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.