UNIVERSITI PUTRA MALAYSIA

CRUDE OIL DEGRADING BACTERIA: ISOLATION, GROWTH AND BIODEGRADATION STUDIES

FARAG M. ALI

FSAS 1998 22

CRUDE OIL DEGRADING BACTERIA: ISOLATION, GROWTH AND BIODEGRADATION STUDIES

By

FARAG M. AlI

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the

f�culty of Science and Environmental Studies Universiti Putra Malaysia

April 1998

I Dedicate This Work To My Mother­

KADEGA A. BUBAKAR

She who has over prayed for my success ! ! !

ACKNOWLEDGEMENT

In the name of ALLAH the most Benevolent and Merciful .

First and foremost al l praise be to ALLAH for giving me the

opportunity, patience, help and guidance for the completion of this thesis.

I am indebted to the chairman of my supervisory committee, Assoc.

Prof. Dr. Che Nyonya Abdul Razak for her dedicated efforts, invaluable

advice and intellectual guidance throughout the study. I am also thankful to

Prof. Dr. Abu Bakar Salleh, Assoc. Prof. Dr. Mahiran Basri and Mr.

Mohammed Pauzi Zakaria, members of the supervisory committee for their

comments, encouragement and constant support during the period of this

study and mostly availability, show of concern and criticism during the

preparation of this work.

Also the financial support from the University of Omar AI Mukhter

Elbedia-Libya and the Ministry of Science, Technology and Environment

Malaysia ( IRPA No. : 09-02-04-043) is gratefully acknowledged.

SpeCial thanks should go to my friends, Kak Yam, Praba, Kak

Ruhaidah, Palson, Su, Shida, Moon, Kak Hal , Aiza, Yap, Kak Yati, and all

the department staff for their encouragement and support. It was wonderful

to work with all of them, and I would like to express my gratitude in these

simple word: THANKS.

i i

I \NOuld l ike to extend my appreciation to all those who have

contributed to the success of this study. May ALLAH bless them all. Amin.

Last but not least, my deepest gratitude and love goes to my mother,

my wife and other family members who constantly pray for my success.

i i i

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . . , . . . . . . xi LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi i ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii i ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

CHAPTER

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

II LITERATURE REVIEW. . . . . . . . . . . . . .... . . ... . . . .. .. . . . . . . . . . .. . . . . 6

Oil Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 6 Composition of Crude Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Source of Oil Inputs into Environment. . . . . . . . . . . . . . .. . . . . . . 1 3 The Fate of Oi l. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3

Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 Photochemical Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 Dissolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 8 Emulsification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9

Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . 21 Bioremediation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Microbial Degradation of Hydrocarbons. . . . . . . . . . . . . . . . . . 25

Aliphatic Hydrocarbons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Aromatic Hydrocarbons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Factors Affecting Oil Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . 32 Substrate Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :. 35 Nutrients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 37 Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Salinity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 pH . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

iv

III MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Materials . . . . . . . , . . . . . . . . . . . . . . . , . . . . . . . , . . . . . . . . . . . . . . .. . . . . . . . . . 45 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Preparation of Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 49 Hydrocarbon Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Screening and Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Growth of Different Concentrations of Crude Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Growth in Bushnell Hass with 1 % Crude Oil . . . . 53 Bacteria Preparation . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Substrate Specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Optimization Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Growth of Bacteria at Optimal and Normal Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Growth of Mono and Mixed Cultures at Optimal Condition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Identification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Laboratory Scale Bioremediation Studies Using Isolate 1 70. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Bioremediation of Crude Oil Contaminated in Liquid Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Bioremediation of crude Oil Contaminated in Sandy SoiL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Extraction of Residual Hydrocarbon for Infrared Spectrometry ( IR) Analysis. . . . . . . . . . . . . . . . 62 Extraction of Residual Hydrocarbon for Gas Chromatography (GC) Analysis . . . . . . . . . . . . . . . . . . . . . . . 62 Gas Chromatography (GC) Analysis. . . . . . . . . . . . . 64

IV RESULTS AND DiSCUSSiON................................. 65 Isolation and Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Growth in Different Concentrations of Crude oiL. . . . . . . 68 Growth of the Five Isolates in Bushnell Hass with 1 °1(, Crude OiL. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Substrate specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Optimization Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Effect of Nutrient Supplements on Growth . . . . . . 81 The Effect of Various Physical Factors on Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Effect of Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Effect of pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Effect of Sal inity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. 89 Effect of Agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Growth Curve of Bacteria at Optimal and Normal Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Infrared Spectrometry ( IR) Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . 93 Growth of Mono and Mixed Cultures at Optimal Condition. . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Identification. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

v

Laboratory Scale Bioremediation Studies. . . . . . . . . . . .. . . .. 1 07 Bioremediation of Liquid Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07 Bioremediation of Sandy Soil. . . . . . . . . . . . . . ... . ........... . .... . . 1 1 0

V CONCLUSiON . .. .. .. .... ............................ . .. . . ............. . 1 16

BIBLIOGRAPHy... . . . . ... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 20

APPENDICES ... .. . . ..... '" .,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 32

VITA............................................................. ... ... .......................................... 1 52

vi

UST OF TABLES

Table Page

1 Average Amounts of Major Classes of Hydrocarbons and Related Compounds in Different Crude Oils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 1 1

