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Isolation and screening of diesel degrading bacteria from ship dismantling facility in Tanjungjati coastal,

Madura, Indonesia

Harmin Sulistiyaning Titah1,*, Herman Pratikno2, Atiek Moesriati1, Muhammad Fauzul Imron1, and Rizky Islami Putra1

1Department of Environmental Engineering, Faculty of Civil Engineering and Planning, Institut Teknologi Sepuluh Nopember, Surabaya 60112, Indonesia

2Department of Ocean Engineering, Faculty of Marine Technology, Institut Teknologi Sepuluh Nopember, Surabaya 60112, Indonesia

Abstract. Ship dismantling activity is one industry that caused diesel contamination in environment. The objectives of this study were to isolate and to screen the diesel degrading bacteria from diesel contaminated area. Diesel contaminated seawater and soil samples were collected from ship dismantling facility in Tanjungjati coastal, Madura, Indonesia. Isolation was conducted in aseptic technique and growing the mixture culture was carried out based on pour plate method. After 24 h incubation, thirteen bacteria strains were isolated from that area. Isolated bacteria were identified base on morphological characterization. Gram positive bacteria were mostly found in diesel contaminated seawater and soil sample. These isolated bacteria were screened by using nutrient agar medium containing various diesel concentration (0%, 5%, 10%, and 15% (v/v)). The result of screening test showed that bacteria coded EL and CT showed the best resistance and highest growth in diesel polluted medium. It showed that both of them have potentially high capability to utilize diesel as carbon and energy source than others.

Keywords: Diesel, Isolation, Ship dismantling facility, Tanjungjati.

1. Introduction

Ship dismantling activity is one source of diesel pollution in coastal area. The main activity is structure dismantling and the part cutting of ship. Indirectly, the diesel, as engine fuel of ship, will be released into seawater and soil (Panda et al., 2013). One of ship dismantling facility in Indonesia located in Tanjungjati coastal, Madura (Kusuma, 2015). That activity caused seawater and soil pollution. It was indicated by the changing of seawater and soil color to become muddy and black. Every year, about 1500 tons of diesel were released into seawater from this activity.

Diesel is a kind engine fuel that contains rich hydrocarbons ranged C8 – C26 and polyaromatic hydrocarbons (PAHs) (Ramasamy et al., 2017). Diesel was reported as seawater and soil contaminant (Palanisamy et al., 2014). About 1.7 – 8.8 million metric tons of diesel were released to aquatic environment and soil (Dadrasnia and Agamuthu, 2013). Ministry of Environment Indonesia (2004) said that the maximum concentration of PAH in seawater is 0.003 mg/L. Diesel spillage causes serious effects to marine ecosystem because the component of diesel are potentially carcinogenic and toxic for environment (Ramasamy et al., 2017). One of the method that can be used to reduce diesel pollution is bioremediation (Palanisamy et al., 2014).

Bioremediation is one of biotechnology that utilized the metabolism of microorganism to reduce diesel contamination in seawater and soil. In previous study, many bacterias were isolated from diesel contaminated area such as Psuedomonas, Micrococcus, Staphylococcus,

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Bacillus, Flavobacterium, Acromobacter, Klebsiella, Actinomycetes, Acetobacter, Rhodococcus (Bhuvaneswar et al., 2012). Olawale et al. (2017) said that bioremediation have many advantages for detoxification of toxic substances because its environment friendly and simple method. Bioremediation process depends on the ability of microorganism to degrade diesel pollutant (Ibrahim, 2016). Some bacteria isolated from diesel contaminated area have ability to producing enzyme to degrade and utilize diesel as carbon and energy source (Patil et al., 2012). Furthermore, microorganism has capability to produce biosurfactant in order to increase the solubility of diesel.

Indigenous microorganisms isolated from diesel contaminated area have more efficient capability in degrading diesel (Prathyusa et al., 2016). The objectives of this study were to isolate the diesel degrading bacteria from ship dismantling facility in Tanjungjati coastal, Madura, Indonesia and determining resistant bacteria based on bacteria growth capability that screened in various diesel concentration.

