isolation and selection of thermal tolerant toluene degrading bacteria

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 BACTERIA SUPERVISOR Dr. NGUYEN HUU HIEP STUDENT VO NGOC THAO NGUYEN Student code: 3082615 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPPERVISOR Dr.NGUYEN HUU HIEP STUDENT VO NGOC THAO NGUYEN Can Tho, May , 2013 PRESIDENT OF EXAMINATION COMMITTEE 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. i 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 ii 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. 1 2. MATERIALS AND METHODS 2.1. Materials Soil sample from four potential sites in exposuring to Toluene in Can Tho city Medium: MSB (mineral salt basal) medium (Na et al., 2005) : K2HPO4 : 4.5 g/l, KH2PO4 : 3.4 g/l, (NH4)2SO4 : 2 g/l, MgCl2.6H2O : 0.34 g/l, MnCl2.4H2O : 0.1 g, FeSO4 : 0.6 g, CaCl2.2H2O : 2.6 g, Na2MoO4 : 0.02 g, ZnCl2.7H2O : 0.01 g, CoCl2.6H2O : 0.01 g, CuSO4 : 0.01 g, NiSO4.6H2O : 0.001 g, NaSeO4 : 0,001 g per liter of deionized water 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 carbon sources. The main principle to determine degrading Toluene capability was comparing the change of optical density of MSB liquid media containing Toluene at 0%, 1%, 1.5%, 2% of different bacterial strains after 0, 2, 4 day. One percentage of bacteria were cultured in MSB liquid media containing Toluene and incubated at 45oC, shaking at 120 rpm. 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. 3 2.2.4. Identification of selected bacterial strains at molecular level Based on the result of the ability to degrade Toluene and produce enzyme, three strains were selected for the identification by molecular technique at Biological Molecular Laboratory of Biotechnology Research and Development institute, Can Tho University. 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 4 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 5 T12 T38 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 6 solvent concentration was lower than 0.3% (Moriya and Horikoshi, 1993). Note: OD(600nm) Time Figure 5. Optical density of bacterial strains through days T14 and T50 strains had different growing pattern because their OD increased in day 2 and decreased in day 4 at the concentration of 2% (Figure 1). Maybe, these two strains could adapt quickly and used Toluene as a carbon source to grow. The decrease might be caused by the inhibition among bacteria. According to Sardessai and Bhosle (2002), some bacteria 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 7 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) 01 T3 0.5c 02 T4 0d 03 T9 1.2ab 04 T12 0d 05 T14 1.3ab 06 T34 0.3c 07 T38 0d 08 T50 1.1b 09 T51 1.4c (Note: F= 54.42, means followed by the same letters in the same column were not significantly different (P[...]... According to Sardessai and Bhosle (2002), some bacteria 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 7 06 out of 09 strains were able to produce enzyme protease Among these T14, T9, T50 and T51 strains... 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):... while T38 could only grow in Toluene at 1% and 1.5% 4.2 Suggestions Due to limitation of time, several studies should be done in the future to determine the capacity of these isolated strains in degrading Toluene in the polluted environment 19 REFERENCES Abe, A., A Inoue, R Usami, K Moriya and K Horikoshi 1995 Degradation of polyaromatic hydrocarbons by organic solventtolerant bacteria from deep sea Bioscience... Kato 2005 Isolation and characterization of benzene -tolerant Rhodococcus opacus strain Journal of Bioscience and Bioengineering, 99: 378-382 Ogino, H., K Miyamoto and, H Ishikawa 1994 Organic solventtolerant bacterium which secretes organic solvent-stable lipolytic enzymes Applied Environment Microbial, 60: 3884-3886 Ogino, H., K Yasui, T Shiotani, T Ishihara and H Ishikawa 1995 Organic solvent -tolerant. .. 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 16 of two-, three-, four-, and five-ring cycloalkanes and mono-, di-, tri-, tetra-, and. .. (Al-Sharidah et al., 2000) Therefore, it was very promising for this bacterial strains to be able to grow in medium containing Toluene as a sole carbon source The results of the experiment showed that T38 could only survive at Toluene 1% and 1.5% This was in accordance with the result of selection with OD as a parameter Table 13 Bacterial density of T38 strain (CFU/ml) Treatments Day 0 Day 2 Day 4 Treatment... 0 0 T38 strain, bacterial density increased slightly in day 4 and equal 0 after 6 days It only grew in 1% and 1.5% of Toluene (Table 6) This might be because T38 strain could not adapt to Toluene at high level This was in agreement with previous study of Abe et al (1995) which reported that Bacillus subtilis could not grow in media containing 10% of organic solvents 18 4 CONCLUSIONS AND SUGGESTIONS... 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 12 3.4 Result of identification of selected bacterial strains at molecular level Three strains T14, T38, T50 were identified based on 16S rRNA gene sequencing Using BLAST search tool for analysis of these isolates The results showed that T14... Dierstein and M H Tadros 2000 Isolation and characterization of two hydrocarbondegarding Bacillus subtilis strains from oil contaminated soil of Kuwait Microbiological Research, 155: 157-164 Lau, P.K.W and A Koenig 2001 Management, disposal and recycling of waste industrial organic solvents in Hong Kong Chemosphere, 44: 9-15 Na, Kyung-su, Akio Kuroda, Noboru Takiguchi, Tsukasa Ikeda, Hisao Ohtake and Junichi... (Moriya and Horikoshi, 1993) Note: OD(600nm) Time Figure 5 Optical density of bacterial strains through days T14 and T50 strains had different growing pattern because their OD increased in day 2 and decreased in day 4 at the concentration of 2% (Figure 1) Maybe, these two strains could adapt quickly and used Toluene as a carbon source to grow The decrease might be caused by the inhibition among bacteria