bioremediation of crude oil contaminated sediment using slow release fertilizer: hydrocarbonoclastic...

ILMU KELAUTAN. Februari 2010. Vol. 2. Edisi Khusus: 462-476____________________ISSN 0853 - 7291

www.ik-ijms.com Diterima / Received: Januari 2010 © Ilmu Kelautan, UNDIP Disetujui / Accepted: Februari 2010

Bioremediation of Crude Oil Contaminated Sediment Using Slow Release Fertilizer: Hydrocarbonoclastic Bacteria

Population Dynamics

Yeti Darmayati Microbiology Laboratory, Marine Dynamics Division

Research Center for Oceanography-Indonesian Institute of Sciences Contact email: [email protected]

Abstrak Penelitian lapangan untuk bioremediasi minyak dengan menggunakan Osmocote© sebagai biostimulator telah dilakukan di Pulau Pari, Kepulauan Seribu, Indonesia. Eksperimen ini dilakukan dengan mesokosm kolom pasir yang ditempatkan di zona pasang surut selama tiga bulan. Kondisi air laut pada air pori di dalam kolom, di dalam mesokosm dan sekitar mesokosm diukur sebanyak lima kali selama 0 hari – 3 bulan. Parameter yang diukur meliputi pH, Oksigen terlarut (DO), Oxidation-Reduction Potential (ORP), suhu dan and Electrical conductivity (EC). Dinamika populasi bakteri selama penelitian diamati dengan menghitung jumlah total sel bakteri dengan menggunakan metode AODC dan bakteri hidrokarbonoklastik dengan metode MPN. Bakteri Hidrokarbonoklastik dihitung secara terpisah untuk bakteri pendegradasi total minyak, alkana dan PAH. Konsentrasi minyak diukur dengan metode pengukuran menggunakan Infra Red (IR). Hasil penelitian menunjukkan bahwa Osmocote© mampu meningkatkan pertumbuhan bakteri, khususnya bakteri hidrokarbonoklastik. Persamaan dan perbedaan dapat teramati pada pola pertumbuhan ketiga kelompok bakteri pendegradasi minyak ini. Persamaannya adalah peningkatan pertumbuhan teramati sampai bulan ke-2, selanjutnya penurunan pertumbuhan terjadi pada bulan ke-3 eksperimen. Perbedaannya adalah bakteri pendegradasi PAH- dan alkana memiliki lag-phase, sedangkan bakteri pendegradasi total minyak tidak memiliki fase ini. Secara alami minyak mentah dapat didegradasi sampai 28 % pada bulan ke-3 setelah pencemaran minyak. Pupuk lepas lambat (Osmocote©) dapat meningkatkan degradasi minyak sejalan dengan peningkatan konsentrasi pupuknya (r2= 0.90). Penggunaan Osmocote© dengan konsentrasi sedang dan ulangan (setiap 2 bulan) memberikan hasil yang lebih baik daripada konsentrasi tinggi dengan satu kali aplikasi

Kata kunci: Bioremediasi, Minyak mentah, Sedimen, Pupuk lepas lambat, Bakteri Hidrokarbonoklastik, Dinamika populasi

Abstract

Field experiment for oil bioremediation by using Osmocote© as biostimulator has been conducted in Pari Island, Seribu Island waters, Indonesia. The experiment was conducted in the sand-column mesocosm placed in the intertidal zone for three months. Seawater conditions in pore water inside column, inside mesocosm and surrounding mesocosm were measured in five times between 0 day – 3 months. Parameters measured were pH, DO, Oxidation-Reduction Potential (ORP), temperature and Electrical conductivity (EC). Bacterial population dynamics were monitored by calculating the total number of bacterial cells using AODC method and hydrocarbonoclastic bacteria by MPN method during experiment. Hydrocarbonoclastic bacteria were calculated separately for total oil-, alkane- and PAH degrading bacteria. Oil concentration was measured by IR method. The result showed that Osmocote© was able to enhanced the bacterial growth, especially hydrocarbonoclastic bacteria. There was similarity and differences on the growth pattern of three oil degrading bacterial groups. The similarity is increasing growth rate until the 2nd months then declining growth rate in the 3th months of experiment. The