2. Materials and methods2.1. Samples collection

Diesel contaminated seawater and soil were collected from ship dismantling facility in Tanjungjati Coastal, Madura, Indonesia. Seawater and soil samples were aseptically collected below surface at a depth of 20 cm (Bhasheer et al., 2014) and randomly selected in three different points. The seawater samples were collected into sterilized bottles and the soil samples were collected into sterilized plastic bags. In location, pH and temperature were measured on seawater and soil samples by pH meter (Cyberscan pH 510, Singapore) and thermometer (OneMed, Indonesia), the seawater salinity measured in laboratory by salinity meter (pH Onlab, USA). All collected samples stored in ice box at 4oC temperature (AlDisi et al., 2016) and then transferred to laboratory for further analysis.

To determine the components of diesel in seawater and soil samples, all samples should be extracted with organic solvent. For seawater samples, 10 mL of seawater samples were extracted with two stages of 20 mL of n – hexane solvent (Fulltime, USA) by using separation funnels (Pyrex, Germany) to remove cellular materials (Palanisamy et al., 2014). For soil samples, 2 grams of soil samples were extracted by using ultrasonic method along with 2 gram of anhydrous sodium sulfate (Merck, Germany) and 25 mL of n – hexane solvent for 30 minutes and then filtered through glass wool (Liu et al., 2011). The sample extracts were transferred to vials and kept at 4oC until analyzed with gas chromatography (HP 6890, USA).

2.2. Isolation of diesel degrading bacteria

Isolation of diesel degrading bacteria were performed using serial dilution method (Harley – Prescoot, 2002). A hundred mL of each seawater samples were mixed into 500 mL sterilized Schott bottle (Pyrex, Germany). Ten grams of each soil samples were suspended in 100 mL of 0.85% NaCl into sterilized 250 mL Erlenmeyer (Pyrex, Germany) (Prathyusa et al., 2016). After that, all samples shaken by rotating shaker (Memert, Germany) at 150 rpm for 1 hour (Pranowo and Titah, 2016). One mL of each samples was transferred into tube containing 9 mL of 0.85% NaCl and followed by serial dilutions up to 10-8. 0.1 mL of 10-6, 10-7, and 10-8 dilutions were spreaded on the surface of nutrient agar plate and then incubated in Incubator (Ogawa Seiki, Japan) at 37oC for 24 hours (Palanisamy et al., 2014). After the incubation period, different colonies appeared on agar surface were counted by bacteria colony counter (Topac, USA) and streaked into nutrient agar plates for purification and identification. After that, the isolated

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colonies were transferred into nutrient agar slants and stored in refrigerator at 4oC for further analysis (Bhuvaneswar et al., 2012).

2.3. Morphological of diesel degrading bacteria

The morphological of isolated bacteria were conducted based on physical characterization (Bhuvaneswar et al., 2012). The physical characterization such as size, shape, form, elevation, margin, appearance, pigmentation and gram staining were determined based on Harley – Prescoot (2002).

2.4. Screening of diesel degrading bacteria

Screening of diesel degrading bacteria were carried out by using streak plate method (Mujahid et al., 2015). Nutrient agar medium containing various concentration of diesel were streaked with isolated bacteria onto the surface of nutrient agar medium. Diesel concentrations used for screening of diesel degrading bacteria were 0%, 5%, 10%, and 15% (v/v) (Palanisamy et al., (2014); AlDisi et al., (2016)). Streaked diesel contaminated nutrient agar then incubated in incubator at 37oC for 24 – 48 hours (Mujahid et al., 2015).

The result of screening was determined by qualitative and quantitative analysis (Pranowo and Titah, 2016). The qualitative analysis was done based on physical characteristic appearance of isolated bacteria. The quantitative analysis was done based on the surface area percentage of bacterial growth in various diesel concentration contaminated nutrient agar medium that compared to nutrient agar plate surface area by using bacteria colony counter.

