Efikasi berbagai Metode Bioremediasi untuk Pantai Terkontaminasi Minyak di Cilacap, Jawa Tengah, Indonesia
EFFICACY OF BIOREMEDIATION METHODS FOR OIL
CONTAMINATED SANDY BEACH IN CILACAP, CENTRAL
JAVA, INDONESIA
YETI DARMAYATI
POST GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATMENT OF THE DISSERTATION AND SOURCES OF
INFORMATION AND DEVELUTION COPYRIGHT*
I hereby certify that the dissertation entitled Efficacy of Bioremediation Methods
for Oil Contaminated Sandy Beach in Cilacap, Central Java, Indonesia is true of
my work with the direction of the supervising commission and has not been
submitted in any form to any college. The source of information originating or
taken from works published or unpublished from other authors have been
mentioned in the text and listed in the Bibliography at the end of this dissertation.
I hereby assign copyright of my paper to the Bogor Agricultural Institute.
Bogor, January 2016
Yeti Darmayati
C561100022
SUMMARY
YETI DARMAYATI. Efficacy of Bioremediation Methods for Oil
Contaminated Sandy Beach in Cilacap, Central Java, Indonesia. Supervised by
HARPASIS S. SANUSI, TRI PRARTONO, DWI ANDREAS SANTOSA and
RUYITNO NUCHSIN.
Oil spills threat Indonesian marine waters. It caused by this waters has a role
as one of oil world transportation main routes and many activities of oil refineries
and exploration are located in near and off shore. The most impacted area is coastal
area with many problems in ecological and economical aspects emerged. The
chemical and physical approaches which are not environmentally friendly have
been functioned practice in addressing the problem of oil pollution in the coastal
areas of Indonesia. Bioremediation is a method that use biodegradation processes
to eliminate environmental pollutants. This method is very promising for the
application of reducing the impact of oil spill in marine waters of Indonesia, due
to the high diversity of bacteria and tropical environmental condition. However,
this technique almost has never been applied in Indonesia coastal water due to it
still to be developed. The reasons are limiting of understanding regarding with
microbial characteristic, microbial selection, metabolism capability, interaction
between indigenous and exogenous microbes, trigger needed etc. Therefore, this
study was conducted to provide data and information needed to develop a
common understanding of bioremediation in the oil contaminated Indonesian
coastal environments.
The aim of this study was to obtain the appropriate and effective
bioremediation method in controlling oil pollution in sandy beach area by
conducting the laboratory and field experiments. Oil spills in Cilacap coastal area
is very often occurred, therefore this area was selected as study area as a
representative of chronic oil contaminated area. Using the indigenous bacteria of
Indonesian waters (7 selected bacterial cultures isolated from Jakarta harbor) and
local slow release fertilizer (3 different brands produced by Indonesian company)
will reduce our dependency on imported products. The study consisted of several
steps such as: the selection of potential bacteria and slow-release fertilizer,
optimization of fertilizer concentration and density of bacteria for the best results
in degrading oil, and the efficacy of various methods of bioremediation for sandy
beaches.
The amendments of oil degrading bacteria (bioaugmentation) and fertilizer
(biostimulation) are common technique in bioremediation. In order to find out the
best treatment for enhancing oil degradation rate, microcosm experiment was
conducted for selecting the bacteria and fertilizer, and also for optimizing
different bacterial density, bacterial composition and fertilizer concentration.
The results showed that single culture of RCO/B/08-08 (Alcanivorax sp. TE-9
with homology 100 %), mixed culture B and Gra slow release fertilizer were the
most potential agent for bioremediation in Cilacap coastal area. In addition, the oil
degradation rate was more influenced by nutrient concentration than the number
of bacteria amended. The efficacy of biostimulation in degrading oil was better
than bioaugmentation, because it increased oil degradation up to 6.4 times higher
than the control experiment. The optimum amount of fertilizer added was in N
concentration of 7.5 mg g-1 (C:N ratio of 1000:75). This concentration increased
depletion rate both in biostimulation-only and the combination of biostimulation bioaugmentation up to 6.4 and 7.5 times higher than the control experiment,
respectively. Therefore, we proposed the bioremediation of oil polluted sandy
beach in Cilacap would be optimal by conducting a combination between slow
release fertilizer Gra at ratio concentration C/N = 1000:75 and single cultured of
RCO/B/08-008 with the density of 0.5 x 108 cells mL-1.
The study of suitable biological remediation methods for anticipating oil
spills in Cilacap sandy beach was conducted by using mesocosm in the intertidal
area near shore of Cilacap. The result showed that after 3 months experiment, the
percentage of oil depletion in control, single cultured and mix cultured treatments
were 60.4 %, 74.5% and 73.5% respectively. Bioaugmentation could increase
oil biodegradation in tropical sandy beach. However, bioremediation efficacy
on the amendment of single cultured and mixed cultured bacteria was not
significantly different.
Evaluation of using biostimulation and a combination between
biostimulation and bioaugmentation by using mesocosm showed that the oil
depletion rate of biostimulation-bioaugmentation combination treatment was
faster and higher than biostimulation only. At the 16th day treatment, the
biostimulation treated by adding the consortium and single strain increased oil
depletion of 2.2 and 1.6 times higher than the control respectively. For a longer
period of treatment, both the combination treatments showed a similar efficacy in
degrading oil contamination. Therefore, combination of biostimulationbioaugmentation with the consortium was proposed for combating oil-pollution
for a short period.
This study concluded that bioremediation through bioaugmentation,
biostimulation or the combination of both techniques could alter the oil
degradation. Combination of bioaugmentation and biostimulation showed the
highest oil depletion rate. The indigenous microbial agent and t h e locally
product fertilizer had potency to be used in combating oil spilled. The single
cultured of RCO/B/08-008 or mix cultured of RCO/B/08-008, RCO/B/08-004 and
RCO/B/08-015 could be proposed as potential bioremediation agents, as well as
Gra slow release fertilizer as a stimulant. Introduced single or mix cultured
bacteria for oil-chronic polluted area did not cause different effect in increasing
oil degradation rate. C:N ratio should be considered carefully before adding the
fertilizer in oil spilled area. In Cilacap coastal area, adding the fertilizer Gra at
C/N ratio of 1000:75 and intoduction of bioremediation agent at density of 0.5
x 108 cell mL-1 was appropriate to increase oil depletion rate.
