universiti putra malaysia degradation of benzene, … · contaminated soils from petronas gas plant...
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UNIVERSITI PUTRA MALAYSIA
DEGRADATION OF BENZENE, TOLUENE, ETHYLBENZENE, AND XYLENE BY ISOLATED MICROORGANISMS FROM PETRONAS
GAS PLANT AT KERTIH, TERENGGANU
HAMID REZA SALSALI
FK 1999 16
DEGRADATION OF BENZENE, TOLUENE, ETHYLBENZENE, AND XYLENE BY ISOLATED MICROORGANISMS FROM PETRONAS GAS PLANT AT
KERTIH, TERENGGANU
HAMID REZA SALSALI
MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA
1999
DEGRADATION OF BENZENE, TOLUENE, ETHYLBENZENE AND XYLENE BY ISOLA TED MICROORGANISMS FROM PETRONAS GAS PLANT AT
KERTIH, TERENGGANU
By
HAMID REZA SALSALI
Thesis submitted in Fulfillment of the Requirements for the Degree of Master of Science in the Faculty of Engineering
U niversiti Putra Malaysia
November 1999
ACKNOWLEDGEMENT
I would like to express my great appreciation and gratitude to my principal
supervisor, Dr. Azni Hi Idris and Co-supervisor Dr. Abdul Manaf Bin Ali and Dr. P.K.
Roychoudhury, for their close supervision and the endless hours of discussion and
dedication.
I must also extend my gratitude to Petronas Gas Berhad, Kertih Terengganu for
their assistant in collecting my sample. Thanks to Mr. Majeed Ishaqe for his helpful
advice and comments, also to all my colleges in Microbiology and Environmental Lab.
I'm also grateful to the opportunity given to me by the Faculty of Engineering,
Universiti Putra Malaysia, to enroll and work as a postgraduate student at the department
of Chemical and Environmental Engineering.
My great appreciation goes to my parents, brothers and sister for their moral
encouragement. I'm also greatly indebted to the patient of my wife who supported me
throughout all stages of my studies and suffered a great deal from my dedication to this
work. In return to her care, support and encouragement, I present this thesis with my
deepest love ...
iii
TABLE OF CONTENTS
Page ACKNOWLEDGEMENT... .. . ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 111
LIST OF TABLES ...... ... ... ... ... ... ...... ... ... ...... ...... ... ... ... ... ... .............. vii
LIST OF FIGURES ........ ...... ... ............... ...... ...... ... ... ... ...... .............. ix
LIST OF PLA1'ES ... ... .. . . .. . .. ... . . . .. . ... ...... ... ... ... ...... ... ... ... . . . . .. . . . .. . ... ... xi
LIST OF ABBREVATION . . . ... ... . .. ... ... . . . . .. ...... . .. .. . ... . . . ... ... .. . ..... . . . . ... xii ABS1RACT . . . . .... . ... . . . ..... .. .. . ... . . . . . . . . . .. . . . . . . . . . . . . . . . . ... . . . ... .. .. . . .. . . . . . .. . . xiii ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
CHAP1'ER
I IN1'RODUCTION . . . . .. ... . .. . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . ... . .. . . . . .. . .. ... 1
Objective ofthe Study ..... . . ..... ... ...... . . . .. .. ... . .... . .. '" ...... ........... 3
II LITERATURE REVIEW ... . . . ... ... . . . ... ... .. . . . . . . . .. . . ... . .. . . . . ... . .... ... .. . 4 Hydrocarbon of Interest . .. ... ... .... .. ... ... ... ... ... ... ... . .. ... ..... .. . ....... .4
Benzene ............... ... ... ...... ... ...... ... ... ............ ...... ...... ... 6
Benzene Exposure Routes and Health . .. . . . .. . ...... ... . .. .. . . .. '" ..... 7 Toulene . . . . .. ........ . ..... . . . . . .. ... ... ... . . . ... . .. .. . ... . .. ... . .. . . . .. . ... 7 Toluene Exposure Routes and Health ......... .... ..... ... ... .. . ........ 8 Ethylbenzene . . ...... .. ..... . ... . . . ... . .... .. ... . . . ... ... . . . . .. . .. ... . .. .. . ... 9 Ethylbenzene Exposure Routes and Health . . . . . ...... . . .. . . . ... . ....... 9 Xylene ... ... . . . . . . . .. . . . . . . . . . . .... . . . . . . . . .. . . . . .. . . . . .. . . . . . . . . . . .. . . . . .. .. 10 Xylene Exposure Routes and Health . . . . . . . . . . . . . . . . . . . . . . . . ... .... . . . .1 0
