNA gene sequence of T38 train (784 nucleotides): TCACCGGGGAGCTAAATGCAGTCGAGCGGAAGAGGGAGCTTGCTCCCTGTATGTTAGCGGCGGACGGGTGAGTAAGACGTGGGAAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTTTGCTATCTCTTTTAGATGGACCCCCCGCGCATTTGCTAGTTGGGGAGGGAAAGGCTCCCCCAAGGAACGATGCGGAGCCGACCTGAGAGGGGGATCGGCCCCACTGGGGGTGAGAAACCGGCCAGACTCCTACGGGGGGGAGCAGTGGGGAATCTTCCCCA

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ATGGGCGAAAGTCTGACCGAGCGACCCCCCGTGGGTGATGAAAGGTTTTGGATCTTAAAGCTCTGTTGTTAGGGGAGAACAAATACCGTTCGAATAGGGCGGGACCTTGACGGGACCCAACCAGAAAGCCCCGGCTAACTACCTGCCCACCACCCCGGGAATACGTAAGGGGGAAGGGTTTTCCGGAATTTTTGGGCGTAAAGGGCTCGCAGGGGGTTTTTTTAATTTGATGTGAAAACCCCCGGCTCCACCCGGGAGGGTCATTGGAAACTGGGGAACTTTAGTGGAAAAAAAGAAAGTGGAATTTCCCCTGTAGCGGTGAAATGGGTAAAGATGTGGAGGAACACCCGTGGCCAAAGGGACTTTTTGGGCTGTAACTGACACTGAAGAGCGAAAACGTGGGGAGCGAACAGGATTTAATACCCTGGGTAT

T38 was identified as Bacillus subtilis at the identical level

of 89% Although Bacillus subtilis was reported to be unable to

grow in the presence of organic solvents at concentration of 10% (Abe et al (1995) Recently study of Navacharoen and Vangnai

(2011) proved that Bacillus subtilis could use diethyl phthalate as

a sole carbon source In addition, Bacillus subtilis also had the

ability to degrade crude oil (Das and Chandran, 2010) and Dimethylformamide (Vidhya and Thatheyus, 2013)

*16s rRNA gene sequence of T50 strain (957 nucleotides): CGGTGGGCCGGCCAAAAATGCAGTCGAGCGGGCGAGGTTGCTTCTGTTCTGAGCTAGCGGCGGAGGGTGAGTAATGAATAGGAATCTGCCTATTATGGGGGGAGGCATTCCTTAAGGGAAGCTAATACCACATACGTCCTACTGGAGAAAGCCAGGGCTCATTATGAACTTGCGCTAATAGATGACCCTTACTCAGATTCCCTAGGTGGTGGGGTTAAGGGCTAC

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CAAGGCGACTATCTGTAGCGGGTCTGAGAGGATGATCCGACAGGGTGGGACTGAAACACGGGCCAGACTCCTACCGGAGGCTCCTACGGGGAATATTGGACAATGGGCGGAACCCTGATCCAACCATGCCACGGGTGTGAAGAACGTCTTTTGGGTGTGAATTACTTTAAGAGAGGAGGAGGGTTACCTGAGAAATACCTGGGCTAAGTGGACGTTACCCACAAAATAACCACCGGCTAACTCTGTGCCAAACTCCGCGGTAATACAGAGGGTGCGAGCGTTAATCGGATTTACTGGGCGAAAAGCGCGCGTAGGTGGTTAATTAAGTCAAATCTTAAATCCCCGAGCTTAACTTGGGAATTGCATTTGGAACTGGTTGGAAACTGTATGGGAGAGGATGGTAGAATTCCAGGTGTAATTCCACAAATGCCTATAAAATTGGAAGAATTCCGAAGGAACACGCATGCCTCTGGCCTAATTCTGACCCTTAAGTGACACTGAATGGGGAGCACACAGGAATAAAAACCGAGGAAATACATGGCGTAAACAATGCCTACTAGACGATTGCTGATTGGAACATGTAGTCCGCCCTCTAACGCTGATAAGTAAACCATCTGGACAGTACCGTGCGCAAGTACTAACTCAGACTGAAATGACAGGGGCTCGCACGAGCCCCGCACCATGTGGTTTAACATCGTATGTTTACGTCAAAACCACTGACGCTAGGAACTT

After being aligned using BLAST, T50 strain was

identified as Acinetobacter sp at the identity of 85% Acinetobacter genera was known as a potential bacteria in

degrading carbonhydrate which could remove up to 70% of medium chain alkanes (Malatova, 2005) Walker et al (1976)

isolated Acinetobacter species from oil-contaminated water and

sediment in which they could yield significant greater degradation