Keywords: Bioremediation, Bioaugmentation, Biostimulation, Effication, Slow release
fertilizer, Consortium, Single strains, Microcosm, Crude Oil
RINGKASAN
YETI DARMAYATI. Efikasi berbagai Metode Bioremediasi untuk Pantai
Terkontaminasi Minyak di Cilacap, Jawa Tengah, Indonesia. Dibimbing oleh
HARPASIS S. SANUSI, TRI PRARTONO, DWI ANDREAS SANTOSA and
RUYITNO NUCHSIN.
Perairan pesisir Indonesia sangat rentan terkena tumpahan minyak karena
merupakan salah satu jalur utama minyak dunia dan memiliki banyak fasilitas
pengeboran/ pengilangan minyak yang tersebar di pesisir ataupun lepas
pantainya. Dalam mengatasi tumpahan minyak di perairan laut dan pesisir,
Indonesia masih mengandalkan pendekatan fisik dan kimia yang kurang ramah
lingkungan Pendekatan biologis (Bioremediasi), lebih ramah lingkungan dan
lebih murah. Teknik ini sangat menjanjikan diterapkan karena iklim Indonesia
yang tropis dan keragaman mikroba yang cukup tinggi. Namun demikian,
metode ini hampir tidak pernah diterapkan di perairan pesisir karena masih
belum siap. Hal ini disebabkan pemahaman yang masih sangat terbatas terkait
pemilihan/penentuan mikroba, karakter/sifat mikroba, interaksi antara mikroba
eksogenous dan indigenous, faktor pemacu yang dibutuhkan, dan lain-lain. Oleh
karenanya studi ini dilakukan untuk mengisi kekurangan data dan informasi
tersebut dia atas yang diharapkan dapat meningkatkan pemahahaman umum
tentang bioremediaasi di lingkungan pesisir yang terkontaminasi minyak.
Studi ini difokuskan untuk menentukan metode bioremediasi yang tepat dan
efektif untuk menangani tumpahan minyak di perairan pesisir pantai berpasir
dengan menggabungkan eksperimen laboratorium dan lapangan. Tumpahan
minyak di perairan pesisir Cilacap sering kali terjadi, oleh karenanya perairan ini
dipilih untuk mewakili pesisir yang tercemar minyak secara kronis. Penggunaan
bakteri asal perairan Indonesia (7 isolat terpilih dari pelabuhan di Teluk Jakarta)
dan pupuk produk lokal (3 pupuk terpilih produksi dalam negeri) dipilih untuk
mengurangi ketergantungan kita terhadap barang impor. Ada beberapa langkah
penelitian yang telah dilakukan mencakup: seleksi bakteri potensial dan pupuk
lepas lambat, pemilihan strategi aplikasi, optimalisasi konsentrasi pupuk dan
kepadatan bakteri untuk hasil terbaik dalam mendegradasi minyak, juga
efektivitas berbagai metode bioremediasi untuk lingkungan pantai berpasir.
Pemberian bakteri pendegradasi minyak (bioaugmentasi) dan pupuk
(biostimulasi) adalah pendekatan praktis yang umum dilakukan. Untuk
mendapatkan teknik terbaik dalam meningkatkan laju degradasi minyak,
eksperimen secara mikrokosmos telah dilakukan untuk menyeleksi bakteri dan
pupuk. Disamping itu, juga dilakukan uji optimasi dengan menambahkan
kepadatan bakteri, komposisi bakteri, dan konsentrasi pupuk yang berbeda. Hasil
studi menunjukkan bahwa kultur tunggal RCO/B/08_008 (Alcanivorax sp. TE-9,
homologi 100%), kultur campuran dan pupuk Gra adalah agen yang paling
potensial untuk bioremediasi cemaran minyak di Cilacap. Ditemukan pula bahwa
konsentasi nutrient lebih berpengaruh terhadap laju degradasi minyak daripada
jumlah bakteri ataupun komposisi bakteri yang ditambahkan. Efikasi teknik
biostimulasi dalam meningkatkan laju degradasi minyak lebih baik daripada
bioaugmentasi, yaitu sampai 6.4 lebih tinggi dari kontrolnya. Jumlah optimum
pupuk yang ditambahkan adalah dengan konsentrasi N 7.5 mg g -1 (Rasio C:N
= 1000 : 75). Konsentrasi ini mampu meningkatkan laju penurunan konsentrasi
minyak baik pada perlakuan biostimulasi dan kombinasi biostimulasi-bioaugmentasi
masing-masing sampai 6.4 dan 7.5 kali lebih tinggi dari kontrolnya. Oleh karena itu,
dapat diusulkan untuk bioremediasi pantai berpasir tercemar minyak di Cilacap akan
optimal dengan pemberian perlakuan kombinasi penambahan pupuk lepas lambat
dan bakteri. Kombinasi tersebut adalah penambahan pupuk lepas lambat pada
konsentrasi dengan perbandingan C:N = 1000:7.5 dan kultur tunggal bakteri
RCO/B/8-008 (homologi dengan Alcanivorax sp. TE-9 100%).