Risk Assessment . . . ... ... ... . . .. . . . , .. . . . . . . . . . . . . . . . . . . . .. . . . , . . , ... . ...... . . . .. . .. . 1 0 Hazardous Waste Treatment Methods ... . . . . .. . . . . . . . . . . . . . . . . . . . . . ... . . . .. . ..... 11
Chemical Treatment of Hazardous Waste ..... . ... .. . ......... . . . . . . ... 12
Chemical Techniques .. . ... ... '" ...... ... ...... . .. .............. .... . .... 13
Physical Treatment of Hazardous Waste ....... ...... ... ...... ..... . .. . 14 Physical Techniques . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ... . .. . . . 15
Biological Treatment of Hazardous Waste .. . . . . ... . . . . . . . . . . . . . . . . . . . . . ... ..... . . 16
Biological Techniques ...... ..................... ............ ............. 16 Microbial Growth and Metabolism .... . .... .... . . . .. . .. ...... ... . . . ... .. . . . . . .. 18
Microbial Reactions ... .... . . ... .. . ... ...... .. . .. . ......... . . . . .... , ...... 19 Biodegradation Kinetics and Rates . ......... . . .. . .... . . ...... ... ....... 23
IV
PERPUSTAKAAN UNlVEUITI P'UTItA MALAYSIA
Mineralization..................................................................... 26 Biodegradation of Benzene, Toluene, Ethylbenzene, and Xylenes In Gas-Condensate Contaminated Ground Water . .... ... ... .......... .... .. 27 BTEX Metabolism Pathway...... ... ... ... ...... ... ... ......... ... ... ...... ..... 28
Aerobic Metabolism ......... . . ..................... ................ ..... . 29 Anaerobic Metabolism ........ .... . . . ... ................. ............ . . .. . 30
Benzene Pathway . ..... ..................... ..... .... .......... .... . . . ..... 32 Toluene Pathway ...... ..... . ............. ..... ............ ...... ......... 33 BenzoatePathway . .. . ... " .................................................. 36 EthylbenzenePathway ... ........ . . . . ........ ..... . '" .. , .................. 38 m-XylenePathway ..... . .............. . ...... . . . ... ...... . . . ...... ...... ... 42 o-Xylene Pathway ........... ...... . .. ................ ..................... 44 p-Xylene Pathway . . . . ..... .............................. ... . . .......... . .. 46
Bioremediation .. . ....... . . . . . . .. .. . . . . . . . ... . . ....... . .. ....... . ......... . . .. ...... 49 Microbial Cultures ... . . . ................. . .... . . ... .. . . .... ....... ... ............... 52 Factors Affecting Microbial Growth ... ... .... .. . .................... ..... . ...... 53
Effect of Temperature on Growth . . ... ....... ........ . . . .. ....... . ....... 54 Effect of Microbial Population on Growth. . ... ..... . ........... . . ..... 54 Effect of Oxygen Availability on Growth............... .. .......... 54 Effect of Nutrient Concentrations on Growth ............... ......... .54
III MATERIALS AND METHODS ................. . . . . . ........ .................. 55 Site Study ......... . ........ . . ....... . . . . . . . . . ...... .. . . .. . . . ... . . . .. . . . . . . . . .. . . . . ... 55
Equalization Basin .... . . .... . . .. . . . . . .............. ...... ... ... . ..... . . . ... 55 Aerated Lagoon . .. . . . . . . . .... ...... . . . . .. . ... ......... . . . ... ... . ...... . . . ... 55 Site Problem ......... . . . . .. . .. ... . .. ..... . . .. . . . . ... .. .... . . ... ....... . ..... 56
Sample Collection and Handling... . ................................. , .... '" ... 56 Hydrocarbon Preparation . . ... . . . . .... ..... . . . . . . ... .. . ... . ..... ... .... . . . . . . . . . . .. 57 Chemicals and Instruments . . . . . . ... . .. .... .... . . . . . . . ....... .. . , ................ . 57
Chemical ..... . . .. .... . . . . . . . ........ ..... ......... . . . ... ... . ... . ... . .... ..... 57 Instnunents .... ... .. ... ..... . ... .. . . . . .. . ..... . ..... . .. . ... . . . . ..... . .. ...... 57
Media Preparation ...... . .. .. . .. .. . . . .. . . . . . .. . ..... . . .... . . ..... .... . . . . . . .......... 59 Reagent Preparation . . . . .... . . . . . . . . .. . ... .. . .. . . . . . . . .. .. . . . . . . . . . . ..... , .......... 60 Isolation and Screening .. ....... . . . .. . ... ... . .. . . . . . . . . . .. . . . . .. . . . . . . . .. . .. . . . . . . . 60