ILMU KELAUTAN. Februari 2010. Vol. 2. Edisi Khusus: 462-476 ___________________________________

Bioremediation of Crude Oil (Yeti Darmayati) 463

difference is PAH- and alkane-degrading bacteria had lag-phase, however, “total “oil degrading bacteria was not. Crude oil can be degraded in 28 % naturally after the 3rd months of crude oil contamination. Slow-release Fertilizer (Osmocote©) can increase oil degradation in accordance with fertilizer concentration (r2= 0.90). Application of Osmocote© in moderate concentration and repetition (in 2 month) created a better impact than high osmocote concentration in one time application.

Keywords: Bioremediation, crude oil, sediment, slow release fertilizer, hydrocarbonoclastic bacteria, population dynamics Introduction

Oil pollution is one of the most serious

threats for Indonesian marine waters. It is due to many offshore explorations, coastal oil refineries and a main route for oil world transportation. In addition, oil spillage from domestic waste introduced to the sea via river runoff is also may not be ignored. Therefore, it is important to find a method for reducing the impact of oil spills on marine life. An evidence of oil spill close to the coastline will always impact significantly living organisms compared with that observed in the open ocean. This is simply because coastal areas are habitats for very concentrated and diversified populations of marine life. Nevertheless, oil spills from Open Ocean does not only affect to marine organisms, but it can also end up and contaminate beaches hundreds of miles away. Oil spills can harm marine life in three different ways, by poisoning after ingestion, by direct contact and by destroying habitats.

Bioremediation is an alternative method that promising for cleaning up oil spills. Some research and application of bioremediation techniques in Indonesia have been conducted in soil environment (Sugoro and Aditiawati, 2003). However, bioremediation study in marine environment is still limited (Ruyitno, 1997) although some studies have showed that oil degrading acteria was available in Indonesian marine waters (Thayib, 1978; Feliatra, 1999; Darmayati, 2003; Darmayati 2008a; Darmayati, 2008b).

Bioremediation in marine environment, in general, limited by the supply of inorganic nutrients mainly nitrogen and phosphorus (Atlas and Bartha, 1972; Prince, 1997). Some studies conducted have proved that growth of oil-degrading bacteria and oil degradation can be strongly enhanced by fertilization with inorganic N and P (Brag et al. 1994, Swannell, et al., 1996; Röling et al., 2002; Boyd et al., 2007). Our previous study in laboratory showed that Osmocote©, slow-release fertilizer, enhanced oil degradation. However, the efficacy of this fertilizer in the field was still questionable for Indonesian marine waters. Therefore, the objective of this field experiment were to evaluate water quality dynamics in the porewater, to evaluate the effect of nutrient supply on oil degrading microbial communities, and to evaluate potential of slow release fertilizer on enhancing oil degradation in contaminated sediment. Materials and Methods

Experimental field

The column experiment was

conducted at intertidal zone of Pari Island reef flat in Jakarta Bay of Indonesia. The experimental field was situated about 1 km away from main land of Pari Island (Figure 1) with the tidal ranges about 1.2 m during high tide, whereas 0.3 m during low tide. Five concrete wells were constructed to protect the columns from direct sunlight and damage by sea waves. The height and inner diameter of wells are 1.7 m and 0.8


464 Bioremediation of Crude Oil (Yeti Darmayati)

m, respectively. The study was conducted from June to December 2008 .

For preparation of column, sand was collected from Pari Island seashore. After collection of the sand, it was sieved in range of 0.25 - 4 mm size and washing by seawater from surrounding.