Untuk mengetahui pendekatan remediasi yang tepat dalam menanggulangi
minyak di pantai berpasir Cilacap, evaluasi efikasi metode bioremediasi di
lapangan telah dilakukan dengan pendekatan mesokosme di daerah pasang surut
Cilacap. Hasil penelitian menunjukkan bahwa penurunan konsentrasi minyak
mencapai 60.4 %, 74.5% dan 73.5 % dalam kurun waktu 3 bulan, masing-masing
pada kontrol, perlakuan kultur tunggal dan kultur campuran. Biaugmentasi dapat
meningkatkan laju biodegradasi di pantai berpasir tropis. Namun perbedaan
efikasi dari penambahan kultur tunggal dan kultur campuran secara statistik tidak
nyata
Evaluasi biostimulasi dan kombinasi biostimulasi-bioaugmentasi telah
menunjukkan bahwa efektivitas penurunan konsentrasi minyak pada perlakuan
kombinasi biostimulasi-bioaugmentasi lebih cepat teramati dan lebih tinggi
daripada perlakuan biostimulasi saja. Pada hari ke-16 setelah aplikasi pupuk
yang ditambah kultur campuran dan kultur murni persentasi penurunan
minyaknya masing-masing mencapai 2.2 dan 1.6 kali lebih tinggi daripada
kontrol Namun untuk periode yang lebih panjang, kedua perlaku an
kombinasi ini memiliki efikasi yang relatif sama. Untuk itu, penggunaan
kombinasi biostimulasi dengan kultur campuran lebih disarankan untuk
menangani pencemaran dalam periode yang pendek .
Kesimpulan dari kajian ini adalah tumpahan minyak di lingkungan pesisirtercemar minyak yang kronis, dapat terdegradasi secara alami tetapi dalam tingkat
yang rendah. Bioremediasi baik melalui bioaugmentation, biostimulation maupun
kombinasi keduanya terbukti secara signifikan mampu meningkatkan laju
degradasi minyak. Kombinasi bioaugmentation dan bistimulation menunjukkan
tingkat penurunan konsentrasi minyak paling tinggi. Hal itu terbukti dalam
percobaan in vitro dan mesocosm. Ada agen mikroba indigenous dan pupuk
produk lokal yang prospektif untuk digunakan dalam menanggulangi tumpah
minyak. RCO/B/08-008 dalam bentuk kultur tunggal atau dalam kultur campuran
dengan RCO/B/ 08-004 dan RCO/B/ 08-015 dapat diusulkan sebagai agen
bioremediasi, serta pupuk lepas lambat Gra sebagai agen stimulan potensial.
Pemberian kultur tunggal atau campuran untuk daerah tercemar minyak kronis
tidak menimbulkan efek yang berbeda dalam meningkatkan laju degradasi
minyaknya. Perbandingan nilai C:N rasio harus dipertimbangkan dengan hati-hati
sebelum menambahkan pupuk di wilayah tercemar minyak. Untuk pesisir Cilacap,
penambahan pupuk G pada konsentrasi N dengan perbandingan C:N = 1000:75
dan kultur bakteri dengan kepadatan 0.5 x 108 sel mL-1 cukup untuk
meningkatkan laju degradasi minyak.
Kata Kunci: Bioremediasi, Bioaugmentasi, Biostimulation, Efikasi, Mikrokosmos,
Pupuk lepas lambat, Konsorsium, Kultur tunggal, Minyak mentah
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EFFICACY OF BIOREMEDIATION METHODS FOR OIL
CONTAMINATED SANDY BEACH IN CILACAP, CENTRAL
JAVA, INDONESIA
YETI DARMAYATI
A dissertation
submitted in partial fulfillment of the requierements for the degree of
Doctoral
in Marine Science Major
POST GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
Penguji pada Ujian Tertutup : Prof Dr Ir Dietriech Geoffrey Bengen DEA
Dr Ir Agung Dhamar Syakti DEA
Penguji pada Ujian Terbuka : Prof Dr Ir Dietriech Bengen DEA
Dr Ir Zainal Arifin MSc
Dissertation Entitle : Efficacy of Bioremediation Methods for Oil Contaminated
Sandy Beach in Cilacap, Central Java, Indonesia
Name
: Yeti Darmayati
NIM
: C561100022
Approved
by
Graduate Committee
Prof Dr Ir Harpasis S. Sanusi, MSc.
Chairman
Dr Ir Tri Prartono, MSc.
Member
Prof Dr Ir Dwi Andreas Santosa, MSc.
Member
Prof Drs Ruyitno Nuchsin, MSc.
Member
Acknowledged by
Head of Marine Sciences Major
Dean of Graduate School
Dr Ir Neviaty P. Zamani, M.Sc.
Dr Ir Dahrul Syah, M.Sc.Agr.
Date of Close Examination : 29 January 2016
Date of Open Examination : 5 February 2016
Date of Graduation :
PREFACE
Praise and gratitude author prayed to Allah SWT for His abundant
blessings and gifts so that this dissertation was completed. The dissertation
entitled Efficacy of Bioremediation Methods for Oil Contaminated Sandy
Beach in Cilacap, Central Java, Indonesia, is conducted to develop an
effective clean-up method for reducing the impact of oil spills in tropical marine
environment.
I am immensely grateful to those individuals who have helped to complete
my doctoral study in Bogor Agricultural University. As the outset, I would like
to express my great appreciation to Prof Dr Ir Harpasis S. Sanusi MSc, for his
enthusiastic support, guidance, being always there, and the time spent in guiding
me through the writing process. As my supervisor, his observation and
comments helped me to establish the overall direction of the research and to
move forward with its investigation in depth. I would like also to express my
gratitude to my supervisors Prof Drs Ruyitno Nuchsin MSc, Dr Ir Tri Prartono
MSc, and Prof Dr Ir Dwi Andreas Santosa MSc for their help, support and
guidance both during the study and in the writing of a dissertation
I acknowledge Prof Dr Ir Dietriech Geoffrey Bengen DEA, Dr Ir Agung
Dhamar Syakti DEA, and Dr Ir Zainal Arifin MSc as external examiner
commission has been testing, analyzing and providing input for the perfection of
this dissertation. Many thank to Dr Ir Neviaty P. Zamani MSc as the Chairman
of Marine Science study program who has directed and encouraged me for
implementing education in Marine Science Program SPs IPB. I will not forget Dr
Dirhamsyah MA, and Dr Suhartati M. Natsir for their great help and granted
permission during my doctoral program.