Primary Screening ... . .. . . . ..... . . .. . . ... ... . ........ . .. . . .... ... . .... . . .. . 60 Enrichment Culture Method . . . . . . .. . .... . . .......... .. ... . .. . ... . . . . ... 60 Screening of Isolates ... . ........ . . ........ . . ...... ... . ..... .. . ... ........ . 61 Secondary Screening . . ........ ........... ..... . .... . .... . . . ......... " .... 61
Inoculwn Prepration ... ... ... . . . . ... . .. . . . . . ... . . . . . . .. . . . . . . . . .. . . ... . .. , ......... 62 Effect of Different Hydrocarbon Concentration . . . . . . . ... .. .... .. .. . ... . .. . .. 62 Growth Rate Study . ............ ............. . ... ........... . . . .. .. .... . .... ..... . 63 Degradation Study ofBTEX . . . ... .. . . . . . . .. . .... . . . . . . . . . .......... ....... . . ... . . 63 High Performance Liquid Chromatography .. .. ..... . . . . .. . . . ..... . . . . .. . .. . .. 65
v
Reagents .. . . .. '" . . . . . ... , ... .. .. ..... ... ... . .. ... '" ... . .. .. . ... ... .. . . . ... 65 Analysis of Data ... ...... ... ..... . . . . . .. ... . . . . .. ... . . . . . . . . . . .. . .. . . . ... .. 66
Gas Chromatography Mass Spectra (GC-MS) Procedure ... . .. . .. .. . . . . .. ... 65 Apparatus .. . . . . . . . .. . . . . . . . .. . . .. . . . . . . . .. .. . . .. . . . . . .. . . . . . .. . . . . . .... . . . . 65 Analysis .. . ... .. . . . . . .. .. . . .. . . ... . . . . . .. . . . . .. . . . . . . . . . . . . . .. . . . .. . . . . .. . .. . 66 GC-MS Parameters .. .. . . . . . . . .. . .. .. .. .... . .. .. . . . . ... ... . .. . . . .. . ... ..... 66
Mass Spectrometer Parameters . .. . .. . . . ... . .. . . . . . . . . .. . . . " .. . ... ....... 66
N RESULTS AND DISCUSSION ... . . . '" ... ... ...... ... ............ ... ... ... .... 67
Isolation and Screening ... ...... ... ............ ... ... ...... ... ......... ... ....... 67 Primary Screening . . . .. . . .. .. . ... . . . . . . . . . . . . .. . . . . .. . . . . . .. ... . . . . . . . . . . . . 68
Secondary Screening . . . . . . . .. . .. . . . . . .. .. . . . . . . .. , ... ... ... . .. ... ... ... ... 7 0 Effect ofBTEX Concentrations on Bacterial Growth ... . ... . .... . . . . .. ... ... 7 3 Growth of Bacteria on Formaldehyde . . . . . . . . . . . . .. . . . . . . . ...... . . . .. . .... . . . . . . 78 Growth Curve ofIsolated No.4... ... .. . ... ... . . . .. . .. . . . . . . . . .. . .. .. . ... . . . . . . .. 80 Biodegradation ofBTEX . .. . . . . . . . . . . .. '" ... ... .. . ......... ... ...... ...... ...... 82
V CONCLUSION .. . ... . . . .. . . . . . . . . . ... . .. . . . . . . . . . . . . . . ... .. . .. . . .. . . . . . . . . . . . ... .. . 1 01
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03
APPENDICES . . . . .. . . . . .. .. . . . . . . . . . . . . . . . , ... ..... , ... ... ... ... ... ... ... . .. .. . ... ... ... ... 1 09
A Benzene Irritation and Inhalation Data . .. . . . . ... .. . . . . . . . . . . .. . . . . . . . . . . . . . . . 1 1 0 B Toluene Irritation and Inhalation Data . . . . .. .. . . . . . .. . . . ... .. . .. . .. . .... .. .... .1 1 1 C Ethylbenzene Irritation and Inhalation Data .. . . . . . . . ... . . . ... . .. . . . . . . .. . . .. 1 12 D Example of Growth Rate Calculation . . . .. . . . . .. , ...... ....... .. ... . , . ......... 1 13 E GC-MS Results ... ... . . . . . . . " ...... ...... ........................... ............... 114
BIODATA OF AlJTfIOR . . . .. . . . .. . .. .. . . . . . . . .. . . .. . .. .. . . . . . . . ... . . . .. .. . . . .. . .. ... .. . . . . 1 1 7
vi
LIST OF TABLES
�� � 2.1 Physical properties of selected aromatic hydrocarbons (BTEX) . . . . . . . . . . . . . . . . .. .5
2 2 Composition (Mass fraction) ofBTEX in gasoline .. .... ... ...... . . . ... . , . ......... .. 6
2.3 Summary of benzene exposure routes and health .. . ... ... . . . . . . . .. .. . ... . .. . . . . . . . . . . . 7
2.4 Summary of toluene exposure routes and health .. . . . . . . . . . . . . . . . . . . . . , . . .... . .... . .. .. 8
