Isolation, Screening and In Vitro Mutational Assessment of Indigenous Soil Bacteria for Enhanced Capability in Petroleum Degradation

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1 Isolation, Screening and In Vitro Mutational Assessment of Indigenous Soil Bacteria for Enhanced Capability in Petroleum Degradation Naveenkumar.S 1, Manoharan.N 1, Ganesan. S 1, Manivannan.S.P 2, Velsamy. G 1 1 Department of Marine Sciences, Bharathidasan University, Tiruchirappalli 2 Department of Biotechnology, Bharathidasan University, Tiruchirappalli biomano@gmail.com ABSTRACT A total of 25 bacterial isolates from petroleum contaminated soil in various niches were screened for degradative ability. Out of which three strains showed increased activities. Standard morphological and biochemical analysis identified up to generic level show that they belong to Micrococcus sp, Staphylococcus sp and Pseudomonas sp. The isolated strains were enriched and assessed for degrading activity after UV mutagenesis. Optimization parameters were standardized in various temperature, ph, Nitrogen source and Carbon source. Prominent degradation was found for Micrococcus sp and Staphylococcus sp at 37 0 C, ph 7.0, Carbon source as Petrol 15uL and Nitrogen source as Calcium nitrate tetra hydrate, whereas Pseudomonas sp showed a significant elevation in activity compared to Micrococcus and Staphylococcus sp. The parameters optimized for Pseudomonas sp depict that they are viable at 43 0 C, ph 8.0, Carbon source as Petrol 15uL and Peptone as Nitrogen source. Optimization and mutagenesis show that UV mutant Pseudomonas sp showed prominent activity and most promising in the field of petroleum degradation. The study is novel as we propose that UV mutagenesis inducts an increased activity in indigenous bacteria possessing petroleum degradative activity. Keywords: Degradation, Bacteria, Mutational assessment, degradative studies. 1. Introduction Nature takes thousands of years to form the fossil fuel. All fossil fuels primarily consist of a complex mixture of molecules called hydrocarbons (Alexander, 1994). Petroleum is a complex mixture of many thousands of compounds mainly consisting of carbon and hydrogen. These can be divided into four major groups: alkanes, aromatics, resins, and asphaltenes. In general, an alkane fraction is the most biodegradable, whereas the polar fraction (i.e., resins and asphaltenes) is resistant to biological degradation. The aromatic compounds, especially the polycyclic aromatic hydrocarbons (PAHs), are of intermediate biodegradability, but these are of most concern owing to their toxicity and tendency to bioaccumulate (Wrenn et al., 1995). The utilization of crude petroleum all over the world serves as the sources for fuel and energy. Lubricants and other petrochemical products cause a lot of serious problems in oil contamination. These sources serve as the major contributor to the environmental pollution problems especially in the soil and water. Contamination of the environment by petroleum products is known to be toxic and hazardous to the environment. As industrialization expands, petroleum hydrocarbons become a greater potential source of soil contamination. Soil contamination with hydrocarbons causes extensive damage of local ecosystem since accumulation of pollutants in animals and plant tissues may causes progeny s death or mutation. It has become one of the major environmental pollution that is becoming more stringent and to 498

