Isolation of marine hydrocarbonoclastic bacteria from petroleum contaminated sites in Dumai

. The Dumai Sea is vulnerable to pollution, especially oil spills. Oil pollution in Dumai seawater causes a decline in the population of aquatic organisms. Isolation and screening of marine hydrocarbonoclastic bacteria is one effort to find potential local agents. This research aims to isolate marine hydrocarbonoclastic bacteria from the Petroleum Contaminated Site in Dumai. This research uses a survey method, and it was in four stages such as, isolation, screening, morphological and physiological characteristics, bacterial identification, and data analysis. Water samples were taken using purposive sampling at seven different locations. The samples were analyzed in situ, including water characteristics, pH, temperature, salinity, and brightness. Furthermore, the water samples were further analyzed at the Marine Microbiology Laboratory, University of Riau. Isolation and screening results showed that ten isolates of marine hydrocarbonoclastic bacteria can grow in SSMS liquid media with a petroleum content of 5%. The ten isolates in sequence are DS21a, DS22b, DS42c, DS52d, DS61e, DS62f, DS63g, DS71h, DS72i and DS73j. The characterization and identification results obtained four different genera from petroleum-contaminated sites in Dumai, sequentially Micrococcus, Rhodococcus, Marinobacter , and Bacillus .


Introduction
The Dumai Sea is a coastal area on the east coast of Sumatra Island, which directly borders the Malacca Strait.The Dumai Sea has a strategic location and is an international trade route.In addition, activities around the Dumai sea are also diverse, such as mining, processing and distribution of petroleum, palm oil (CPO), coconut oil (VCO), tourist areas and residential areas [1].A research report that the pollution index in Dumai seawaters was categorized as heavily polluted [2].It is also in accordance with the Decree of the Minister of State for the Environment No. 51 of 2004 regarding the pollution in Dumai waters, especially oil, has exceeded the specified threshold, namely <5 ppm.
In time, efforts to control the number of pollutants in the Dumai seawaters are not easy to solve.It is due to the large area polluted and the high costs required in the recovery process.
Therefore, the research for potential bacteria that can release the oil spills in the sea be an alternative.In the other hand, the use of bacteria in bioremediation technology has been carried out, and it still uses bacteria resulting from Genetically Modified Organisms (GMO) [3].Whereas, the indigenous bacteria are safer to use as bioremediation agents because they are able to produce extracellular enzymes that can modify the structure of pollutants into simple compounds that are safe in the environment [4].
In Indonesia, the implementation of bioremediation technology has been regulated and has standards.Especially in overcoming environmental problems due to mining, petroleum activities, and other forms of pollution.The Ministry of Environment for Ministry decision letter No. 128 of 2003 stated that the procedures and technical requirements for treating petroleum waste by bioremediation must use local (indigenous) microbes.It is because indigenous microorganisms are considered safer and more effective to develop.The development of bioremediation technology in reducing pollutant levels in waters can also use as an alternative to control pollution in the marine environment [3,5].
One type of microorganism can used as a bioremediation agent to control the oilcontaminated waste is marine hydrocarbonoclastic bacteria.An oil-degrading bacteria are a group of microorganisms that can utilize complex hydrocarbon compounds for metabolic processes and convert them into other compounds such as water and oxygen [6].Besides that, hydrocarbonoclastic bacteria can also oxidize aliphatic and aromatic hydrocarbon compounds by using several enzymes to break down carbon chains, such as monooxygenase and dioxygenase enzymes [7].Hydrocarbonoclastic bacteria are able to produce biosurfactant compounds and make hydrocarbon compounds to be the only source of carbon and energy [6,8].
In the water, several organisms produce biosurfactant compounds, including bacteria, microalgae, and fungi.Many research reports that marine bacteria have the potential to produced biosurfactants and degrade hydrocarbon compounds, such as Marinobacter spp [9], Rhodococcus erythropolis [10], Bacillus subtilis [11], and Oleispira antarctica [12].Thus, isolation and screening of hydrocarbonoclastic bacteria from petroleum-contaminated sites in Dumai can be found as new sources of biosurfactants and used to be degrading oil pollution in the sea.

Sampling location
The sample used in the research was sea water from petroleum contaminated sites in Dumai.Samples were taken from seven different stations with characteristics of locations that were oily/affected by oil spills.Then, water samples were put into sterile dark bottles at a depth of ± 10-15 cm from the sea surface.

Measurement of environmental conditions
The Measurements of environmental conditions were carried out by in-situ methods based on the physical and chemical aspects, such as temperature, pH, salinity and water transparency.

Enrichment and Isolation
The enrichment and isolation processes were carried out in Marine Microbiology Laboratory at Department of Marine Science Universitas Riau.A total of 10 ml of water sample was inoculated into 100 ml of liquid SMSS medium and then homogenized.Then, the samples were incubated at room temperature with stirring at 120 rpm for seven days.After the marine hydrocarbonoclastic bacteria grew, all isolates were cultured on solid SMSS medium to obtain pure bacterial cultures using the pour plate method.

Morphological and physiological characteristics
After the isolates were pure cultured, all isolates were characterized based on morphological and physiological characteristics.Morphological features observed, included colour, shape, size, elevation and edge of colony.Physiological properties were determined by Gram staining and biochemical assays medium, such as Triple Sugar Iron Agar (TSIA), Simmons's Citrate Agar (SCA), Sulfide Indole Motility (SIM), and Catalase Enzymes.