I would like to express my thanks to Ministry of Research and
Technology and Indonesian Institut of Sciences which has provided me
scholarships for Doctoral Program in IPB and financial support for the
implementation of this research, respectively. Also, to PERTAMINA Refinery
Unit IV Cilacap and BPLHD Cilacap for their cooperation to conduct this
research in Cilacap.
I would like to express my sincere thanks to all my best friend Dr Dwi
Hindarti, Dr Woro Hastuti, Dr Hayati Dr Etty Riani, Noor Farida AMd, and Dr
Aleh Human Saleh for their help, prayer, encouragement, enthusiasm and for
being very good friends. I will not forget to Fellow Major Marine Science Post
Graduate School-IPB: Dr Yunita F. Noya, Dr Rhozirwan, Dr Noverita, Khalid,
Tumpak Sidabutar, Ani, Denti, Dr Nurul Dhewani, Femi Hukom, for your help,
support and togetherness. I appreciate Surahman, Bella S. Rosepta, Wahyu
Utomo, Tia D. Martianingsih, Dwi Astuti, Linda Safitri and all member of
bioremediation research group Research Center for Oceanography – LIPI for
their technical assistance in this research.
Last, but not least, I would like to dedicate this dissertation to my beloved
husband, Mochamad Erawan, my dearly loved daughters, Marina Irawati
and Mazida Fidelia Darmawati, and my adored sons, Gwyn Walesa and
Muhammad Ardy Makarim for your prayers and sacrifices outstanding
during the author completed education. Also, My mother and father (alm.), my
sisters, Tuti Darmawati, Eli Darmalingganawati, Darmawidyawati, and my
brothers, Dudung Dumadi Fachrudin and Dadang Darmayana. Without dear
support and encouragement, I could not have completed my doctoral study.
I Hope this scientific work will be useful.
Bogor, January 2016
Yeti Darmayati
C561100022
xi
TABLE OF CONTENT
LIST OF TABLE
xii
LIST OF FIGURE
xiii
1 GENERAL INTRODUCTION
Background
Logical Framework
Objective and Purpose
Goal
Novelty
1
1
2
4
5
5
2 LITERATURE REVIEW
Oil Pollution in Marine Environments
Biodegradation of Oil
Bioremediation in Oil Polluted Shoreline
6
6
10
17
3 GENERAL METHODOLOGY
Time and Study Site
Reseach Steps
Materials Preparation
Laboratory Experiment
Field Experiment
Sample Analysis
Data Analysis
26
26
27
28
31
33
36
37
4 POTENTIAL MICROBIAL AGENT FOR CRUDE OIL
DEGRADATION
Introduction
Materials and Methods
Result and Discussion
Conclusion
38
38
39
42
45
5 ALTERNATIVE SLOW RELEASE FERTILIZER ENHANCES
BIODEGRADATION OF OIL CONTAMINATED TROPICAL
SANDY BEACHES
Introduction
Materials and Methods
Result and Discussion
Conclusion
47
47
48
50
55
6 OPTIMAL BACTERIAL DENSITY AND FERTILIZER
CONCENTRATION FOR BIOREMEDIATION OF OIL
CONTAMINATED SANDY BEACH
Introduction
Materials and Methods
Results and Discussion.
Conclusion
56
56
57
59
65
xii
7 THE EFFICACY OF BIOAUGMENTATION ON REMEDIATING OIL
CONTAMINATED SANDY BEACH USING MESOCOSM
APPROACH.1
Introduction
Materials and Methods
Results and Discussion
Conclusion
66
66
66
70
75
8 THE EFFECT OF BIOSTIMULATION AND BIOSTIMULATIONBIOAUGMENTATION ON BIODEGRADATION OF OILPOLLUTION ON SANDY BEACHES USING MESOCOSMS
Introduction
Materials and Methods
Results and Discussion
Conclusion
76
76
77
81
89
GENERAL DISCUSSION
900
CONCLUSION
92
REFERENCES
93
CURRICULUM VITAE
104
LIST OF TABLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Shoreline clean-up options, benefits and limitations
Common methods in chemical analysis of oil and oil constituent
Common methods in microbiological monitoring
Parameters measured and methods can be used in monitoring oil
bioremediation
Selected cultured used for the experiment
Physicochemical characteristics of Arabian Light Crudeoil used
(provided by PERTAMINA 2011)
Composition of slow release fertilizer content
Treatments for bacterial and fertilizer selection experiments
Treatment for optimization bacteria and fertilizer experiment
Treatment employed in field experiment
Measurement of physicochemical parameters
The composition of bacterial mixed culture
Environmental parameters condition over 28 days experiment
Soluble Total Nitrogen and Phosphate during experiment
Treatments performed for optimalization study
Environmental parameters in the range during 28 days experiment
Pore water quality in average and range value during 90 day
sexperiment
First order rate constant (k) value in different treatment at various
periode
10
23
24
25
29
30
31
31
32
35
37
39
52
54
58
65
70
72
xiii
19 k value in each treatments at different time periods
20 Pore water quality over time experiment inside and outside mesocosm
82
84
LIST OF FIGURE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Logical Framework
Major weathering processes after an oil spills (ITOPF 2009)
Representative organic compounds found in crude oil
Locationof field experimental site in Cilacap Coastal
Flow chart of research steps
Bioreactor
Experiment in microcosm system
Plot lay out on the inter-tidal foreshore of Cilacap Coastal
Schematic design of mesocosm
Porewater sampler and capiler hose forsucking the sample
Research design for bacteria selection
Residual oil content in single (A) and mix culture (B) treatment
Bacterial growth of single (A) and mix culture (B)
Oil depletion percentage in different fertilizer treatment during 28 days
experiment
Bacterial growth in 4 different SRF treatments over 28 days
experiment. Standard deviations of the replicates (n = 3) are denoted by
the error bars. Columns marked by the same letter (a to d) are not
significantly different (p
CONTAMINATED SANDY BEACH IN CILACAP, CENTRAL
JAVA, INDONESIA
YETI DARMAYATI
POST GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
STATMENT OF THE DISSERTATION AND SOURCES OF
INFORMATION AND DEVELUTION COPYRIGHT*
I hereby certify that the dissertation entitled Efficacy of Bioremediation Methods
for Oil Contaminated Sandy Beach in Cilacap, Central Java, Indonesia is true of
my work with the direction of the supervising commission and has not been
submitted in any form to any college. The source of information originating or
taken from works published or unpublished from other authors have been
mentioned in the text and listed in the Bibliography at the end of this dissertation.