Basic design and construction of columns

Columns was constructed with 6

segments of poly vinyl chloride pipe in diameter of 10.0 cm. Height of each column segment was 20 cm except top segment (50 cm). First, a glass filter and a punched flange were mounted at the bottom of the base column segment by bolts and screws. Then, base column segment was filled up with pre-treated sand and be saturated by adding seawater. Thereafter, intermediate column segments were connected followed by filling up with sand, sequentially. The top column segment left without sand was mounted with sand filled column segments. Finally,

the top opening of column was covered using a flange. Total 35 columns were constructed, which were labeled as of Table 1. Thirty columns were constructed for sand sampling and other 5 columns for pore water sampling. Five different treatments were imposed on sand sampling column with 6 replicates. Different replicate columns with same treatment were used to collect soil sample at different sampling time (Table 1). Five pore water sampling columns were also imposed with different treatments like soil sampling column. For pore water sampling plastic tubes were installed in each segment. In one of the columns were installed a punched poly vinyl chloride pipe to measure the water level in the column. Sand filled column segments are labeled as P-1, P-2, P-3, P-4 and P-5, sequentially from base column segment to upper segments. Thermo sensors (MDS-MKV/T, Alec Electronics Co. Kobe, Japan) were installed at each segments of column labeled with T1S6 to measure sand soil temperature.

Figure 1. Location of the experimental field



Table 1. List of columns with various different treatments and sampling time

Column ID

Treatments Sand sample Porewater sample Crude oil (g)

Fertilizer No. of column

Sampling Schedule

No. of column

Sampling Schedule

Amount Application schedule

Case-A 200

0 - 6 0 10 days 1 months 2 months 3 months

1 0 10 days 1 months 2 months 3 months

Case-B 2g x 2 0 and 60 days

6 1

Case-C 5 g x 2 0 and 60 days

6 1

Case-D 30 g x 2 0 and 60 days

6 1

Case-E 60 g x 1 0 day 6 1 Crude oil = Arabian light crude oil (ALCO) Fertilizer = Osmocote @ (14 -14-14)

Installation of columns in wells Six columns for sand sampling and one

column for pore water sampling with same treatment were installed in one well as shown in Figure 2 and Table 1. For mechanical support and provide dark condition to the columns, interstitial blank spaces were filled up with small sand bags in

the wells. The bottom column segment (P-1) was always submerged by tide level. Whereas, the column segment P-5 was only submerged during the highest tide and P-4 during high tide. P-4 was the highest port that always get a full tidal cycle everyday. Therefore, P-4 has been chosen as our samples during 4 months study to represent a tidal beach.



Figure 2. Arrangement of each column in the wells

Figure 3. Arrangement of each segment in the columns. Fertilizer, oily sand and sand only were presented in orange, dark brown and brown color respectively. Components of treatments

Arabian light crude oil was used as a source of oil contamination in sand. 200 g

ALCO was applied at the top of the sand column. On the other hand a slow release fertilizer Osmocote© (14-14-14) has been applied as biostimulator for indigenous microbes in beach sand. Basal dose of



fertilizer was applied at the top of sand columns except Case E where total amount (60 g) of fertilizer was mixed with sand of P5 segment during construction of columns. Rest amount of fertilizer was applied on the top of the sand column according to the schedule as shown in Table 1. Composition of Osmocote14-14-14 is shown in Table 2.

Table 2. Nutrient composition of slow

release fertilizer osmocote 14-14-14

Component Quantity (%) Total nitrogen (T-N) 14.0 Ammoniacal nitrogen (NH3-N)

8.2

Nitrate nitrogen (NO3-N)

5.8

Available phosphate (P2O5)

14.0

Soluble potash (K2O)

14.0

Preparation of sand soil samples

Columns were taken out from the wells at 1 day, 10 days, 1 month, 2 months and 3 months. They were carried to the laboratory of Indonesian Institute of Sciences (LIPI) at Pari Island. Each column segment was separated and kept in the cooler box (Fig.3). Column segments were transported to laboratory of LIPI-RCO at Jakarta for further preparation and analysis.