2 be given a lot of attention. The elevated loading of petroleum hydrocarbons in soil cause a significant decline in soil quality and these soils have become unusable. (Alvarez et al., 1991). Physical, chemical and biological methods can be used for cleaning up the polluted sites. Biodegradation is most often the primary mechanism for contaminant destruction including petroleum contaminants. It has been proposed for cleanup of oilspills as cost effective technology of removing contaminants (Leahy et al., 1990). Microorganisms survive in contaminated habitat because they are metabolically capable of utilizing its resources and can occupy a suitable niche. Contaminants are often potential energy sources for microorganisms. It shows that microorganisms have broad range of enzymes that enable them to degrade many chemicals (Madigan et al., 1998). Hydrocarbon degrading microorganisms are widely distributed in marine, freshwater and soil ecosystems. The microbial degradation of oil pollutants is a complex process and the environmental factor have a great influence on the fate of spilled oil, but with an understanding and studying this process in the environment, it is possible to develop strategies for utilizing microbial hydrocarbon degradation activities for the removal of oil spills from contaminated areas. These strategies appear to be the most environment friendly method of removal of oil pollutants since other method such as surfactant washing and incineration lead to introduction of more toxic compounds to the environment (Atlas et al., 1973). Research is being focused to investigate novel degradative treatments and technologies for the degradation of petroleum compounds. Based on these problems and to control the environmental risk caused by petroleum products the present study was designed to optimize degradative ability of petroleum degrading soil bacteria and evaluate UV mutagenesis as a tool to enhance degradation by indigenous soil bacteria. 1.1 Materials and Methods Soil samples 100gm of three different petroleum contaminated soil samples were collected using sterile bags from three different petrol bunks at Tirupur, India. And they were brought to the laboratory without further contamination. They were mixed together and used as a single sample for further studies. 1.2 Isolation and enumeration of bacteria Isolation and enumeration of bacteria were performed by plate technique. Three media were used for finding the best utilization patterns. The media used are Bushnell Haas broth, Mineral Salt broth and Basal Salt broth, Bushnell Haas broth showed maximum activity. 100 ml of each broth media were prepared in 250 ml Erlenmeyer flask and sterilized at 121 C for 15 lbs/sq.inch for 15 minutes. After sterilization, the broth media were incorporated with 0.1 ml of petrol as a carbon source and 1 gm of each soil samples were inoculated. They were incubated at 170 rpm at 30 C for a week. After a week of incubation, the turbidity of the broth cultures was observed by spectrophotometer (JASCO) at 600nm. 499

3 1.3 Screening of petroleum degrading bacterial isolates The enriched bacterial isolates were purified by sub culturing. They were screened for the ability to degrade the petroleum compounds present in the soil samples collected as follows. 100ml of Bushnell Haas Agar medium was prepared and sterilized at 121 C for 15lbs/sq.inch for 15 minutes. After sterilization, the medium and 1ml of each enriched broth cultures were mixed poured into petriplates. After solidification, four wells were created within the plates. Then the wells were loaded with different concentrations of petrol (5µl, 10µl 15µl and 20µl) was incubated at 37 C for 24 days. After incubation colonies were obtained around the wells and they were used for further studies. 1.4 Characterization of Bacterial isolates Each isolate was examined many times for its size, shape, margin, consistency, opacity, elevation, pigmentation, gram reaction and cell morphology as described by Cowan et al. The isolates were characterized as described by Holt et al., Diagnostic properties used include motility, catalase production, indole, urease, oxidase, coagulate test, oxidative fermentation of sugar, methyl red, nitrate reduction test and Gelatin liquefaction test. 1.5 UV Mutagenesis of the bacterial isolates The isolated strains were improved mutationally to enhance their degradative ability. They were UV irradiated as follows; Nutrient agar was prepared and sterilized at 121 C 15 lbs/sq.inch for 15 minutes. After sterilization the medium were poured into petridishes. The purified cultures were streaked onto the agar medium and incubated at 37 C for 24 hrs. After incubation the cultures were exposed to UV radiation by placing under UV light for 30 minutes. Optimized Bushnell Haas agar medium was prepared and sterilized for 121 C 15 lbs/sq.inch for 15 minutes. After sterilization the medium was added with 1ml of mutant and wild type broth cultures in each flask and allowed to cool. Then the medium were poured into petridishes and allowed for solidification. Wells were created in the plates and loaded with increased concentrations of petrol (10µl, 20µl and 30µl) as carbon source and incubated for 37 C for 24 days. After incubation the colonies were counted for enhanced growth. 1.6 Optimization of growth parameters on the growth of isolates After identification, the bacterial strains were optimized under different growth parameters such as temperature, ph, carbon source and nitrogen sources. 1.7 Effect of temperature 100 ml of Bushnell Haas broth was prepared and sterilized at 121 C under 15 lbs/sq.inch for 15 minutes. After sterilization, the media was incorporated with 0.1ml of petrol and 1ml of purified cultures were inoculated in each flask and incubated at different temperatures (4 C, 28 C, 37 C and 43 C) for 24 hrs. OD was read at 600nm and observed for maximum growth. 500

