Abstract

IPTC 15316

Enhancing Bioremediation of Oily Waste by Bioaugmentation Juli Rusjanto, Gayatri Asmaradewi, Dian Safitri, Total E&P Indonesie, and Agus Jatnika, Bandung Institute of Technology

Copyright 2011, International Petroleum Technology Conference This paper was prepared for presentation at the International Petroleum Technology Conference held in Bangkok, Thailand, 7–9 February 2012. This paper was selected for presentation by an IPTC Programme Committee following review of information contained in an abstract submitted by the author(s). Contents of the paper, as presented, have not been reviewed by the International Petroleum Technology Conference and are subject to correction by the author(s). The material, as presented, does not necessarily reflect any position of the International Petroleum Technology Conference, its officers, or members. Papers presented at IPTC are subject to publication review by Sponsor Society Committees of IPTC. Electronic reproduction, distribution, or storage of any part of this paper for commercial purposes without the written consent of the International Petroleum Technology Conference is prohibited. Permission to reproduce in print is res tricted to an abstract of not more than 300 words; illustrations may not be copied. The abstract must contain conspicuous acknowledgment of where and by whom the paper was presented. Write Librarian, IPTC, P.O. Box 833836, Richardson, TX 75083-3836, U.S.A., fax +1-972-952-9435

Total E&P Indonesie (TEPI) has an ongoing bioremediation facility located in Senipah which is called IPOC (Industrial Process of Oily waste Composting). IPOC started its first phase in 2001 using drill cutting and contaminated soil with initial Total Petroleum Hydrocarbon (TPH) content maximal 15000 mg/kg, and currently it is being used to treat oily waste from production activities such as produced sand and sludge from emulsion treatment with initial TPH around 40000-60000 mg/kg using composting method. A study program has been developed to accelerate bioremediation process through the use of more selective microorganisms with identified capability to degrade hydrocarbon. The use of this technique are key strategy to minimize operating time and cost per unit of volume of oily waste to reach end point TPH criteria (10000 mg/kg) within less than 8 months as regulated. This paper presents the results of treatability tests designed for enhancing bioremediation process. Introduction Oil and gas production activities in TEPI generate hydrocarbon contaminated waste such as produced sand entrained in desander units at well platforms, sludge from vessel and tank cleaning, and residual solid decanter from emulsion treatment facility. These types of waste are categorized as hazardous waste and needs to be managed and treated properly according to prevailing regulations. Bioremediation using natural biological activity to degrade hydrocarbon is among the most useful and cost-effective method that is used for managing these types of waste.

Initiated as pilot project in 2001, IPOC has been used to compost drill cuttings and aged contaminated soil to reach end result of around 10000 mg/kg TPH content within 365 days, with removal rate of 65% - 75% (Chaineau, 2003). After demonstration of the successful pilot scale to authorities, the company has decided to enlarge the bioremediation facility to be used in industrial scale. Starting from 2006, IPOC has been used in industrial scale as licensed facility for biotreatment of hydrocarbon contaminated waste from oil and gas production activities of the company.

The evaluation of implementation of bioremediation process in IPOC after 4 years of implementation in industrial scale has resulted in decision to perform further study to optimize the hydrocarbon biodegradation process using bioaugmentation method, i.e. by addition of isolated and cultured microorganisms capable of biodegrading or transforming hydrocarbons to the treated media in order to achieve cost-savings and accelerate bioremediation.

A treatability study was conducted to evaluate the efficiency of cultured microorganisms to stimulate bioremediation of hydrocarbon contaminated waste. The microorganism had been isolated from the hydrocarbon contaminated waste. The rate of degradation of petroleum hydrocarbons by the indigenous microorganism and in the presence of cultured microorganism was assessed with the addition of bio-surfactant and various type of bulking agents for a period of about 70 days in laboratory.

Indonesian Regulation for Treatment of Oily Waste and Contaminated Soil According to the Ministry of Environment Decree No. 128 Year 2003 (MoE Decree No.128/2003) about Technical Guideline and Requirement for Biological Treatment of Oily Waste and Contaminated Soil, the treatment of oily waste can be conducted with biological method as one of several treatment options. Several allowed biological methods in treating oily wastes are landfarming, biopile, and composting. The technical guideline for biological treatment of oily waste and composting covers the technical requirement, analysis of treatment process, end criteria of treatment process, handling of treatment result, and monitoring and control of treatment result. All of those technical requirements are further specified in permit of biological treatment which shall be validated prior the beginning of treatment process.