I hereby assign copyright of my paper to the Bogor Agricultural Institute.
Bogor, January 2016
Yeti Darmayati
C561100022
SUMMARY
YETI DARMAYATI. Efficacy of Bioremediation Methods for Oil
Contaminated Sandy Beach in Cilacap, Central Java, Indonesia. Supervised by
HARPASIS S. SANUSI, TRI PRARTONO, DWI ANDREAS SANTOSA and
RUYITNO NUCHSIN.
Oil spills threat Indonesian marine waters. It caused by this waters has a role
as one of oil world transportation main routes and many activities of oil refineries
and exploration are located in near and off shore. The most impacted area is coastal
area with many problems in ecological and economical aspects emerged. The
chemical and physical approaches which are not environmentally friendly have
been functioned practice in addressing the problem of oil pollution in the coastal
areas of Indonesia. Bioremediation is a method that use biodegradation processes
to eliminate environmental pollutants. This method is very promising for the
application of reducing the impact of oil spill in marine waters of Indonesia, due
to the high diversity of bacteria and tropical environmental condition. However,
this technique almost has never been applied in Indonesia coastal water due to it
still to be developed. The reasons are limiting of understanding regarding with
microbial characteristic, microbial selection, metabolism capability, interaction
between indigenous and exogenous microbes, trigger needed etc. Therefore, this
study was conducted to provide data and information needed to develop a
common understanding of bioremediation in the oil contaminated Indonesian
coastal environments.
The aim of this study was to obtain the appropriate and effective
bioremediation method in controlling oil pollution in sandy beach area by
conducting the laboratory and field experiments. Oil spills in Cilacap coastal area
is very often occurred, therefore this area was selected as study area as a
representative of chronic oil contaminated area. Using the indigenous bacteria of
Indonesian waters (7 selected bacterial cultures isolated from Jakarta harbor) and
local slow release fertilizer (3 different brands produced by Indonesian company)
will reduce our dependency on imported products. The study consisted of several
steps such as: the selection of potential bacteria and slow-release fertilizer,
optimization of fertilizer concentration and density of bacteria for the best results
in degrading oil, and the efficacy of various methods of bioremediation for sandy
beaches.
The amendments of oil degrading bacteria (bioaugmentation) and fertilizer
(biostimulation) are common technique in bioremediation. In order to find out the
best treatment for enhancing oil degradation rate, microcosm experiment was
conducted for selecting the bacteria and fertilizer, and also for optimizing
different bacterial density, bacterial composition and fertilizer concentration.
The results showed that single culture of RCO/B/08-08 (Alcanivorax sp. TE-9
with homology 100 %), mixed culture B and Gra slow release fertilizer were the
most potential agent for bioremediation in Cilacap coastal area. In addition, the oil
degradation rate was more influenced by nutrient concentration than the number
of bacteria amended. The efficacy of biostimulation in degrading oil was better
than bioaugmentation, because it increased oil degradation up to 6.4 times higher
than the control experiment. The optimum amount of fertilizer added was in N
concentration of 7.5 mg g-1 (C:N ratio of 1000:75). This concentration increased
depletion rate both in biostimulation-only and the combination of biostimulation bioaugmentation up to 6.4 and 7.5 times higher than the control experiment,
respectively. Therefore, we proposed the bioremediation of oil polluted sandy
beach in Cilacap would be optimal by conducting a combination between slow
release fertilizer Gra at ratio concentration C/N = 1000:75 and single cultured of
RCO/B/08-008 with the density of 0.5 x 108 cells mL-1.
The study of suitable biological remediation methods for anticipating oil
spills in Cilacap sandy beach was conducted by using mesocosm in the intertidal
area near shore of Cilacap. The result showed that after 3 months experiment, the
percentage of oil depletion in control, single cultured and mix cultured treatments
were 60.4 %, 74.5% and 73.5% respectively. Bioaugmentation could increase
oil biodegradation in tropical sandy beach. However, bioremediation efficacy
on the amendment of single cultured and mixed cultured bacteria was not
significantly different.
Evaluation of using biostimulation and a combination between
biostimulation and bioaugmentation by using mesocosm showed that the oil
depletion rate of biostimulation-bioaugmentation combination treatment was
faster and higher than biostimulation only. At the 16th day treatment, the
biostimulation treated by adding the consortium and single strain increased oil
depletion of 2.2 and 1.6 times higher than the control respectively. For a longer
period of treatment, both the combination treatments showed a similar efficacy in
degrading oil contamination. Therefore, combination of biostimulationbioaugmentation with the consortium was proposed for combating oil-pollution
for a short period.
This study concluded that bioremediation through bioaugmentation,
biostimulation or the combination of both techniques could alter the oil
degradation. Combination of bioaugmentation and biostimulation showed the
highest oil depletion rate. The indigenous microbial agent and t h e locally
product fertilizer had potency to be used in combating oil spilled. The single
cultured of RCO/B/08-008 or mix cultured of RCO/B/08-008, RCO/B/08-004 and
RCO/B/08-015 could be proposed as potential bioremediation agents, as well as
Gra slow release fertilizer as a stimulant. Introduced single or mix cultured
bacteria for oil-chronic polluted area did not cause different effect in increasing
oil degradation rate. C:N ratio should be considered carefully before adding the
fertilizer in oil spilled area. In Cilacap coastal area, adding the fertilizer Gra at
C/N ratio of 1000:75 and intoduction of bioremediation agent at density of 0.5
x 108 cell mL-1 was appropriate to increase oil depletion rate.