2.5 Summary of ethylbenzene exposure routes and health . . . ... . .. . . . . . . .. . ... . . . .. . .. . .. 9
2.6 Summary of xylene exposure routes and health . . . . .. . . . .. . . . . . . . . .. ... .. . . . . . . . .... . 10
2.7 Summary of previous key papers .. . . . . . . . . . . . . .. . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 8
2.8 Advantages ofbioremediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.9 Disadvantages ofbioremediation .. . ... ... ... . . . .. . ... . ........ . .. ... .. . . . . ....... .. . ... 51
3.1 list of chemical used in this study... . . . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . ... . . . . .. ... . . . 5 7
3.2 list of instrument used in this study . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3. 3 Equipment used in GC-MS analysis . . . . . . . . . . . . . . . . . . . .. . . . . .. . .. .. . .. . . . . . . . . . . . . . . . . 65
3.4 GC-MS parameters . . .... . . . . .. . . , .. , ... ............ ... ...... ...... ... ... ... .............. 66
4.1 Total bacteria population of the different samples (Media used was NA) . . . . . . . 68
4.2 Growth of bacterial isolated in primary screening after 3 days (in 0.1% VN) . . 6 9
4.3 Growth of bacterial isolates in lo/o(VN) BTEX ......... .. . ... ... ...... ... ...... .. ... 71
4.4 Growth of bacterial isolates in different concentration of benzene . . . . . . . .. .. . . . . . 74
4. 5 Growth of bacterial isolates in different concentration of toluene ... . . . . .. .. . . . . . . 75
4.6 Growth of bacterial isolates in different concentration of ethylbenzene . . . . . . . .. . 76
4.7 Growth of bacterial isolates in different concentration ofx.ylene . . . .. . .. . . . ....... 77
4.8 Growth of good isolates on formaldehyde . . . . . . . . . . . . . . . . . . . .. . . ... .. .. .. . . . . . . . . .. . . 79
vii
4.9 Calculated specific growth rates at 50,60 and 70% (v/v) ofBTEX . . . .. . . .. . . . . . . . 81
4.10 Rate of benzene, toluene, ethylbenzene, and xylene degradation . . . . . . . . . . . . .. . .. 94
viii
LIST OF FIGURES
B� p.
2.1 Pathways ofbenme, toluene, ethylbenzene,and xylene degradation . . . . . . . .. . .. . .21
2.2 Disappearance curves . . . ... . . . '" ...... .. . .... .... .... ... ........... ............. ... . ..... 25
2.3 Aerobic degradation of the BTEX ... ...... ... ... ... ... ... '" ... ...... ... ... ...... ... ... 30
2.4 Anerobic degradation of the BTEX . .. .. . ... ... . . . . . .... ... ... .. . . . . . . . .. . . . . . . . . . . .. .. 31
2.5 Benzene degradation pathway . . . .. . . . . . . . . .. . . . . . . .. . .. . . .. .. . . . . ... . . . . . . .. . . .. . ..... . .32
2.6 Aerobic degradation ofbenzene . . . .. . . ... . .. . . .. . .. . ...... ... ... ... ... . . . ... . .. ....... .33
2.7 Toluene degradation pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.8 Anaerobic degradation of toluene ... ... ...... ...... ...... ...... ...... ...... ... ..... , .... 35
2.9 Benzoate degradation (anaerobic) pathway ...... ...... ...... ... ............ ... ... .... 38
2.10 Anaerobic degradation of ethylbenzene ...... ...... ...... ...... . , . ... . , .... '" ... ..... .40
2.11 Ethylbenzene degradation pathway ........ , ... ... ... ... ... ...... ......... ... ... ... ..... 41
2.12 m-xylene degradation pathway ........... , . . . . . . . . . . .. . .. . . . . . . .. . . . . . . . . .. ... . ... . . . . .. 42
2.13 Anaerobic degradation of the m-xylene ... ......... ......... ... ............ ...... ...... 43
2.14 o-xylene degradation pathway ... ... ...... ...... ... ...... ... ... ...... ............ ........ 44
2.15 Anaerobic degradation of the o-xylene ... ... ... ... ... ... ... ... ... ... ... ... ...... ... ... 45
2.16 p-xylene degradation pathway ...... ... ...... ... ......... ... ... . " ........ , '" ... ....... 47
2.17 Aerobic degradation of the p-xylene . . . ... . . . ... . . . ... . .. . . . .. . . . . . . . . . . .... . . .. . ... . . . 48
2.18 Bioremediation treatment systems .. . ....... ..... ...... ...... ...... ... ......... ... ..... .50