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Before biological treatment could be conducted, the incoming oily wastes must be analyzed to show the waste composition and characteristics. The mandatory analyses consisting of TPH and heavy metal Toxicity Characteristic Leaching Procedure (TCLP) tests.

Certain requirements must be fulfilled for the waste to be treated. The maximum TPH concentration of the incoming waste shall not exceed 150000 mg/kg (before treatment process), the TCLP tests results shall fulfill the standards stated in Head of BAPEDAL Decree No. 04/Bapedal/09/1995, and any other requirements will specifically be reviewed according to the characteristics and composition of the waste. The treatment procedures could be either aerobic or anaerobic. However, there are several technical specifications that must be fulfilled, such as:

Added material used for mixing must not exceed 1:1 ratio between waste and materials. Bulking agents can be added to enhance porosity of the waste-and-material mixture. If the treatment process is aerobic, the aeration can be carried out by mechanical aeration or through perforating

pipes. Optimum humidity must be maintained with watering. Optimum pH (near neutral) must be maintained. Nutrients addition can be carried out, considering other factors, such as the possibility of other contamination to occur

and the appearance of certain odours. The microorganisms used to degrade the contaminants must not be of genetic-manipulation origin. Surfactants, if used, must be easily biodegradable and attached with Material Safety Data Sheet (MSDS). Leachate collected in storm basin could be circulated to help maintain moisture.

The effectivity of bioremediation process in reducing TPH shall be evaluated with maximum treatment process time of 8 months. If the treatment process exceeds 8 months, re-evaluation shall be carried out to improve performance of treatment.

The parameters which are required to be analysed during the process are described in Table 1.

Table 1: Treatment process parameters to be analyzed (MoE Decree No.128/2003) Parameters Frequency/ Sampling

Time

Sampling Technique Analysis Method

TPH Once every 2 weeks,

minimum during

process

- 2 composites Spectrofotometry

- 5 lateral points Gravimetric

- 3 vertical points for biopile or composting

BTEX* At the end of Process -See Above- Chromatography

TOTAL PAH** At the end of Process -See Above- Chromatography

Heavy Metals TCLP At the beginning and

the end of process

-See Above- TCLP extractor,

AAS*** * Benzene, Toluene, Ethylbenzene, Xylene **PAH : Polycyclic Aromatic Hydrocarbon ***AAS : Atomic Absorption Spectrofotometry

In order to ensure bioremediation process occurs effectively, some parameters are required also to be monitored i.e. soil

moisture, nutrient content (NPK) and bacterial counts. End product shall fulfill the requirement listed below.

Table 2. End point criteria of biological treatment of oily waste (MoE Decree No.128/2003) No Parameters Unit Standard

A. Waste Analysis

1 PH - 6-9

2 TPH (mg/kg) 10000

3 Benzene (μg/g) 1

4 Toluene (μg/g) 10

5 Ethylbenzene (μg/g) 10

6 Xylene (μg/g) 10

7 Total PAH (μg/g) 10

IPTC 15316 3

B. TCLP Analysis

1 Pb mg/L 5

2 As mg/L 5

3 Ba mg/L 150

4 Cd mg/L 1

5 Cr mg/L 5

6 Cu mg/L 10

7 Hg mg/L 0.2

8 Se mg/L 1

9 Zn mg/L 50

The end product of the bioremediation treatment process must be managed after the treatment process is conducted.

Several management options of the final result of bioremediation treatment process are listed below.

Table 3: Post-treatment management options (MoE Decree No.128/2003)

No. TPH Concentration Management Option Note

1. > 20000 mg/kg Continue treatment process Until standard has been fulfilled

2. 10000 – 20000 mg/kg Landfill According to Head of BAPEDAL Decree No.