Keywords: Bioremediation, Bioaugmentation, Biostimulation, Effication, Slow release
fertilizer, Consortium, Single strains, Microcosm, Crude Oil
RINGKASAN
YETI DARMAYATI. Efikasi berbagai Metode Bioremediasi untuk Pantai
Terkontaminasi Minyak di Cilacap, Jawa Tengah, Indonesia. Dibimbing oleh
HARPASIS S. SANUSI, TRI PRARTONO, DWI ANDREAS SANTOSA and
RUYITNO NUCHSIN.
Perairan pesisir Indonesia sangat rentan terkena tumpahan minyak karena
merupakan salah satu jalur utama minyak dunia dan memiliki banyak fasilitas
pengeboran/ pengilangan minyak yang tersebar di pesisir ataupun lepas
pantainya. Dalam mengatasi tumpahan minyak di perairan laut dan pesisir,
Indonesia masih mengandalkan pendekatan fisik dan kimia yang kurang ramah
lingkungan Pendekatan biologis (Bioremediasi), lebih ramah lingkungan dan
lebih murah. Teknik ini sangat menjanjikan diterapkan karena iklim Indonesia
yang tropis dan keragaman mikroba yang cukup tinggi. Namun demikian,
metode ini hampir tidak pernah diterapkan di perairan pesisir karena masih
belum siap. Hal ini disebabkan pemahaman yang masih sangat terbatas terkait
pemilihan/penentuan mikroba, karakter/sifat mikroba, interaksi antara mikroba
eksogenous dan indigenous, faktor pemacu yang dibutuhkan, dan lain-lain. Oleh
karenanya studi ini dilakukan untuk mengisi kekurangan data dan informasi
tersebut dia atas yang diharapkan dapat meningkatkan pemahahaman umum
tentang bioremediaasi di lingkungan pesisir yang terkontaminasi minyak.
Studi ini difokuskan untuk menentukan metode bioremediasi yang tepat dan
efektif untuk menangani tumpahan minyak di perairan pesisir pantai berpasir
dengan menggabungkan eksperimen laboratorium dan lapangan. Tumpahan
minyak di perairan pesisir Cilacap sering kali terjadi, oleh karenanya perairan ini
dipilih untuk mewakili pesisir yang tercemar minyak secara kronis. Penggunaan
bakteri asal perairan Indonesia (7 isolat terpilih dari pelabuhan di Teluk Jakarta)
dan pupuk produk lokal (3 pupuk terpilih produksi dalam negeri) dipilih untuk
mengurangi ketergantungan kita terhadap barang impor. Ada beberapa langkah
penelitian yang telah dilakukan mencakup: seleksi bakteri potensial dan pupuk
lepas lambat, pemilihan strategi aplikasi, optimalisasi konsentrasi pupuk dan
kepadatan bakteri untuk hasil terbaik dalam mendegradasi minyak, juga
efektivitas berbagai metode bioremediasi untuk lingkungan pantai berpasir.
Pemberian bakteri pendegradasi minyak (bioaugmentasi) dan pupuk
(biostimulasi) adalah pendekatan praktis yang umum dilakukan. Untuk
mendapatkan teknik terbaik dalam meningkatkan laju degradasi minyak,
eksperimen secara mikrokosmos telah dilakukan untuk menyeleksi bakteri dan
pupuk. Disamping itu, juga dilakukan uji optimasi dengan menambahkan
kepadatan bakteri, komposisi bakteri, dan konsentrasi pupuk yang berbeda. Hasil
studi menunjukkan bahwa kultur tunggal RCO/B/08_008 (Alcanivorax sp. TE-9,
homologi 100%), kultur campuran dan pupuk Gra adalah agen yang paling
potensial untuk bioremediasi cemaran minyak di Cilacap. Ditemukan pula bahwa
konsentasi nutrient lebih berpengaruh terhadap laju degradasi minyak daripada
jumlah bakteri ataupun komposisi bakteri yang ditambahkan. Efikasi teknik
biostimulasi dalam meningkatkan laju degradasi minyak lebih baik daripada
bioaugmentasi, yaitu sampai 6.4 lebih tinggi dari kontrolnya. Jumlah optimum
pupuk yang ditambahkan adalah dengan konsentrasi N 7.5 mg g -1 (Rasio C:N
= 1000 : 75). Konsentrasi ini mampu meningkatkan laju penurunan konsentrasi
minyak baik pada perlakuan biostimulasi dan kombinasi biostimulasi-bioaugmentasi
masing-masing sampai 6.4 dan 7.5 kali lebih tinggi dari kontrolnya. Oleh karena itu,
dapat diusulkan untuk bioremediasi pantai berpasir tercemar minyak di Cilacap akan
optimal dengan pemberian perlakuan kombinasi penambahan pupuk lepas lambat
dan bakteri. Kombinasi tersebut adalah penambahan pupuk lepas lambat pada
konsentrasi dengan perbandingan C:N = 1000:7.5 dan kultur tunggal bakteri
RCO/B/8-008 (homologi dengan Alcanivorax sp. TE-9 100%).