Figure 4. Column segment separation

The column segments has been opened and dispensed sand on bowl made of stainless steel and mixed vigorously by a stainless steel made scoop. Thereafter, sand will be divided in to two parts for each column segment and preserved in two polypropylene bottles at 4oC temperature for further analysis. Measurement of parameters related with sand Bacterial counting

Subsample sediments from each column at P-4 segment were processed for most probable number (MPN) analysis of PAH, alkane and oil degrading bacteria by using separate 96-well microtiter (Wrenn and Venosa, 1996). In addition, total cell bacterial number was counting by Acridine Orange Direct Count (Hobbie et al. 1977). Sediment sample was prepared by mixing 1 g wet weight of sediment into dilution water containing 9 ml seawater sterile. This solution was then placed in vortex at 300 rpm for 15 minutes to detach bacteria from sediment.

Since the MPN procedure is considerably determined by the substrate used to bacterial growth, several carbon sources as substrate were applied to evaluate the number of total oil-, alkane- and PAH-degrading Carbon source for PAH degraders, alkane degraders, and oil degraders were PAH mixture (10 µL/well), n-hexadecane and Arabian Light Crude oil (each 5 µl/well), respectively. PAH mixture consisting of (10 g phenanthrene (95%)/L, 1 g anthracene (99%)/L, 1 g fluorene (99%)/L and 1 g dibenzothiophene (97%)/L in hexane) and n-hexadecane (99%) sterilized by filtration, while crude oil was sterilized using autoclave. Chemicals used were obtained from Wako Pure Chemical Industries, Ltd. Media of the



bacteria was previously-filtered and sterilized seawater collected from P Pari, and it was subsequently added with N and P (180µL/well). PAH degraders were incubated in 29oC for 3 weeks, while the other degraders were incubated for 2 weeks. INT (Iodonitrotetrazolium violet) was added to alkane- and oil- degrading wells to identify positive wells (Haines et al., 1996). PAH degraders will exhibit yellow color when bacteria show positive result. Calculation data of MPN result was using a computer program (Klee, 1993). Total hydrocarbon measurement

Sample was prepared by taking 5 g of 3 subsamples from P-4 sediment samples. Oiled samples were extracted at least twice with 977 solvent (solvent provided by HORIBA) Water was absorbed with Na2SO3. Total petroleum hydrocarbons (TPH) in contaminated sediment were measured by IR method using HORIBA oil content analyzer OCMA-355.

Water sample collection

Water samples have been collected from outside each well, inside each well and inside column (pore water) at high tide during sediment sampling. 50 ml of pore water samples were collected from each column at P-4 segment by syringe. Sampling was conducted cautiously to avoid oxygen contamination. The electrical conductivity (EC), dissolved oxygen (DO), pH and oxidation-reduction potential (ORP) were measured immediately after sampling by using EC meter DO meter , pH meter and ORP meter, respectively.

Result and Discussion Environmental condition

Seawater quality monitored during bioremediation experiments in outside of mesocosm was relatively constant such as EC, DO and pH, except for ORP value (Table 3). The average value of EC, DO, pH and ORP were 4.4 S/m, 5.8 mg/L, 8.1 and 106.8 mV, respectively. This was due to protection mesocosm system from sea wave of outside and that of oil leakage from the columns. And hence produce the stability of water quality within inside the well compared with the outside. Furthermore, only minor contamination of fertilizer on the seawater quality inside wells was observed.

Zhu et al. (2001) reviewed that salinity of the environment is an important factor in oil bioremediation, particularly in estuarine environments or in marine shorelines where regional seaward ground water flow exists. Changes in salinity may affect oil biodegradation through alteration of the microbial population. The salinity can be measured by a conductivity or density methods. In this study, the EC value in pore water during experiment showed relatively stable and similar with seawater (Table 3), except in Case-E (Fig. 5). It was indicated that seawater salinity during experiment was stable, it may caused by a minor supply of fresh water into the environment. Study site was located in pseudo-atoll without any river, about 1 km from Pari Island (Fig 1). The EC value in Case-E was abnormal, it may come from human error on the measurement; there was no agreement with the other related data.



Table 3. The quality of seawater monitored inside and outside wells during 3 months of experiment.