04/Bapedal/09/1995

3 ≤ 10000 mg/kg Removal to limited area Planted with non-consumable vegetatation

Utilization Mixing material for road or building construction

Bioremediation Operation in IPOC The method used for bioremediation in IPOC is composting using windrows. Windrow piles are set up from oily waste mixed with clean soil or re-used treated soil and bulking agent (rice hulls) with maximum composition of 50 : 50. Fertilizers are supplied to the mixture to obtain C : N : P ratio of 100 : 10 : 1. Piles with volumes between 100 – 200 m3 are disposed on a concrete base on Treatment Area. The height of piles is maintained at 1-1.2 m. Oxygen supplies are maintained with regular mixing once per week, and optimum soil moisture (15-18%) is maintained by watering the piles or covering the piles with terpauline during rain to prevent leaching. Two concrete-based storage pits with 200 m3 capacity are used for temporary storage of incoming waste. The facility is completed with drainage system which routes rainwater from the area to storm basin where the effluent is regularly controlled (Figure 1). The facility is also completed with monitoring wells upstream and downstream of the facility which is also monitored regularly to ensure that there is no contamination to groundwater.

Figure 1. IPOC Bioremediation Facility

Monitoring of bioremediation process are performed as per below method and schedule.

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Table 4. Bioremediation monitoring method and schedule

Parameter Method Beginning

Stage

Weekly

Basis

Biweekly

Basis

Quarterly

Basis

End of

Process

TPH IR Spectrofotometry (US EPA SW-846) • • •

Soil Moisture ISRIC Technical Paper 9 2002 • • •

pH ISRIC Technical Paper 9 2002 • • •

TCLP Heavy

Metal

AAS (US EPA 1311) • •

N, P

K

ISRIC-6 Ed (2002)

APHA 3120B (2005)

• •

Microbiology Plate Count (SNI 19-2897-1992 point 1.1) • •

BTEX Chromatography •

Total PAH Chromatography •

LD50 Bio-assay •

Each windrow is sampled in 5 sampling points lengthwise to obtain 3 composite samples at 3 depths (0-20 cm, 60-80 cm,

100-120 cm) and 1 composite sample for all depths. Therefore there are 4 samples taken for each windrow. After TPH reaches 20000 mg/kg, the windrows are moved to maturation area located on compacted clay with maximum

permeability of 10-5 cm/s and monitoring is continued until the TPH of 10000 mg/kg is reached. Windrows which has reached 10000 mg/kg TPH is then moved to dedicated controlled area in Senipah and re-used as top soil for replantation or re-used as mixing material for subsequent composting process.

Between 2006 - 2010, approximately 2000 m3 of oily waste have been treated in IPOC. Average initial contents of TPH in the windrow piles were between 29000 mg/kg to 56000 mg/kg (48000 mg/kg as average). The result of initial TPH and TCLP test conducted prior starting bioremediation process has shown that all parameters fulfilled the standard limit as regulated in MoE Decree 128/2003.

The performance of TPH degradation of several windrows in IPOC (2007 – 2008) is shown in Figure 2 below.

Figure 2. Biodegradation of hydrocarbon over time of several windrows in IPOC (2007 – 2008)

The figure above indicates that the time required to reach end point criteria (<10000 mg/kg TPH) varied between 270 - 800 days. The removal rates for TPH were from 78% to 85%. The analysis result of TCLP, pH, BTEX, and PAH conducted at the

IPTC 15316 5

end of bioremediation process have shown that there is no parameter which exceeded the standard limit as regulated in MoE Decree 128/2003.

The result of bioremediation above has shown that although the end result criteria of 10000 mg/kg TPH content was achieved, the duration of the process sometimes took longer than 8 months which is the time limit set by MoE Decree No.128/2003, therefore further analysis and evaluation to optimize bioremediation process was required to be conducted.

Process Optimization: Bioaugmentation Study In order to improve the bioremediation process in IPOC, research on the process of bioaugmentation has been conducted with cooperation of university (Bandung Institute of Technology, ITB). Some studies performed by ITB have successfully isolated local bacteria capable of degrading oil. This bacteria has been tested and posses a high activity in degrading crude-oil. The objective of the study is to improve the biodegradation process by using more selective microorganisms with identified capability to degrade hydrocarbon in order to accelerate bioremediation process to minimize operating time and cost per unit volume of waste. The works carried out consisting of: Identification of naturally existing microorganisms and major contributing microorganisms for hydrocarbon degradation

in the field. Isolation of the major hydrocarbon degrading microorganism from oily waste and treated soil in IPOC area. Culture and acclimatization of the isolated microorganisms in large quantity Determination of the performance of isolated petroleum degrading microorganisms for bioremediation activity in

laboratory scale. Performing analysis of hydrocarbon degradation rate by using isolated microorganism including analysis on finger print of

treated soil using GC-MS (beginning process, middle and end of process), n-C17/pristine and n-C18/phytane ratio, extent of biodegradation and other related parameters.