Untuk mengetahui pendekatan remediasi yang tepat dalam menanggulangi
minyak di pantai berpasir Cilacap, evaluasi efikasi metode bioremediasi di
lapangan telah dilakukan dengan pendekatan mesokosme di daerah pasang surut
Cilacap. Hasil penelitian menunjukkan bahwa penurunan konsentrasi minyak
mencapai 60.4 %, 74.5% dan 73.5 % dalam kurun waktu 3 bulan, masing-masing
pada kontrol, perlakuan kultur tunggal dan kultur campuran. Biaugmentasi dapat
meningkatkan laju biodegradasi di pantai berpasir tropis. Namun perbedaan
efikasi dari penambahan kultur tunggal dan kultur campuran secara statistik tidak
nyata
Evaluasi biostimulasi dan kombinasi biostimulasi-bioaugmentasi telah
menunjukkan bahwa efektivitas penurunan konsentrasi minyak pada perlakuan
kombinasi biostimulasi-bioaugmentasi lebih cepat teramati dan lebih tinggi
daripada perlakuan biostimulasi saja. Pada hari ke-16 setelah aplikasi pupuk
yang ditambah kultur campuran dan kultur murni persentasi penurunan
minyaknya masing-masing mencapai 2.2 dan 1.6 kali lebih tinggi daripada
kontrol Namun untuk periode yang lebih panjang, kedua perlaku an
kombinasi ini memiliki efikasi yang relatif sama. Untuk itu, penggunaan
kombinasi biostimulasi dengan kultur campuran lebih disarankan untuk
menangani pencemaran dalam periode yang pendek .
Kesimpulan dari kajian ini adalah tumpahan minyak di lingkungan pesisirtercemar minyak yang kronis, dapat terdegradasi secara alami tetapi dalam tingkat
yang rendah. Bioremediasi baik melalui bioaugmentation, biostimulation maupun
kombinasi keduanya terbukti secara signifikan mampu meningkatkan laju
degradasi minyak. Kombinasi bioaugmentation dan bistimulation menunjukkan
tingkat penurunan konsentrasi minyak paling tinggi. Hal itu terbukti dalam
percobaan in vitro dan mesocosm. Ada agen mikroba indigenous dan pupuk
produk lokal yang prospektif untuk digunakan dalam menanggulangi tumpah
minyak. RCO/B/08-008 dalam bentuk kultur tunggal atau dalam kultur campuran
dengan RCO/B/ 08-004 dan RCO/B/ 08-015 dapat diusulkan sebagai agen
bioremediasi, serta pupuk lepas lambat Gra sebagai agen stimulan potensial.
Pemberian kultur tunggal atau campuran untuk daerah tercemar minyak kronis
tidak menimbulkan efek yang berbeda dalam meningkatkan laju degradasi
minyaknya. Perbandingan nilai C:N rasio harus dipertimbangkan dengan hati-hati
sebelum menambahkan pupuk di wilayah tercemar minyak. Untuk pesisir Cilacap,
penambahan pupuk G pada konsentrasi N dengan perbandingan C:N = 1000:75
dan kultur bakteri dengan kepadatan 0.5 x 108 sel mL-1 cukup untuk
meningkatkan laju degradasi minyak.
Kata Kunci: Bioremediasi, Bioaugmentasi, Biostimulation, Efikasi, Mikrokosmos,
Pupuk lepas lambat, Konsorsium, Kultur tunggal, Minyak mentah
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EFFICACY OF BIOREMEDIATION METHODS FOR OIL
CONTAMINATED SANDY BEACH IN CILACAP, CENTRAL
JAVA, INDONESIA
YETI DARMAYATI
A dissertation
submitted in partial fulfillment of the requierements for the degree of
Doctoral
in Marine Science Major
POST GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016
Penguji pada Ujian Tertutup : Prof Dr Ir Dietriech Geoffrey Bengen DEA
Dr Ir Agung Dhamar Syakti DEA
Penguji pada Ujian Terbuka : Prof Dr Ir Dietriech Bengen DEA
Dr Ir Zainal Arifin MSc
Dissertation Entitle : Efficacy of Bioremediation Methods for Oil Contaminated
Sandy Beach in Cilacap, Central Java, Indonesia
Name
: Yeti Darmayati
NIM
: C561100022
Approved
by
Graduate Committee
Prof Dr Ir Harpasis S. Sanusi, MSc.
Chairman
Dr Ir Tri Prartono, MSc.
Member
Prof Dr Ir Dwi Andreas Santosa, MSc.
Member
Prof Drs Ruyitno Nuchsin, MSc.
Member
Acknowledged by
Head of Marine Sciences Major
Dean of Graduate School
Dr Ir Neviaty P. Zamani, M.Sc.
Dr Ir Dahrul Syah, M.Sc.Agr.
Date of Close Examination : 29 January 2016
Date of Open Examination : 5 February 2016
Date of Graduation :
PREFACE
Praise and gratitude author prayed to Allah SWT for His abundant
blessings and gifts so that this dissertation was completed. The dissertation
entitled Efficacy of Bioremediation Methods for Oil Contaminated Sandy
Beach in Cilacap, Central Java, Indonesia, is conducted to develop an
effective clean-up method for reducing the impact of oil spills in tropical marine
environment.
I am immensely grateful to those individuals who have helped to complete
my doctoral study in Bogor Agricultural University. As the outset, I would like
to express my great appreciation to Prof Dr Ir Harpasis S. Sanusi MSc, for his
enthusiastic support, guidance, being always there, and the time spent in guiding
me through the writing process. As my supervisor, his observation and
comments helped me to establish the overall direction of the research and to
move forward with its investigation in depth. I would like also to express my
gratitude to my supervisors Prof Drs Ruyitno Nuchsin MSc, Dr Ir Tri Prartono
MSc, and Prof Dr Ir Dwi Andreas Santosa MSc for their help, support and
guidance both during the study and in the writing of a dissertation
I acknowledge Prof Dr Ir Dietriech Geoffrey Bengen DEA, Dr Ir Agung
Dhamar Syakti DEA, and Dr Ir Zainal Arifin MSc as external examiner
commission has been testing, analyzing and providing input for the perfection of
this dissertation. Many thank to Dr Ir Neviaty P. Zamani MSc as the Chairman
of Marine Science study program who has directed and encouraged me for
implementing education in Marine Science Program SPs IPB. I will not forget Dr
Dirhamsyah MA, and Dr Suhartati M. Natsir for their great help and granted
permission during my doctoral program.