Parameter Range Average + SD

Out side Mesocosm EC (S/m) 3.7 - 4.6 4.4 + 0.3

DO (mg/L) 4.4 - 7.4 5.8 + 0.1 pH 8.0 - 8.2 8.1 + 0.1 ORP (mV) 52 – 181 106.8 + 49.5

Inside Mesocosm EC (S/m) 3.9 - 4.6 3.5 + 1.5

DO (mg/L) 1.9 - 7.3 3.5 + 3.5 pH 7.7 - 8.1 7.9 + 0.1 ORP (mV) 30 – 191 110.1 + 63.0

Figure 5. Effect of fertilizer application on pore water quality (EC, pH, DO and ORP) during 3

months bioremediation experiment.



The pH value in seawater is generally stable and slightly alkaline. Study has shown that degradation oil increase with increasing pH, and that optimum degradation occurs under slightly alkaline condition (Dibble and Bartha, 1979). In this study, the process of oil degradation slightly changed the pH value in the pore water of all experiment. The pH value declined may caused by CO2 and acid metabolites production during oil mineralization by microbes. The declining of pH value in the case A was apparently had slower rate than the others. It was indicating that degradation rate of oil in case A was lower than others. Besides, Osmocote© has successfully increased oil degradation rate and finally reduced the time for remediation. It was confirm by a good correlation between pH and oil reduction (r2 = 0.91).

Aerobic conditions are generally considered necessary for extensive degradation of oil hydrocarbons in the environment since major degradative pathways for both saturates and aromatics involve oxygenases (Zhu et al, 2001). However, in the last decade, it has become abundantly clear that the anaerobic biodegradation of hydrocarbons is also widespread phenomena (Atlas and Philp, 2005). During this study, oxygenase enzymes play an important role in the early steps. After that, anaerobic biodegradation has been observed to take a part since 10 days of experiment. Anoxigenic condition occurred in treated column faster than control. It may caused by stimulation bacterial growth by osmocote© affected the number of bacteria and their oxygen consumption.

It was in a good agreement with oxygen availability, ORP value also has been decreased since 10 days of experiment and sharply dropped at the 26th day (Fig. 4). It indicated that biodegradation mostly may undergo in reducing condition. Atlas and Philp (2005) mentioned that small water-soluble aromatic compounds such as benzene and toluene have been shown to under take

biodegradation under sulfate-reducing, nitrate-reducing, perchlorate-reducing, ferric ion–reducing and methanogenic condition. The lag time on reaching reduction condition was observed in control and treatment B. It was also may caused by oxygen availability. The ORP value was observed to rise at Case D. It may indicated that recovery condition will begin caused by substrate (oil) concentration has been finish.

Microbial population dynamics

Total Bacterial cell counted no specific bacterial groups; it is included all kinds of bacterial cells. Therefore the number can be kept increase due to many bacterial group in the environment still be alive as long as their nutrient and environment condition is favorable (Fig. 5). The addition of fertilizer (Osmocote) have been enhanced the growth of bacterial cells. It was supported by the data of bacterial density in control during experiment which was lower than the others. The density was only (2 – 3) x 107 cells/g. In the other hand, the density of treated columns at the end of experiment reached (2 – 3) x 108 cells/g. The lag time was observed at the early stage in all experiments; however, it seemed that the control (Case A) and Case B need more time for adaptation which was about 1 month (Fig. 6). It may due to the limitation of nutrient.

Figure 7 summarized the result of the oil-, alkane- and PAH- degrading bacterial population data for all sampling events. The oil-degrading bacteria were increased their population since the beginning until the 2nd month of experiment in all columns. The initial density was about 2 x 102 MPN/ g wet weight sand. At the second month, the density in each treatment reached 108 MPN/g wet weight sand and then decreased at the end of experiment into 106 MPN/g wet weight sand. It was different phenomenon occurred on control. The growth steadily increased until



Figure 6. Effect of oil and fertilizer addition on bacterial total cell number during 3 months bioremediation experiment

the 3rd months; however, the rate was slower than others. The density of 108 MPN/g wet weight sand just been achieved at the end of experiment (3rd month). This suggests that oil and the fertilizer synergistically give a positive impact on the growth of oil-degrading bacteria. The concentration of fertilizer seemed give a different impact, however, it was only on the early stages.