Table 1. Seawater and soil sample analysis

Physicochemical Properties

Seawater samples Soil Samples1 2 3 1 2 3

pH 8.1 7.3 7.6 < 3 3.2 3Temperature

(oC)< 3 3,2 3 33.7 38.6 36.8

Salinity (‰) 15.8 16 17.8 - - -

Soil Colour - - -Thick brown

Thick brown

Thick brown

Table 2. Components of diesel in seawater and soil

No Components of diesel1 Naphthalenane (C10)2 Cyclohexane (C6)3 Capnellane (C15)4 Tricosane (C23)5 Octadecane (C18)6 Tetracosamethylcyclododecasiloxane (C24)7 Hexadecane (C16)8 Heptadecene (C17)9 Octadecamethylcyclononasiloxane (C18)

10 Isochiapin B11 Bergamotane (C15)

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3. Results and discussions3.1. Physicochemical Properties of Diesel Contaminated Seawater and Soil

The physicochemical properties of diesel contaminated seawater and soil such as pH, temperature, salinity, and soil colour were analysed and shown in Table 1. The components of diesel in seawater and soil were analysed by using gas chromatography and shown in Table 2. Table 1 showed that seawater salinity in Tanjungjati coastal, Madura, Indonesia is brackish water. The salinity range of brackish water is 5‰ - 30‰.

Table 2 showed that the components of diesel in seawater and soil were hydrocarbons ranged C6 – C24 such as cyclohexane, naphthalenane, hexadecane, octadecane, and etc. Ramasamy et al. (2017) said that diesel contains rich hydrocarbons ranged C8 – C26 and polyaromatic hydrocarbons (PAHs). Bhuvaneswar et al. (2012) also said that diesel contains a large amount of alkanes hydrocarbon chains from C10 – C20.

3.2. Isolation of diesel degrading bacteria

Thirteen bacterial strains were isolated from diesel contaminated seawater and soil by serial dilution method. The isolated bacteria were identified based on morphological characterization (Table 3). According to Table 3, the percentage of isolated gram positive bacteria (54%) is higher than gram negative bacteria (46%). Gram positive bacteria dominate in diesel contaminated areas (Bhasheer et al., 2014). It was due to gram positive bacteria have stronger cell than gram negative bacteria and this allow them to grow in various condition. Different with observation by Mujahid et al. (2015) said that amount of gram negative bacteria isolated from diesel contaminated soil is higher than gram positive bacteria. Furthermore, Pranowo and Titah (2016) also said that amount of gram negative bacteria isolated from diesel contaminated seawater is higher than gram positive bacteria.

Table 3. Morphological Characterization of Isolated Bacteria

Bacteria code

Morphological CharacterizationGram

stainingShape Form Elevation Margin Appearance Pigmentation

Diameter (mm)

Soil SamplesAT - Coccus Circular Convex Entire Shiny White 2BT - Rod Circular Raised Entire Shiny White 1CT + Coccus Circular Convex Entire Shiny White 3ET - Coccus Punctiform Flat Entire Shiny White 1FT + Rod Punctiform Flat Entire Dull White 0.5GT - Coccus Circular Convex Entire Dull White 0.5HT - Rod Circular Convex Undulate Dull White 1

Seawater SamplesAL - Rod Circular Convex Entire Dull White 0.5BL + Rod Circular Convex Undulate Dull White 0.1CL + Rod Punctiform Convex Undulate Shiny White 0.1DL + Rod Circular Convex Entire Shiny White 1EL + Coccus Circular Convex Entire Shiny Yellow 0.1FL + Rod Circular Convex Entire Dull White 1

Thus, the distribution of bacteria isolates was obtained from different point indicated that there was active metabolism from microorganism in diesel contaminated area (Bhasheer et al., 2014). That was strongly suggested that the isolated bacteria from diesel contaminated area

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should be used to degrade diesel in seawater and soil because that bacteria isolates can utilize the diesel as carbon and energy source.

3.3. Screening of Diesel Degrading Bacteria

The capability of isolated bacteria utilizing diesel as carbon source and energy was identified by using screening method. Thirteen isolate bacteria were inoculated onto nutrient agar plate containing various diesel concentrations (0%, 5%, 10%, and 15% (v/v)). Every microorganism has different capability to degrade diesel depended on the condition, diesel concentration, and metabolism (Bhuvaneswar et al., 2012). The purpose of screening test was to select two bacteria with great growth in diesel contaminated nutrient agar medium. Two selected bacteria will be used in further diesel degradation test by using single or consortium culture. The result of screening test was determined by qualitative and quantitative analysis (Table 4 and Figure 1).