3.1 Site sample collection station .. . . .. .. . . . . . . . . . ... . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . .. . . . .. 58
4.1 Growth curve of bacteria No.4 in 50, 60.and 70 % benzene... . . .... .............. 83
ix
4.2 Growth curve of bacteria No.4 in 50, 6O.and 70 % toluene ... '" ..... , ...... ..... 84
4.3 Growth curve of bacteria No.4 in 50, 60.and 70 % ethylbenzene... ... ... . .. .... 85
4.4 Growth curve of bacteria No.4 in 50, 6O.and 70 % xylene ... ... ...... .. , ... ... ... 86
4.5 Rate of benzene elimination under anoxic condition ... ... ... ... ... '" ... ... ... ...... 88
4.6 Rate of toluene elimination under anoxic condition ... '" ... '" .,. '" '" .. , ... ..... , .88
4.7 Rate of ethylbenzene elimination under anoxic condition ..... , ...... ... ...... ... ... 89
4.8 Rate of xylene elimination under anoxic condition ...... ...... ...... ... . , . ........... 89
4.9 Removal of benzene under anoxic condition ... ... ...... ... ...... ... ... ... ...... ...... 90
4.10 Removal of toluene under anoxic condition ......... ... ... ... ... ... ... ................ 90
4.11 Removal of ethylbenzene under anoxic condition ...... ... ...... ... '" ......... ... ... 91
4.12 Removal of xylene under anoxic condition ...... ... ...... ...... ... ... ... . , . ... ... .... 91
4.13 Mass spectra of I -phenylethanol.. . ... ...... ...... ...... ... ...... ... ... . " ... .......... 95
4.14 Mass spectra of acetophenone ...... ... ... ... . " ...... ... ...... ... ...... .... , . ... ...... 96
4.15 BTEX chromatogram at time zero . . . . . . ... ... ... ... ... ... ... ... ... ... ... . . . ... ... ... .. 97
4.16 BTEX chromatogram after 2hrs ... ........... , ...... ......... ... ...... ... .. , ........... 98
4.17 BTEX chromatogram after 48 hrs ... ... ... ... ... . . . ... ... ... ... .. . . . . . . . ... ... . " ..... 99
4.18 Proposed pathway of initial reaction in anoxic ethylbenzene oxidation... ... ... 100
x
LIST OF PLATES
p�� p�
1 Site samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' " .............. 56
2 Me Farland standards . . . . . . . . . . . . '" ...................................................... 62
3 Viable cells of isolate No.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4 Growth of isolate No.4 on 10 up to 80% (VIV) benzene .............. .............. 74
5 Growth of isolate No.4 on 10 up to 80% (VIV) toluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6 Growth of isolate No.4 on 10 up to 80% (VIV) ethylbenzene . . . . . . . . . . . . . . . . . . . . . . 76
7 Growth of isolate No.4 on 10 up to 80% (VIV) xylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Xl
BTEX
CO2
cfu/rnL
g
GC
GC-MS
HPLC
mgIL
v/v
w/v
TLV
IDLH
M
LIST OF ABBREVIA nONS
Benzene, toluene, ethylbenzene, xylene
Carbon dioxide
Colony forming unitlrnL
gram
Gas chromatograph
Gas chromatograph mass spectra
High Performance Liquid Chromatography
Milligram/litre
volume/volume
weight/volume
Threshold limit value
Immediately dangerous to life and health
Mixture of benzene, toluene, ethylbenzene, and xylene
XlI
Abstract of thesis submitted to the Senate ofUniversiti Putra Malaysia in fulfillment of the requirements for the degree of Master of Science
DEGRADATION OF BENZENE, TOLUENE, ETHYLBENZENE, AND XYLENE BY ISOLATED MICROORGANISMS FROM PETRONAS GAS PLANT,
KERTIH TERENGGANU
By
HAMID REZA SALSALI
November 1999
Chairman Assoc Prof Dr Azni Hj Idris
Faculty Engineering
A total of 38 isolates originating from five different sources of sediments, and
contaminated soils from Petronas Gas Plant at Kertih, Terengganu were obtained through
enrichment culture method using 0.1 %(v/v) of benzene, toluene, ethylbenzene, and
xylene or mixture of them as their sole energy and carbon sources. Most of the isolates
were from S5 (Residential area) and the least from Sl(Equalization basin). Out of this, 11
good isolates were able to grow in varying hydrocarbon concentration up to 50% (v/v)
BTEX. Only isolate No.4 showed extremely good growth in concentration up to 80 %
(v/v) of BTEX.