The alkane-degrading bacterial populations increased coincide with time until the 2nd months of experiment in all columns. The population then decreased at the 3rd months of experiment, except in the Case-D. The alkane-degrading bacteria required a lag phase in their growth, especially when there was no fertilizer addition (Fig 7). The phase was observed on the 10th days of experiment.

The PAH-degrading bacterial population was found in very limited number. The initial density was 2 – 8 MPN/g wet weight sand. The highest density was achieved mostly at the 2nd months of experiment, except in the Case-D. The lag phase was also observed in different time at different experiment. It may caused by the differences of nutrient and C source availability.

Although the number of total cell bacteria was higher than oil degrading bacteria, the growth rate of total bacteria was lower than crude oil and alkane degrading bacteria. In addition, total bacterial growth rate tend to decrease coincide with time. The average value of µ (growth rate coefficient) for total cell bacteria was 0.042, whereas, the value of oil and alkane degrading bacteria were 0,169 and 0,068 respectively. Initial density of total cell bacteria was in the range of 2 – 4 x 107 cells/g and the density at the end of experiment was in the range of 0.6 – 2 x 108 cells/g. The initially density of oil and alkane degrading bacteria were 13 – 23 MPN/g and 9 – 32 MPN/g. The highest density of both group of bacteria were 0.02 – 200 x 107 MPN/g and 0.2 – 1 x 107

MPN/g.

The growth rate of total bacteria correlated positively with the amount of fertilizer added at 1st month (r = 0.50) and 2nd months (r = 0.99) of experiment. However, it was negative correlation observed at the end of experiment (r = - 0.60). It showed that osmocote © released N and P slowly and the effect can be detected at the first and second months of experiment



Figure 7. Effect of fertilizer on crude oil, alkane and PAH degrading bacteria during 3 months experiment . There was repetition in the same amount of fertilizer application to the C column at the second months. However, the impact in the third months was not similar with the first month of experiment. It may caused by the C source was not as much as before and the micro environment was also different (Figure 6).

Similar with some previous studies (Atlas and Bartha, 1972; Prince and Atlas, 2005), in this experiment showed that crude oil degrading bacteria in oil unpolluted sand was available in very small portion (4 – 9 x 10-5 %) . This bacterial group may increase into 2.91% in sand beach after the 3rd month of oil application (Table 4). Osmocote©

gave positive impact on oil degrading bacterial growth. It enhanced the proportion into 16 – 21 % in low concentration and 34.3 % in moderate and 34.8% in high concentration. It answered why oil degradation in moderate and high concentration of osmocote© application (Case D and E) showed a higher rate than low concentration.

There was similarity and differences on the growth pattern of three oil degrading bacterial groups. The similarity of crude oil, alkane and PAH degrading bacteria was increasing growth rate until the 2nd months then declining growth rate in the 3th months of

Table 4. Percentage of oil degrading bacteria during bioremediation experiment

0 d 10 d 1 month 2 months 3 months Case-A 0.00005 0.00019 0.00149 0.16159 2.91223 Case-B 0.00009 0.00027 0.00402 21.02779 0.52739 Case-C 0.00006 0.00018 0.00621 16.56735 0.96405 Case-D 0.00004 0.00171 0.00302 34.33365 1.12238 Case-E 0.00006 0.00320 0.00170 34.81487 0.91886



experiment. The growth pattern differences of crude oil and alkane degrading bacteria was in the 10th day of experiment. Alkane and PAH degrading bacteria need adaptation for growing but the other was not; it was indicated by declining slightly of the MPN of alkane and PAH degrading bacteria at the 10th days after fertilizer application.