Methods Isolation of Petrophilic Bacteria The process of isolation of bacteria capable of using crude oil as carbon source was performed by taking inocula sources originating from three sources in IPOC, namely from the oily waste existing in the storage, fresh untreated soil and treated soil from bioremediation processes. The isolated bacteria had been sub-cultured in solid media that use crude oil as carbon sources several times. The isolated bacteria were also maintained in liquid culture containing crude oil as its substrate in order to determine its capability to use crude oil as carbon source. Naturally existing microorganisms and major contributing microorganisms for hydrocarbon degradation had also been identified. Growth Kinetic Determination The aim of growth kinetic experiments is to determine the ability of isolated microorganisms in the degradation of petroleum hydrocarbon. The kinetics parameters determined were Specific Growth Rate, Half Saturation Constant (Substrate Affinity Constant), and Maximum Specific Growth Rate.

Specific Growth Rate, μ, 1/time µ is the rate of bacterial growth in the exponential phase. The change in biomass (dx) with time (dt) has a positive correlation with the initial biomass (x). Thus:

0

0lnln

)0(0lnln0

tt

xx

ttxx

textx

xdt

dx

xdt

dx

−

−=

−=−

=

=

∝

μ

μ

μ

μ

Since a growth curve was obtained by plotting several data points on semilog plot paper, µ was calculated using least square correlation analysis. Once a linear equation was drawn, then the slope of the line was the specific growth coefficient (µ).

Half Saturation Constant (Substrate Affinity Constant), Ks, % (v/v) and Maximum Specific Growth Rate, μmax, 1/time Experiments to obtain Ks and µmax values were established by growing culture on various substrate concentrations ranging from 0.1% – 0.5% or depending on the ability the isolated bacteria to grow in certain concentration. The

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specific growth rate (µ) at each substrate concentration (C) was calculated using least square and double reciprocal plots drawn to obtain Ks and µmax values. According to Monod (1949):

)(max CsK

C

+= μμ

The double reciprocal plot was derived from this equation:

max

1

max

1

μμμ+=

CsK

This equation is liner when μ

1 was applied as y (ordinate) and

C

1 as x (abscissa). When

C

1= 0, so

μ

1is equal to

max

1

μand when

μ

1= 0, so

C

1is equal to abs

sK

1.

Laboratory Treatability Test To determine the performance of isolated bacteria in degrading petroleum hydrocarbon, oil-contaminated soil was taken from IPOC storage pit. The soil was then used as the target of this treatability test. The mix culture of isolated bacteria was used in this test. Laboratory scale of reactor for windrow technique has been developed and set up.

There were five reactor conditions employed in this test with variations of the type of bulking agent and addition of bio-surfactant:

1. Control reactor; indigenous bacteria (A) 2. Reactor with the addition of Isolated Bacteria + Palm Shells Bulking Agent (B) 3. Reactor running with the addition of Isolated Bacteria + Palm Shells Bulking Agent + Bio-surfactant (C) 4. Reactor with the addition of Isolated Bacteria + Shredded Grass Bulking Agent (D) 5. Reactor with the addition of Isolated Bacteria + Shredded Grass Bulking Agent + Bio-surfactant (E)

The composition of oil-contaminated soil : bulking agent : bacteria was 100:1:1. To the reactor C and E, bacteria Azotobacter sp which was isolated from other research was added. In certain conditions, these bacteria have the ability to produce the kind of surfactant which is known as a bio-surfactant. Thus, with the addition of these bacteria, TPH emulsification process occurred. Emulsified TPH should be easier to be degraded by petrophylic bacteria because of the size of oil droplets are much smaller than before the addition of Azotobacter sp. RESULTS Isolation of Petrophilic Bacteria The petrophilic bacteria from the oily waste existing in the storage, fresh untreated soil and treated soil from bioremediation processes in IPOC had been isolated and cultured in solid media that use crude oil as carbon source. There were three dominant bacterial consortia grown from each of the above inoculums source. Individually these bacteria have shown their capability to grow on media containing petroleum hydrocarbon. It indicated that they use petroleum hydrocarbon as carbon source for growth.