Using benzene at 60% (v/v), the growth rate was observed to be highest during
the first 2 hours, while degradation rate and percentage of removal by using isolate No.4
showed 1 .5E-04 mMis and 75 % respectively. For toluene at 60% (v/v), the growth rate
Xlll
was highest during the first 3 hours, while degradation rate and percentage of removal
were SE-OS rnMIs and 70 % respectively. In the case ethylbenzene at 60% (v/v) , the
growth rate was highest during the first 7hours, while degradation rate and percentage of
removal by using same isolate showed lE-04 rnMIs and 98% respectively. For xylene at
60%(v/v), the growth rate was highest during the first 5hours, while degradation rate and
percentage of removal achieved were 5.35E-04 rnMIs and 8.9 % respectively. In general,
all the isolates had tolerated the organic solvent (BTEX), which may be due to the
adaptability of the cell membrane through a metabolic or enzymatic properties.
Both High- Performance Liquid Chromatography and Gas Chromatography
Mass-Spectra profile had clearly showed a BTEX-peaks area ratio reduction, where the
isolates were shown to biodegrade the hydrocarbon either by mineralization or
biotransformation. However, based on the almost complete peak reduction as well as
appearance of unidentified peaks, it may be suggested that BTEX can be degraded by
naturally present microorganisms which was identified at Petronas Gas Berhad site.
xiv
Abstrak tesis yang dikemukakan kepada Senat Universiti Puta Malaysia sebagai memenuhi keperluan untuk Ijajah Master Sains
PEREPUTAN BENZENE, TOLUENE, ETILBENZENE, DAN EXYLENE
DENGAN MENGGUNAKAN PERGASINGAN MIKROORGANISMS MENGGUNAKANPETRONAS GASKllANG, �ERT1HTERENGGANU
OLEH HAMID REZA SALSALI
November 1999
Pengerusi Prof.Madya.Dr. Azni Hj Idris
Fakulti Kejuruteraan
Terdapat lima jenis "inocular" ia termasuk pemendapan, enapan pas!r, hasil
keluaran proses yang digunakan untuk mengefektifkan kaedah kultur bakteria bagi
benzene, toluene, etilbenzene, dan exylene. Sejumlah 38 bahan tersingkir yang diperolehi
menggunakan 0.1 %(v/v) samada benzene, toluene, etilbenzene, dan exylene atau
campuran bahan-bahan tersebut sebagai sumber utama tenaga dan karbon kebanyakkan
singkiran adalah daripada S,(Luas Penempatan) dan yang lain daripada Sl(Tangki
Penyamarataan )dari kaedah kultur.
Daripada jumlah ini, hanya 38 bahan pemendap diskrinkan dalam 1 % (v/v)
BTEX. Di antaranya, 1 1 pemendapan yang baik dibiakkan dalam berbagai kepekatan
sehingga mencapai kepekatan 50 %(v/v) BTEX, hanaya pemendapan No.4 menunjukkan
perkembangan ekstrem yang boleh menampung kepekatan sehingga 80 % (v/v) BTEX.
Kedua-dua '1Iigh Performance kromotografi cecair" dan gas profil spektra jisim
telah jelas menunjukkan BTEX-pengurangan nisbah luas puncak, di mana pemendapan
secara amnya membolehkan proses biodegradasi hidrokarbon samada melalui
mineralisasi atau biotransformasi. Walaubagaimanapun, berdasarkan kepada puncak
pengurangan, dan puncak penonjolan yang tidak dapat dipastikan, adalah dicadangkan
bahawa BTEX boleh didegradasikan oleh organisma asli di Petronas Gas Berhad.
CHAPTER I
INTRODUCTION
Aromatic hydrocarbons are components in petroleum products, which have been
used extensively as degreasing agents. These compounds are widespread use common
contaminants in ground water and soils. Both aerobic and anaerobic biodegradations of
aromatic compounds have been studied. The microorganisms responsible for degradation
have been identified and many of the enzymatic and genetic regulatory mechanisms have
been determined (Smith, 1994).
Hazardous waste generated from three major sources, which are industrial,
agricultural and domestic sectors. Potential entry points of pollutants into the
environment are effluent from domestic, industrial and municipal waste treatment plants,
emissions and solid wastes produced by the manufacturing sector. However chemical
products and chemical wastes undergo different routes into the environment, either may
enter the ecosystem directly or after passing through some sort of waste treatment system.
The wide use of chemical products by consumers makes their release difficult to control,
while the stream of concentrated waste chemicals released by manufacturing plants
justify the development of sophisticated treatment techniques. A great variety of pollutant
are released into environment by illegal dumping.
However organic compounds can be a major pollution problem in ground water. Their
presence in water can create a hazard to public health and the environment. The BTEX
group of contaminants consists of benzene, toluene, ethyl benzene, and three isomers of
xylene. Previous reports have been shown that BTEX was accumulating in the
2
ground water for over 20 years. There is now a two - foot thick layer floating on ground
water at approximately twenty feet below the surface ( Gossett, 1 987; Almedia, 1 995).