I would like to express my thanks to Ministry of Research and
Technology and Indonesian Institut of Sciences which has provided me
scholarships for Doctoral Program in IPB and financial support for the
implementation of this research, respectively. Also, to PERTAMINA Refinery
Unit IV Cilacap and BPLHD Cilacap for their cooperation to conduct this
research in Cilacap.
I would like to express my sincere thanks to all my best friend Dr Dwi
Hindarti, Dr Woro Hastuti, Dr Hayati Dr Etty Riani, Noor Farida AMd, and Dr
Aleh Human Saleh for their help, prayer, encouragement, enthusiasm and for
being very good friends. I will not forget to Fellow Major Marine Science Post
Graduate School-IPB: Dr Yunita F. Noya, Dr Rhozirwan, Dr Noverita, Khalid,
Tumpak Sidabutar, Ani, Denti, Dr Nurul Dhewani, Femi Hukom, for your help,
support and togetherness. I appreciate Surahman, Bella S. Rosepta, Wahyu
Utomo, Tia D. Martianingsih, Dwi Astuti, Linda Safitri and all member of
bioremediation research group Research Center for Oceanography – LIPI for
their technical assistance in this research.
Last, but not least, I would like to dedicate this dissertation to my beloved
husband, Mochamad Erawan, my dearly loved daughters, Marina Irawati
and Mazida Fidelia Darmawati, and my adored sons, Gwyn Walesa and
Muhammad Ardy Makarim for your prayers and sacrifices outstanding
during the author completed education. Also, My mother and father (alm.), my
sisters, Tuti Darmawati, Eli Darmalingganawati, Darmawidyawati, and my
brothers, Dudung Dumadi Fachrudin and Dadang Darmayana. Without dear
support and encouragement, I could not have completed my doctoral study.
I Hope this scientific work will be useful.
Bogor, January 2016
Yeti Darmayati
C561100022
xi
TABLE OF CONTENT
LIST OF TABLE
xii
LIST OF FIGURE
xiii
1 GENERAL INTRODUCTION
Background
Logical Framework
Objective and Purpose
Goal
Novelty
1
1
2
4
5
5
2 LITERATURE REVIEW
Oil Pollution in Marine Environments
Biodegradation of Oil
Bioremediation in Oil Polluted Shoreline
6
6
10
17
3 GENERAL METHODOLOGY
Time and Study Site
Reseach Steps
Materials Preparation
Laboratory Experiment
Field Experiment
Sample Analysis
Data Analysis
26
26
27
28
31
33
36
37
4 POTENTIAL MICROBIAL AGENT FOR CRUDE OIL
DEGRADATION
Introduction
Materials and Methods
Result and Discussion
Conclusion
38
38
39
42
45
5 ALTERNATIVE SLOW RELEASE FERTILIZER ENHANCES
BIODEGRADATION OF OIL CONTAMINATED TROPICAL
SANDY BEACHES
Introduction
Materials and Methods
Result and Discussion
Conclusion
47
47
48
50
55
6 OPTIMAL BACTERIAL DENSITY AND FERTILIZER
CONCENTRATION FOR BIOREMEDIATION OF OIL
CONTAMINATED SANDY BEACH
Introduction
Materials and Methods
Results and Discussion.
Conclusion
56
56
57
59
65
xii
7 THE EFFICACY OF BIOAUGMENTATION ON REMEDIATING OIL
CONTAMINATED SANDY BEACH USING MESOCOSM
APPROACH.1
Introduction
Materials and Methods
Results and Discussion
Conclusion
66
66
66
70
75
8 THE EFFECT OF BIOSTIMULATION AND BIOSTIMULATIONBIOAUGMENTATION ON BIODEGRADATION OF OILPOLLUTION ON SANDY BEACHES USING MESOCOSMS
Introduction
Materials and Methods
Results and Discussion
Conclusion
76
76
77
81
89
GENERAL DISCUSSION
900
CONCLUSION
92
REFERENCES
93
CURRICULUM VITAE
104
LIST OF TABLE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Shoreline clean-up options, benefits and limitations
Common methods in chemical analysis of oil and oil constituent
Common methods in microbiological monitoring
Parameters measured and methods can be used in monitoring oil
bioremediation
Selected cultured used for the experiment
Physicochemical characteristics of Arabian Light Crudeoil used
(provided by PERTAMINA 2011)
Composition of slow release fertilizer content
Treatments for bacterial and fertilizer selection experiments
Treatment for optimization bacteria and fertilizer experiment
Treatment employed in field experiment
Measurement of physicochemical parameters
The composition of bacterial mixed culture
Environmental parameters condition over 28 days experiment
Soluble Total Nitrogen and Phosphate during experiment
Treatments performed for optimalization study
Environmental parameters in the range during 28 days experiment
Pore water quality in average and range value during 90 day
sexperiment
First order rate constant (k) value in different treatment at various
periode
10
23
24
25
29
30
31
31
32
35
37
39
52
54
58
65
70
72
xiii
19 k value in each treatments at different time periods
20 Pore water quality over time experiment inside and outside mesocosm
82
84
LIST OF FIGURE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Logical Framework
Major weathering processes after an oil spills (ITOPF 2009)
Representative organic compounds found in crude oil
Locationof field experimental site in Cilacap Coastal
Flow chart of research steps
Bioreactor
Experiment in microcosm system
Plot lay out on the inter-tidal foreshore of Cilacap Coastal
Schematic design of mesocosm
Porewater sampler and capiler hose forsucking the sample
Research design for bacteria selection
Residual oil content in single (A) and mix culture (B) treatment
Bacterial growth of single (A) and mix culture (B)
Oil depletion percentage in different fertilizer treatment during 28 days
experiment
Bacterial growth in 4 different SRF treatments over 28 days
experiment. Standard deviations of the replicates (n = 3) are denoted by
the error bars. Columns marked by the same letter (a to d) are not
significantly different (p