Identification of Marine Isolate
Determination of bacteria based on the results of morphological and physiological characteristics of isolates.All of isolate characters were then analyzed using the determination book "Bergey's Manual of determinative Bacteriology", and literature journals.

Environmental conditions
There were seven sampling locations with different characteristics which became isolation points for marine hydrocarbonoclastic bacteria, such as ship routes, oil ship docks, residential areas, marine tourism and mangrove areas.The selection of sample locations was determined based on several aspect including ship activities, community activities and the aquatic environment.Dumai is a sea transportation route that is traversed by small to large ships which cause oil spills along the ship route.Besides that, the activities of people living around Dumai and mangrove ecosystem are also a source of oil pollution.
Based on Figure 1.all sampling locations are close to the oil spill area.It was conducted because marine hydrocarbonoclastic bacteria live in areas contain high levels of hydrocarbon compounds.Oil-degrading bacteria are a group of bacteria that was able to utilize complex hydrocarbon compounds for metabolic process and then convert them into other simple compounds that are less toxic or non-toxic [13].Based on the results of environmental parameter measurements at the seven locations, it can be seen that there are differences in physical and environmental conditions, especially in the level of brightness (Table 1).Water brightness is a condition that shows the ability of light to penetrate the water layer at a certain depth.In seawaters, brightness is the main factor for photosynthesis and the primary productivity in a body of water.The measuring and taking brightness process was carried out in the morning to avoid significant changes at the sampling location.[10] stated that measuring the environmental temperature of waters is greatly influenced by the time of measurement.Therefore, to compare the physical and chemical conditions of the seawater, the measurement time can be determined in advance, such as in the morning, afternoon, evening or at night.Thus, the data obtained is more accurate.According to [14], a brightness under 30 cm indicates that algae blooming has occurred, while a brightness more than 40 cm is a low abundance of diatoms.However, the high brightness values at several sampling locations didn't cause of diatoms abundance but caused of high oil spills on the sea surface.As a result, sunlight did not enter the sea and a number of plankton are lost.Then, it has a negative impact on most marine biota, such as bacteria, plankton, small fish, big fish, shrimp and other organisms [15,16,17].
In other hand, another factor also shown different condition in several location.So, It was necessary to adapted the environment conditions of marine hydrocarbonoclastic bacteria before enrichment and isolation in the laboratory.

Identification of marine hydrocarbonoclastic bacteria
Identification of marine hydrocarbonoclastic bacterial cultures were shows in Tables 2 and  3.There are ten isolates with different morphological characteristics based on colony and cell, including colour, shape, size, elevation and edge.The colonies were found three colours, such as white, cream and yellow.Colony shapes vary, such as circular, irregular and filamentous.In terms of the size, colonies were found varied.Three isolates were largest, three were medium, three were small, and only one were punctiform.Elevation has three types, such as convex, flat and raised.The margins also have three shape, namely undulate, entire, and curled.The observed morphological characteristics indicate possible relationships among the bacterial isolates.Characteristics of the physiological properties of bacteria were performed to assist the process of determining marine hydrocarbonoclastic bacteria based on the book of Bergey's Manual of determinative Bacteriology.All isolates were categorized as Gram-positive bacteria after the isolates was conducted by Gram staining.There were obtained two shapes of bacteria cell including coccus and rod (bacilli).
The biochemical assays results showed that several isolates had the same physiological properties.However, the highest difference in biochemical test results was found in the TSIA (sugar fermentation test) and SCA medium, which showed the production of citrate, iron and H2S after all isolates were cultured within seven days.All isolates were motile, and it is cause they habitat in marine environment.
To ensure that ten isolates are capable of degrading oil, analysis of the growth ability of the isolates can used as an indicator.The growth medium used in the culture has been added 5% hydrocarbon compound.The research showed that ten isolates could grow but with different growth times.Some bacteria also produce pigments, including green, red, yellow, purple, and brown.According to [18], some hydrocarbons degradation can be oxidized under anaerobic conditions when some processing occur, such as nitrate and Fe +3 reduction [15], methane production [14], and bacteria photosynthesis with pigmen produce [1].Thus, several indigenous isolates from the Dumai seawater could degradated hydrocarbon compounds when grown on liquid SMSS medium through cell metabolism, pigmentation, and biofilm formation.Furthermore, all bacteria isolated from petroleum-contaminated sites in Dumai were identified based on morphological and physiological characteristics.Identification was referred to the Bergey Manual of Systematic Bacteriology [19].The results show that four genera of bacteria have similar types, such as Micrococcus, Rhodococcus, Marinobacter, and Bacillus.In addition, some literature reported that the two genera of hydrocarbonoclastic bacteria on the coast of Dumai were identified as Micrococcus and Bacillus.

Conclusion
Based on determination of "Bergeys Manual of Systematic Bacteriology" obtained four genera of bacteria from petroleum contaminated sites in Dumai sequentially, Micrococcus, Rhodococcus, Marinobacter and Bacillus.All of bacteria can growth in SMSS medium with 5% hydrocarbon compounds and some of them produce pigments.

Fig. 1 .
Fig. 1.Sampling location and measuring process of environmental aspects

Table 1 .
The environmental parameter measurement results

Table 2 .
Morphological characteristics of marine hydrocarbonoclastic bacteria

Table 3 .
Physiological characteristics of marine hydrocarbonoclastic bacteria