The isolated bacteria had also been maintained in liquid culture containing crude oil as its substrate in order to determine its capability to use crude oil as carbon source.

The identified dominant bacteria from the culture were Alkaligenes paradoxis, Thermus aquaticus (the presence of this bacterium indicated that the oily waste was coming from high temperature source), and Pseudomonas fluorescence. Growth Kinetic Determination Specific growth rate, μ, was determined by growing the Total Consortium 1, Total Consortium 2 and the mix culture of Consortium 1 and 2 in various TPH concentrations. The growth of bacteria was observed on daily basis in order to determine the growth pattern. The following figures show the result of specific growth rate from isolated bacteria that had been bioaugmented in the laboratory.

The TPH concentration from each sources were significantly different. Thus, variations in the TPH concentration added to the culture would be different depending on the source of the bacteria isolated.

Based on these observations and according to “Michaelis-Menten” equation, the maximum growth rate and half saturation constant could be determined by the following figures.

IPTC 15316 7

Figure 3. Growth Kinetic Determination

Maximum specific growth rate was determined in order to show the maximum rate of isolated bacteria in degrading TPH.

This maximum rate could also indicate the time required to degrade TPH up to certain degree. The other kinetic parameter, half saturation constant, was observed in order to determine the affinity of the isolated bacteria to TPH as shown in the following table.

Table 5. The Maximum Specific Growth Rate & Half Saturation Constant of Isolated Bacteria

Isolates Source µmax, 1/hour Ks, %

Storage 0,035 0,122

Un-treated Soil 0,018 0,010

Treated Soil 0,022 0,010

Based on the above results, it showed that bacteria that had the best ability to degrade TPH were bacteria that came from

treated soil. It can be seen from the value of Ks of these bacteria that was relatively small with a large growth rate when compared with other isolates. These results were in accordance with the initial hypothesis in which the bacteria were grown in the treated soil had a long adaptation phase to allow the ability to use TPH as a carbon source for growth. Bacteria that were isolated from oily waste storage had a relatively high growth rate when compared with other isolates. However, to grow well, these isolates required TPH in a relatively high concentration when compared with other isolates. Thus, this bacterium had relatively a low affinity to TPH. The bacteria that isolated from untreated soil seemed to have a long phase of adaptation. Therefore, when compared with bacteria from treated soil, the growth rate of these bacteria was slightly lower.

8 IPTC 15316

Performance of Isolated Bacteria in Degradation of TPH in Bioreactors The result of the test using 5 bioreactors which had been running for more than 2 months has shown that the isolated bacteria have capability to use TPH as carbon source. It could also indicate that the oily waste taken from IPOC storage was treatable using bioremediation technique. Furthermore, the addition of bio-surfactant produced bacteria affected the biodegradation of TPH by the isolated bacteria. The following figure shows the performance of each reactor.

Figure 4. Treatability Test under Various Conditions (A = Control reactor - indigenous bacteria; B= Reactor with the addition of Isolated Bacteria + Palm Shells Bulking Agent; C = Reactor with the addition of Isolated Bacteria + Palm Shells Bulking Agent + Bio-surfactant; D = Reactor with the addition of Isolated Bacteria + Shredded Grass Bulking Agent; E = Reactor with the addition of Isolated Bacteria + Shredded Grass Bulking Agent + Bio-surfactant)

In the reactors where bio-surfactant-producing bacteria were added, namely the reactor C and E, TPH concentrations below

10000 mg/kg could be achieved within less than one month, relatively faster than in other reactors. This indicated that the emulsification process of TPH gave a significant influence on the biodegradation process conducted by the isolated bacteria. The result was better than reactor B and D in which bioremediation processes only relied on isolated bacteria. Nevertheless the test indicated as well that the isolated bacteria had a strong ability to degrade TPH. The time required to reach TPH concentration below 10000 mg/kg in reactor B & D was less than 1.5 months.

The test has also shown that the addition of bulking agent gave a fairly significant influence on the bioremediation process. In reactor A, which represented the reactor without the addition of bulking agent and bio-surfactant-producing bacteria and also bioremediation process that solely relies on indigenous bacteria, the biodegradation lasted longer (more than two months) when compared with other reactors. Moreover, the palm shells bulking agent gave better results when compared with shredded grass.