4 1.8 Effect of ph 100 ml of Bushnell Haas broth was prepared with different ph (5, 6, 7 and 8) and sterilized at 121 C under 15 lbs/sq.inch for 15 minutes. After sterilization the media was incorporated with 0.1ml of petrol and 1ml of purified cultures were inoculated in each flask and incubated at 37 C for 24 hrs. OD was read at 600nm and observed for maximum growth. 1.9 Effect of concentration of carbon source 100 ml of Bushnell Haas broth was prepared and sterilized at 121 C under 15 lbs / sq.inch for 15 minutes. After sterilization the media was incorporated with different concentrations of petrol (5µl, 10µl, 15µl and 20µl) and 1ml of purified cultures were inoculated in each flask and incubated at 37 C for 24 hrs. OD was read at 600nm and observed for maximum growth. 2. Effect of Nitrogen source 100 ml of Bushnell Haas broth was prepared with 0.1 gm of different nitrogen sources (Ammonium nitrate, Potassium nitrate, Sodium nitrate, Calcium nitrate tetrahydrate and Peptone) and sterilized at 121 C under 15 lbs/sq.inch for 15 minutes. After sterilization the media was incorporated with 0.1ml of petrol and 1ml of purified cultures were inoculated in each flask and incubated at 37 C for 24 hrs. OD was read at 600nm and observed for peak growth. 3. Results 3.1 Isolation of indigenous bacteria from petroleum contaminated soil samples The primarily isolated broth cultures of Bushnell haas broth, Mineral salt broth and Basal salt broth were observed for the growth of bacteria by estimating optical density at 600nm which confirms the degradative ability of the isolates by utilizing petroleum compounds that are present in the soil samples collected. The results are indicative that for Bushnell haas broth show maximum growth. In Enrichment broth too, an average of the values affirms the above statement. As 7 days incubation is the optimal duration, the results prove that Bushnell Haas agar is the optimal media. The result are shown in Table no: 1 & Characterization of isolates As per the characterization the three active degraders are confirmed at generic level as Micrococcus sp, Staphylococcus sp and Pseudomonas sp. The result are shown in Table no: 3a & b. 3.3 Optimization of isolates bacteria Effect of Temperature and ph 501

5 Effect of Temperature and ph hindered the growth of Micrococcus Staphylococcus where as Pseudomonas sp showed maximal activity. Pseudomonas sp optimized at 43 0 C and ph 8.0 shows an increased degradation pattern. The results are shown in Table no: 4, 5 and figure no 1 and figure Effect of Carbon Source and Nitrogen Source The effect of carbon source was analyzed and optimal value is regarded as 15µl. peptone acts as an efficient nitrogen source for optimal degradation using Pseudomonas where as Ammonium nitrate, Potassium nitrate, Sodium nitrate, Calcium nitrate tetrahydrate show a lapsed activity. The results are shown in Table no: 6, 7 and figure no 3 and figure Effect of UV mutation on growth of isolates Mutant varieties of Micrococcus Staphylococcus show increased OD value rather than wild type. Among these Pseudomonas sp, show significant degradative activity. 30 µl of concentration of petrol showed a rise in activity where as µl show an elevated curve. When compared to other two genera in degradation. The result are shown in Table no: 8a&b and Fig no: 5, Discussion The microbial degradation of oil pollutants is a complex process. But with an understanding and studying this process in the environment, it is possible to develop strategies for utilizing microbial hydrocarbon degradation activities for the removal of hydrocarbons from contaminated areas. The objectives of this study are the isolation, identification, optimization of indigenous bacteria from petroleum contaminated soils and their mutagenesis to assess the enhanced degradative ability. A preliminary study was carried out to isolate the indigenous bacteria from petroleum contaminated soil sample using Bushnell Haas broth, Mineral salt broth and Basal salt broth. An increase in turbidity during the growth demonstrates the ability of utilizing of petroleum present in the soil during enrichment (Table no. 1 & 2). Bushnell Haas agar medium exhibited better growth within a short period of incubation ie. 5 days. Hence, it was found to be the best medium and selected for further studies of optimization. Three different isolated strains were identified by using standard procedures. The experimental outcome of morphological and biochemical characterization proved that both gram positive and gram negative bacteria ie., Micrococcus Staphylococcus Pseudomonas is able to survive in such a harsher niche.(table no.3a and 3b). Pseudomonas is the most common bacterial hydrocarbon degrader reported in the literature, which is widespread in nature and can degrade a wide range of xenobiotics (Rusansky et al, 1987; Kiyohara et al, 1992; Jonhson et al, 1996; Barathi and Vasudevan, 2001; Bhattacharya et al, 2002; Pokethitiyook et al, 2003; Van Hamme et al,2003). The present study proved it and Pseudomonas was isolated as one of the isolates. Similar strains were isolated by Sabeen Survery et al., 2004, Mandri and Lin, Studies on optimization of various growth parameters such as temperature, ph, different concentration of petrol as a carbon source and nitrogen sources revealed that the strains of Micrococcus and Staphylococcus showed 502