BTEX makes up one of the main groups of soluble organic compounds that find their
way into our soil and ground water. The release of petroleum products such as gasoline
(Evans, et ai., 1 99 1 ), diesel fuel, lubricating and heating fuel from leaking oil tanks
( Hutchins, 1 99 1 ), and cause of their polarity and very soluble characteristics, these
organic chemicals (BTEX) of petroleum products will be able to enter the soil and
groundwater systems, and cause serious pollution problems.
In peninsular Malaysia alone in 1987, about 26% of the rivers monitored have
been classified as polluted due to organic pollution and approximately 380,000m3 of
hazrdous waste are discharged annually into the environment. The oil and grease accounts
1 9,896m3, sludge, paint, dye, pigment, in the form of solvent about 950m3 and spent
solvent (non-halogenated) about 2,47 1 m3 (Hamid and sidhu, 1 993). However BTEX
group of contaminants make up a significant percentage of petroleum products. The
reason why the BTEX, entering our soil and ground water system, are considered such a
serious problem that have acute and long term toxic effects, especial ly with benzene
which is well known to be a carcinogen.
Great interest has arised in the field of environmental biotechnology to find
microbes capable of biological breakdown or conversion of organic compounds into
simpler compounds of mini mal environmental significance. Microbes are known to carry
out biodegradation, mineralisation on vast array of organic compounds. Studies to be
called as our own should be done owing to the fact that our climate and condition are
different from temperate countries. Research carried out in local conditions would then
3
prove to be more fruitful, practical and promising for our hazardous waste remediation in
the near future.
Objective of the Study
This study will be focused on the degradation of BTEX group of contaminants with the
following objectives:
(i) To isolate and screen of suitable microorganism sampled at Petronas Gas Berhad at
Kertih Terengganu.
(ii) To study of growth performance of native isolate at different BTEX concentration.
(iii) To investigate the biodegradability of BTEX compound using HPLC and GC-MS
respectively.
CHAPTERll
LITERATURE REVIEW
Hydrocarbon of Interest
Organic chemical are widely used in all facets of industries including machine
and electric manufacturing, metal finishing, automotive and engine repair, dry cleaning,
asphalt operations, dye manufacturing, agricultural activities, and food processing. The
usage of organic chemicals is of increasing concern to regulators and the industry in
general because of the contamination of soil and ground water resulting from the
mishandling and disposal of these chemicals.
Typically, these organic chemicals are stored in drums and tanks. Many
industries, especially gasoline fueling stations and trucking companies, store their
chemical in under ground storage tanks. The presence of large quantities of chemicals,
gasoline, and diesel fuel on-site is often an indicator of the potential for soil and ground
water contamination. The specific organic compounds are the petroleum hydrocarbons
and organic functional groups. The majority of sites an engineer has to deal with will
probably involve one of these contaminants, with gasoline being the most common at
the smaller site.
The carbon-containing compounds are classified as organIC compounds. All
organic compound contain carbon; virtually all contain hydrogen and possess at least one
C-H bond in their chemical structure. Hydrocarbons is the most basic organic compound
group contain only the elements hydrogen and carbon.
4
5
Hydrocarbons are divided into two classes, aliphatic hydrocarbon, which is a
straight chain hydrocarbon without a benzene ring. and aromatic hydrocarbon, which
contains benzene ring. A benzene ring contains six carbons joined in a ring structure with
alternative single and double bonds.
Aromatic hydrocarbons, also called alkyl hydrocarbons are characterized by
carbon-carbon ring structures, and as suggested by their name, a distinctive odor.
Benzene, C6a" is the simplest compound of this group of hydrocarbons. Joining
functional groups to the basic benzene ring can form other molecules. If there is only one
functional group is not specified because all six carbon atoms are equivalent.
Table2.1 lists the physical properties of some aromatic hydrocarbons that are
commonly encountered in soil and ground water contamination.
Table2.1: Physical properties of selected aromatic hydrocarbons (BTEX)
Chemical formula
Molecular w�igltl fwmole]
w ilt!:r �!ll!::!ilit:i fmWIl
VaQQr Qressure (at 20 C) [mmHg]
S12�ific Q�nsit3! (at 20 C)
Qganol-�Rt� s;!artitiQn coeff. (at 20 C)
llogKowl Henry's law constant (at
25 C) fkPa*m3/molel
Polarity
Biodegradability
Maximum Contaminant LeveJ(MCL)(mgll)
Benzene Toluene
CA C7�
78 92
1700 515
95.2 28.4
0.8787 0.8669
2.13 2.69
0.55 0.67
Non-polar Non-polar
Aerobic Anaerobic IAerobic
0.005 1
Adapted from (Jimmy et al.,1997).
m-Xylene o-Xylene p-Xylene
C8HW CgHIO CgHw 106 106 106
- 175 198 - 6.6 -
0.8642 0.8802 0.8610
3.20 2.77 3.l5
0.70 0.50 0.71
Non-polar Non-polar Non-polar
Aerobic Aerobic Aerobic
10 10 10
Ethyl benzene
CgHw 106
152
9.S
0.8670
3.l5
0.80
Non-polar
Aerobic
0.7
6
Petroleum is composed chiefly of saturated hydrocarbons, but may also contain
unsaturated hydrocarbons and their derivatives, nitrogen compounds, and sulfur
compounds.Regular gasoline is mixture of more than 100 organic compounds Table 2.2
gives an analysis of fresh and weathered gasoline (Jimmy et ai., 1997).