Crude oil degrading bacteria was positively correlated with alkane and PAH degrading bacteria. However, the correlation was stronger with alkane (r = 0.92) than PAH degrading bacteria (r = 0.50). This phenomenon has been reported by Wrenn and Venosa (1996). In addition, alkane degrading bacteria was available in closer number with crude oil bacteria. It showed that alkane degrading bacteria dominated the group of oil degrading bacteria, whereas, PAH was only available in small number. The number of alkane degrading bacteria was in the range of 8 x 100 – 2 x 107MPN/g (average = 2 x 106 MPN/g) and PAH degrading bacteria was in 1.8 – 20 MPN/g (average = 10) (Figure 6). It may cause alkane is easier to be degraded than PAH in the environment. Moreover, the structure complexity of alkane is also simpler than PAH. Van Hamme et al (2003) reviewed that there was variability in both regulation and clustering of alkane degradation genes between species as well as the realization that a single strain may carry multiple genes that code for different enzymes carrying out similar function.

Maximum growth of PAH, alkane and total oil degrading bacteria was observed at different time. Based on the maximum of µ (growth coefficient) average value, PAH degrading bacteria was occurred at the 2nd months (µ = 0.052) , alkene degrading bacteria at the 1st month (µ = 0.289) and crude oil degrading bacteria at 2nd month (µ = 0.312).

Fertilizer impact on oil degrading bacterial group was observed differently in time also. The strongest positive correlation between fertilizer amount and

crude oil degrading was observed at the 10th day sampling (r = 0.97). However, the strongest positive correlation for alkane and PAH degrading bacteria was in the 3rd months (r = 0.65) and the 1st months (r = 0.82) respectively. It indicated that oil degrading bacteria in general was sensitive on nutrient supply. Effect of fertilizer on oil degradation

This result indicated that Osmocote © fertilizer increased sufficiently oil degradation (r = 0.90). It was about 28 % of crude oil degraded naturally after the 3rd months. However, it can be accelerated between 33 – 50% with the fertilizer treatment (Figure 8). Degradation rate average in control was 0.65 g/day, whereas in fertilizer treatment was 0.99 – 1.56 g/day. The highest rate average of oil degradation was occurred at the first 10 day after treatment (3.80 g/day). This optimum growth and subsequent enhancement of enzymes production to degrade crude oil may be caused by the favorable environment condition in the early stage such as nutrient and oil availability.

Periodic addition of fertilizer in 30 g for 2 months showed a better impact on enhancing oil degradation than single application in 60 g of Osmocote ©, especially at the 2nd month and the 3rd months of experiment (Figure 9). It can be indicated in Case D by the higher degradation rate of 0.22 g/ day and 0.83 g/day at the 2nd month and the 3rd months of experiment, respectively. In the Case E, the rate increased 0.15 g/ day and 0.67 g/day. By time expanse, it is possible to remove oil. Based on linear regression, removal oil is predicted to reach 319 days for A, 192 days for D and 221 days for E. The prediction for Column C and B was not available, due to correlation.



Figure 8. Effect of fertilizer on residual oil in bioremediation process during 3 month experiment

Figure 9. Trend line of oil degradation in different fertilizer application



Conclusion

Oil degradation introducing fertilizer seemed to affect the water quality dynamics in pore water, except on EC parameter. Application of fertilizer has increase nutrient availability, therefore increase the growth of total bacteria and oil degrading bacteria in early stages of bioremediation processes. Regular addition of fertilizer would suggest the enchancement of oil degradation. Acknowledgment

I gratefully acknowledge the funding

of this work by NEDO – Japan and the assistances of Joint Project Committee PA-2 LIPI-NITE, Dr. Yoh Takahata and Dr. B. Kumer Mitra from TAISEI, Dr. Shigeaki Harayama and Dr. A. Yamazoe from NITE, Drs. Djoko H. Kunarso, MSc, Prof. Ruyitno MSc, all Microbiology Lab. Member of Research Center for Oceanography-LIPI and UPT P.Pari -LIPI Staff . References Atlas, R.M., & R. Bartha. 1972. Degradation

and mineralization of petroleum in seawater: limitation by nitrogen and phosphorus. Biotech. Bioeng. 14:309-318.