Table 4. Bacteria growth in various diesel concentration after 48 hours

Bacteria code

Diesel Concentration (%)0 5 10 15

Soil SamplesAT ++++ ++++ +++ +++BT + + - -CT ++++ ++++ ++++ +++ET + - - -FT +++ +++ ++ +GT + ++++ +++ +++HT ++ + + +

Seawater SamplesAL ++ +++ ++++ +++BL +++ + + +CL ++++ +++ ++++ +++DL ++++ ++ ++ ++EL ++++ ++++ ++++ ++++FL + + + -

Notes:+++++ : 81 – 100% growth without color changing++++ : 61 – 80% growth without color changing or 81 – 100 % growth with color changing+++ : 41 – 60% growth without color changing or 61 – 80% growth with color changing++ : 21 – 40% growth without color changing or 41 – 60% growth with color changing+ : ≤ 20% growth without/with color changing or 21 – 40% growth with color changing- : No growth

According to Table 4, the result of qualitative and quantitative analysis for screening test showed bacteria coded EL and CT are the most resistance strain and have highest growth in diesel contaminated medium. Growth percentage of bacteria coded EL and CT were about 61 – 80% and 41% - 60% in diesel contaminated medium without color changing. It was indicated that the capability of bacterial isolates utilizing diesel were different one another (Gayathri et al., 2014). Table 4 also showed that the higher diesel concentration in medium, the lower bacterial growth will be. It was caused by the toxicity of high diesel concentration that makes stress and shock on bacterial growth (Palanisamy et al., (2014); Bhasheer et al., (2014)). By

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using this method, it can be seen that microorganisms have greater growth when the concentration of diesel decreased. Microorganism have its own level of tolerance to diesel because diesel can be their carbon and energy source. In other hand, diesel can be toxic to microorganism.

Figure 1. Result of screening test for bacteria coded EL in various diesel concentration

The results of this study were compared with the previous studies. The result observed by Prakash et al. (2014) showed that there were 59 bacteria strains isolated from petroleum contaminated soil. Only Bacillus sp., Pseudomonas sp., and Micrococcus sp. that can grow in 1% of diesel and capable to utilize and to degrade the diesel. Mijahid et al. (2015) also said that twelve bacterial strains were isolated from petroleum contaminated soil. Only Pseudomonas sp. and Micrococcus sp. can good grow in nutrient agar medium containing 0.25% of hydrocarbon i.e. naphthalene, phenanthrene, biphenyl, anthracene, and xylene. Furthermore, the result observed by Bhasheer et al. (2014) showed the five bacterial strains were isolated from diesel contaminated soil and only Staphylococcus and Pseudomonas were efficiently grow in 10% (v/v) of diesel.

Morphological characteristic of bacteria coded EL and CT were gram positive bacteria with colonies appeared circular, entire, shiny, convex. The difference between bacteria coded EL and CL were the colour pigment and shape. The colour pigment and shape of bacteria coded EL was yellow and coccus with irregular cluster. Meanwhile, the colour pigment and shape of bacteria coded CT was white and coccus with regular ‘grape – like’ cluster. According to Holt et al. (1994), morphological characterization of Micrococcus genus was gram positive bacteria; colonies appeared circular, entire, and convex; shaped coccus with irregular cluster; and the colour pigment was yellow. Furthermore, Holt et al. (1994) also said that the morphological characterization of Staphylococcus genus was gram positive bacteria; colonies appear circular, entire, and convex; shaped coccus with forming regular and ‘grape – like’ cluster; and the colo ur pigment was white.

Based on the result of morphological characterization, bacterial coded EL and CT were predicted as the genus of Micrococcus and Staphylococcus, because the shapes of both bacteria

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were coccus and included in gram positive bacteria. The identification of selected bacteria will be conducted in next step of research by using biochemical test based on Bergey’s Manual of Determination Bacteriology and biomoleculer.

4. Conclusion

It was concluded that diesel degrading bacteria can be isolated from diesel contaminated seawater and soil. In this study, thirteen bacteria strains were isolated which had the potential to degrade diesel. According to the result of screening test, bacterial coded EL and CT were the most resistance strain and showed highest growth in diesel contaminated medium. Those bacteria were predicted as the genus of Micrococcus and Staphylococcus. Both of them have a potential to be used as diesel degrader bacteria in soil or seawater bioremediation.

Acknowledgement

The authors would like to thank Kemenristek DIKTI through skem of Penelitian Unggulan Perguruan Tinggi of PUPT 2017 No. 010/SP2H/LT/DRPM/IV/2017 for funding this research.

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