The following figures show an example of the Gas Chromatography/Mass Spectrophotometer (GC/MS) analyses of TPH for reactor C. These figures show in general that in the first month the heavy fractions of TPH slowly disappeared to become lighter fractions. Although TPH had reached 10000 mg/kg after 1 month for reactor C, there were still some fractions remained in the reactor. It was most likely that these were the aliphatic or aromatic compounds.

IPTC 15316 9

(A)

Prystane

(B)

Prystane

(C)

Figure 5. GC-MS Results (A: t = 0 day, B: t = 15days, C: t = 30 days)

The number of bacteria in the reactors was also observed during the process. This is related to the biological transformation during the process. It was expected that the number of bacteria could be maintained in the same number as an indication of steady state process. Although so far there were no literature mentioned the optimum number of bacteria involved in the

10 IPTC 15316

process, based on experience normally it is around 104 to 107 colony per milligram soil. As shown in Figure 6, the number of bacteria in reactor during the process was around 106 colonies/mg soil.

Figure 6. Bacterial count during the process

The pH of soil taken from IPOC was found to be relatively low. It was around pH 6. Figure 7 shows that on the average, after mixing with bulking agent, the pH during the bioremediation in each reactor was neutral (between pH 7 – 8). Since most hydrocarbon-degrading bacteria are classified as neutrophylic bacteria, these pH conditions supported the growth of these microorganisms under optimum environmental conditions. Lower pH in the reactor A could be also an environmental factor that caused the rate of TPH degradation was lower than other reactor.

Figure 7. pH during Bioremediation Process

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Besides the availability of main substrate as carbon source that was TPH in this study, the growth of microorganisms also depends on the presence of Nitrogen (N) as nutrient. In this study, NPK fertilizers with a concentration of 0.5% were added to the reactors in order to provide nitrogen in a sufficient concentration. Figure 8 shows the presence of N during the process. It could be seen that decrease of TPC was followed by the reduction of N. The presence of N in the bioreactors was also contributed from the soil itself as well as from the addition of bulking agent. The result indicated that N from palm shells bulking agent was higher than from shredded grass. Also, it could be seen that N from palm shells was more readily to be up-taken by microorganisms as indicated by rate of biodegradation and time consumed.

Figure 8. The presence of N-inorganic during bioremediation process

As regulated in MoE Decree No. 128/2003, other parameters which should comply the end point criteria of bioremediation process are benzene, ethyl benzene, toluene and xylene (BTEX). No BTEX was detected by using GC-MS since the beginning of bioreactor started. Toxicity Characteristics Leaching Procedure (TCLP) results showed that no significant heavy metals leached from the pile during the bioremediation process. Moreover, the leaching test results for As, Ba, B, Cd, Cr, Cu, Pb, Hg, Se, Ag and Zn were far below the criteria regulated in the MoE Decree No. 128/2003.

CONCLUSION The result of treatability test of bioaugmentation in laboratory has demonstrated that there were at least 3 consortiums of bacteria successfully isolated and bioaugmented from three sources in IPOC, namely from the oily waste existing in the storage, fresh untreated soil and treated soil from bioremediation processes. Growth kinetics experiment of these isolates demonstrated that these bacteria had ability to degrade and utilize TPH as carbon source at low concentration, even lower than the end point criteria (10000 mg/kg) as regulated in MoE Decree No. 128/2003. This result indicated that the affinity of these bacteria to TPH were relatively high. Using oily waste from Senipah site for treatability test, the mix culture of those isolates demonstrated its ability to degrade TPH. Within less than 1 month the overall TPH concentration could reach below 10000 mg/kg by the addition of bio-surfactant-producing bacteria. The rate of biodegradation in this study was also affected by the type of bulking agent used.

The next step following the result of this treatability test will be to test the efficiency of hydrocarbon degradation using isolated and bioaugmented bacteria in field scale application. The company is continuously striving to enhance the efficiency of bioremediation process in IPOC by continuing research and developments on the subject.

Acknowledgements The authors wish to gratefully acknowledge management of Total E&P Indonesie for support in publishing this paper, Bandung Institute of Technology as partner in this bioremediation research program, as well as all of those who have participated to the implementation of IPOC program for their support.

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