Bragg, J.R., R.C. Prince, E. J. Harner & R.M. Atlas. 1994. Effectiveness of bioremediation for the Exxon –Valdez oil-spill. Nature, 368:413-418.

Darmayati, Y. 2003. Marine oil degrading bacteria: the distribution in Makasar Strait, In D. Natalia (ed.), Proceedings of the Annual Scientific Meeting Indonesian Society for Microbiology 2003, Indonesian Society for Microbiology, Jakarta (In Indonesia). p: 555-562.

Darmayati, Y., S. Harayama, A. Yamazoe, A. Hatmanti, Sulistiani, R. Nuchsin & D.H. Kunarso, 2008 a,

Hydrocarbonoclastic bacteria from Jakarta Bay and Seribu Islands. Mar. Res. Indonesia, 33(I):55-64.

Dibble, J.T. & R. Bartha. 1979. Effect of environmental parameters on the biodegradation of oil sludge. Appl. Environ. Microbiol., 37:729 – 739.

Feliatra. 1999. Isolation, identification and biodegradation of petroleum by bacteria in the Malaka Strait. In S. Soemodihardjo, R.R. Satari and S.Saono (ed.), Proceedings of the First Indonesian Seminar on Marine Biotechnology ’98, Indonesian Institute of Sciences, Jakarta (In Indonesia).p: 291-303,

Haines.J.R., B.A.Wree., E. Holder, E.L., Strohmeir, K.L., R.T.Herrington, & A.D.Venosa. 1996. Measurement of hydrocarbon–degrading microbial population by 96-well most-probable- number procedure. J. Ind. Microbiol., 16(1):36-41.

Hobbie, J.E., R.J. Daley & S. Jasper, 1977, Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl. Environ.Microbiol., 3:1225–1228.

Klee, A.J. 1993. A computer program for the determination of most probable number and its confidence limits. J. Microbiol. Methods. 18:91-98.

Prince, R.C. 1997. Bioremediation of marine oil spills. Trends Biotecnol. 15:58-160.

Prince, R. & R.M. Atlas., 2005. Bioremediation of marine oil spills. (Eds.: R.M. Atlas and J. Philp). In Bioremediation: Applied Microbial Solutions for Real-world environmental Cleanup. ASM Press. Washington D.C. p. 269–292.

Röling, W.F.M., M.G. Milner, D.M. Jones, K. Lee, F. Daniel, R.J.P. Swannell, & I. M. Head. 2002. Robust Hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced



oil spill bioremediation. Appl. Environ.Microbiol., 68:5537-5548.

Ruyitno. 1997. The effect of oil & fertilizer on the growth of microorganisms. In Results on Oceanographic Research in 1996 – 1997. Research Center for Oceanography-LIPI, Jakarta. (In Indonesian). 123p

Sugoro I. & P. Aditiawati, 2003, Analysis of oil sludge biodegradation by hydrocarbonoclastic bacteria using land farming technique in laboratory scale. In D. Natalia (ed.), Proceedings of the Annual Scientific Meeting Indonesian Society for Microbiology 2003, Indonesian Society for Microbiology, Jakarta (In Indonesia).p. 530-536,

Swannell, R.P.J., K. Lee & M. McDonagh. 1996. Field evaluations of marine oil spill bioremediation. Microbiol. Rev.,. 60:342-365

Thayib, S.T., 1978. Notes on hydrocarbonoclastic microorganisms from inshore and offshore waters in Jakarta Bay, Oseanology di Indonesia, 10:1-7. (Indonesian).

Van Hamme, J.D., A. Singh & O.P. Ward. 2003. Recent advances in petroleum microbiology, Microbiol. Mol. Biol. Rev., 67:503-549.

Wrenn, B.A. & A.D.Venosa. 1996. Selective enumeration of aromatic and aliphatic hydrogen degrading bacteria by a most-probable-number procedure. Can. J. Microbiol., 42:252-258.

Zhu, X., A.D. Venosa, M.T. Suidan., & K. Lee. 2001. Guidelines for the bioremediation of marine shorelines and freshwater wetlands. U.S. EPA. Cincinnati. 156 p.

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