Table 2.2: Composition (mass fraction) ofBTEX in gasoline
Aromatic Hydrocarbons Molecular Weight(g) Fresh gasoline % Weathered gasoline%,
B 78.1 0.0076 0.0021 T 92.1 0.055 0.0359 E 106.2 0.0000 0.0156 X 106.2 0.0957 0.0151
Benzene
Benzene (or Benzin) is a member of the closed ring group of aromatic solvent.
Commercially produced from petroleum feed stock reformation and hydrodealkylation of
substituted aromatic. Petroleum naphtha from the craking fractionating of crude oil is
partially converted to benzene and other aromatic by exposing into a platinum catalyst at
elevated temperatures. Some possible reactions are:
CJI14 -+ C6H6 + 4H2
Hexane
(CH3)2Ct>lf1O -+ C� + 2C� + H2
Dimetyl cyclohexane
CH3C9H9 -+ C6� + 3 H2
Metyl cyclopentane
(2.1)
(2.2)
(2.3)
Overall benzene content maybe 5 to 10 percent. This recovered and purified using
processes suc.h as fractional distillation and solvent extraction. Benzene is used in the
production of ethylbenzene/styrene, cumene, cyclohexane, maleic anhydride, vinyl
copolymers and some acrylics and rubber based products (Sedivec , 1976).
7
Benzene Exposure Routes and Health
Benzene exposure routes and health are summarized in Table2.3.
Table2.3: Summary of benzene exposure routes and health
Sources
Human Health
Immediate Action
Irritation and Inhalation
Data
Gasoline, some food, drugs, pesticides, paints, plastic industries. ethylbenzene. styrene, cumene, cyclohexane
and maleic anhydride.
TLV:I0 ppm in the blood, IDLH:2000 ppm,and Carcinogen
Spill Control: Restrict access to spill site. Issue
warning: "FLAMMABLE". Call Fire department and
notify manufacture. Eliminate sources of ignition
including traffic and equipment. Stop the flow and
contain spill. if safe to do so. Avoid contact with liquid
I and vapour; stay upwind of release. Keep contaminated
water from entering sewers or watercourses.
Fire control: Use foam, dry chemical or carbon dioxide
to extinguish. Don't extinguish fire unless release is
stopped. Cool fire- exposed containers with water. Stay
clear of ends to tanks.
Irritation and inhalation data in appendix A
Adapted from Environmental and technology information: Benzene, 1984.
Toulene
Toluene (or metylbenzene) is used in the production of benzene, benzoic acid,
phenol, trinitro toulene, lacquers, resin solutions, lacquer thinners, as a gasoline
component and as a solvent in formulations of rubber cements paints, inks and pesticides.
Toluene is primarily produced by catalytic reformation of naphthene.-Rich petroleum
8
feedstock fractions, generally in conjunction with the manufacture of benzene and
xylenes.
Dehydrogenation of naphtha feed stock is the most common process used to
produce toluene (Sedivec, 1976). The feedstock is preheated vaporized, mixed with
hydrogen-rich recycled gas, and passed through the reactor for a contact time of about 15
seconds. Large quantities of it are consumed in the production of styrene, cumene (for
manufacturing phenol and acetone) and cyclohexane (for manufacturing nylon).
Toluene Exposure Routes and Health
Toluene exposure routes and health are summarized in Table 2.4.
Table2.4: Summary of toluene exposure routes and health
Sources Gasoline additive, rnanufactwing and solvent factories,
benzoic acid/phenoL
Human Health TLV:IOO ppm ,and IDLH:2000 ppm
Immediate Action Spill Control: Restrict access to spill site. Issue warning
;"FLAMMABLE". Call fire department and notify
manufacture. Eliminate sources of ignition including
traffic and equipment. Stop the flow and contain spill, if
safe to do sO.A void contact with liquid and vapour�
stay upwind of release.
Fire control: Use foam,dry chemical or carbon dioxide
to extinguish. Cool fire- exposed containers with water.
Stay clear of ends to tanks.
Irritation and Inhalation Irritation and inhalation data in appendix B.
Data
Adapted from Environmental and technology information: Toluene, 1984.