6 better growth at 37 C, ph 7 and these parameters were found to be the optimum, whereas, 43 C and ph 8.0 were found to be the optimum for Pseudomonas sp. (Table no. 4 & 5 and Fig no. 1 & 2). A related work was carried out with the strains of Flavobacterium Acinetobacterium calcoaceticeum and Pseudomonas aeruginosa by Mandri and Lin, Similar results were obtained. It was evidently found that 15 µl of petrol as carbon source for all the strains and calcium nitrate tetrahydrate as nitrogen source were utilized more efficiently. They served as optimum which provided the maximum growth for the strains of Micrococcus and Staphylococcus sp. (Table no. 6 & 7 and Fig no. 3 & 4). But the strain of Pseudomonas sp, was found to be metabolically different and exhibited better growth in the medium incorporated with peptone as nitrogen source. This shows that the strain of Pseudomonas sp, is able to show the better degradation even by using a very simple and basic nitrogen source. The optimized isolates were utilized for strain improvement by UV mutagenesis. The mutational study revealed that the mutants exhibit considerable improvement in their growth. It was evidenced by increased optical density as well as the degradative ability. The present study confirmed that among all isolates, the strain of Pseudomonas sp. was found to be the better choice for further studies like standardization, ability of producing various extracellular enzymes as well as surfactants and genetic profiling. Table 1: Estimation of growth of bacterial isolates during isolation Incubation period (in days) OD values (at 600 nm) Bushnell haas broth Mineral salt broth 2 Basal salt broth Table 2: Estimation of growth of bacterial isolates during enrichment Incubation period (in days) Bushnell haas broth OD values (at 600 nm) Mineral salt broth Basal salt broth

7 Table 3(a): Gram positive isolates Sl. No Test Isolate No. 1 Micrococcus Isolate No.2 Staphylococcus 1. Microscopic examination: a. Gram s staining b. Shape c. Motility d. Endospore staining 2. Biochemical tests: a. Coagulase b. Carbohydrate fermentation (i) Glucose (ii) Lactose (iii) Mannose (iv) Sucrose Note: : Positive & : Negative Cocci Cocci Table 3(b): Gram negative isolates Sl. Test Isolate No. 3 No. Pseudomonas sp 1. Microscopic examination: a. Gram s staining b. Shape c. Motility d. Endospore staining Bacilli 2. Biochemical tests: a. Indole Production b. Methyl Red c. Voges Proskeur d. Citrate Utilization e. Carbohydrate Fermentation: (i) Glucose (ii) Lactose (iii) Mannose (iv) Sucrose f. TSI 504

8 g. H 2 S Production h. Catalase i. Oxidase j. Urease k. Coagulase l. Nitrate reduction m. Starch hydrolysis n. Gelatin liquefaction AK/A Note: : Positive : Negative AK / A: Alkaline slant / Acid butt Table 4: Effect of temperature Sl. OD values (at 600 nm) No. Temp. Micrococcus Staphylococcus Pseudomonas 1. 4 C C C C Figure 1: Temperature vs O.D 505

9 Table 5: Effect of ph Sl. OD values (at 600 nm) No. ph Micrococcus Staphylococcus Pseudomonas Figure 2: ph vs O.D Figure 3: Carbon source vs O.D 506

10 Table 6: Effect of different concentration of carbon source Sl. No. Carbon source Micrococcus OD values (at 600 nm) Staphylo coccus Pseudomonas 1. 5 μl μl μl μl Table 7: Effect of nitrogen source Sl. No. OD values (at 600 nm) Nitrogen source Micrococc Staphylococc Pseudomonas us us 1. Ammonium nitrate Potassium nitrate Sodium nitrate Calcium nitrate Tetrahydrate Peptone

11 Figure 4: Sources vs O.D Table 8(a): Effect of UV mutation on growth of the isolates Sl. No OD value ( at 600 nm) Isolate Mutant Wild type Micrococcus sp. Staphylococcus sp. Pseudomonas sp Figure 5: Various bacteria vs O.D 508

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