2 Solubilities of Aliphatic and Aromatic Petroleum Hydrocarbons in Seawater and Distilled Water at 25°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3 Some of Petroleum Hydrocarbon Compounds Known to be Oxidised (directly or co-oxid ized) by Hydrocarbonoclastic Microorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4 Some of Aromatic Hydrocarbons Oxidized by Different Species of Bacteria.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

5 Total Bacterial Counts Obtained by Direct Plating and Enrichment Methods During Primary Screening. . . . . . . . . . . . . . . . . . . 65

6 Growth of Selected Bacterial Isolates in Different Concentrations of Crude Oil for 7 days at 37°C . . . . . . . . . . . . . . . . .. . . 71

7 Growth of The 5 Isolates in Various Complex Hydrocarbon for 7 days at 37°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 78

8 Growth of The 5 Isolates in Various Types of Simple Hydrocarbon for 7 days at 37°C. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

9 Bacterial Growth in Different Nutrient Supplements. . . . . . . . . . . . . . 82

1 0 Bacterial Growth i n Different Percentage of Nitrogen and Phosphorus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

1 1 Bacterial Growth in Different Temperatures. . . . . . . . . . . . . . . . . . . . . . . . . . . 86

1 2 Bacterial Growth i n Various pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . :. 88

1 3 Bacterial Growth in Different Concentrations of Sodium Chloride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

14 Bacterial Growth in Different Agitation Rates. . . . . . . . . . . . . . . . . . . . . . . 92

vii

15 Growth of Individual and Mixture Isolates at Optimal Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

1 6-A Cellular Morphology, Growth and Biochemical Test of The Isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . 100

1 6-B Biochemical Tests by Microbact Identification Kit. . . . . . . . . . . . . . . . . 101

17 Total Isolates Obtained from Primary Screening and their Growth in BM + 1 % Crude Oi l . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

viii

UST OF FIGURES

Figure Page

1 The Fate of Oil at Sea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 1 4

2 Number Diversity of Isolates Obtained by Direct Plating and Enrichment Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 67

3 The Growth in Different Isolates in The Secondary Screening Usin� Basal Media Containing 1 % Crude Oil for 7 Days at 37 C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

4 Growth of The Five Selected Isolates in Bushnell Hass Medium with 1 % Crude Oil for 7 Days at 37°C . . . . . . . . . . . . . . . . . 76

5 Growth Curve of Bacteria at Optimal and Normal Conditions in1 % Crude Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . 94

6 Growth Curve of Bacterial Grown in 1 % Crude Oil at Optimum Conditions. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

7 Infrared Spectra of Crude Oil Incubated with the Five Isolates for 7 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 97

8 Growth of Isolate 1 70 in 0.5% Crude Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 09

9 Gas Chromatograms of Crude Oil Incubated with Isolate No. 1 70 in Liquid Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1

1 0 Growth Curve of Isolate 1 70 in Sandy Soil during the Bioremediation. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3

1 1 Gas Chromatograms of Crude Oil Incubated with Isolate No. 1 70 in Sandy Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4

1 2 Infrared Spectra of Crude Oil Incubated with Uninoculated Control after 7 Days Incubation. . . . . . . . . . . . . . . . . . 1 36

1 3 Infrared Spectra of Crude Oil with Isolates 1 70 after 7 Days Incubation. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37

1 4 Infrared Spectra of Crude Oil with Isolates 1 81 after 7 Days Incubation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 38

1 5 Infrared Spectra of Crude Oil with Isolates 1 83 after 7 Days Incubation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 1 39

1 6 Infrared Spectra of Crude Oil with Isolates 2 17 after 7 Days Incubation . . . . . . . . . . . . . . . . . . . . . . . '" . , . . . . . . . . . . . . . . . , '" . . . . . . . . . . . 1 40

ix

1 7 Infrared Spectra of Crude Oil with Isolates 224 after 7 Days Incubation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 41

1 8 Gas Chromatograms of Crude Oil with Uninoculated Control Sample in Liquid Media. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 42

1 9 Gas Chromatograms of Crude Oil with Isolate 1 70 in Liquid Media at 7 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 1 43

20 Gas Chromatograms of Crude Oil with Isolate 1 70 in Liquid Media at 1 4 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 1 44

21 Gas Chromatograms of Crude Oil with Isolate 1 70 in Liquid Media at 21 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 45

22 Gas Chromatograms of Crude Oil with Isolate 1 70 in Liquid Media at 30 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 46

23 Gas Chromatograms of Crude Oil with Uninoculated Control Sample in Sandy SoiL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 47

24 Gas Chromatograms of Crude Oil with Isolate 1 70 in Sandy Soil at 1 4 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 48

25 Gas Chromatograms of Crude Oil with Isolate 1 70 in Sandy Soi l at 30 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 1 49

26 Gas Chromatograms of Crude Oil with Isolate 1 70 in Sandy Soil at 45 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50

27 Gas Chromatograms of Crude Oil with Isolate 1 70 in Sandy Soil at 60 Days. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 1 51

x

LIST OF PLATES

Plate Page

1 An Incubator Showing Growth Vessels Wrapped in 59 Aluminium Foil for Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 Bioremediation of Crude Oil in Sandy Soil with Isolate 1 70 at Room Temperature . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3 Soxhlet Extraction Apparatus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4 Growth of Isolate 1 70 in Basal Media with different Concentrations of Crude OiL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

5 Growth of Isolate 1 70 in 50% Crude Oil After 8 days. . . . . . . . 73

6 The Growth of Isolate 1 70 on Bushnell Haas Agar (BHA) Containing 0.1 % and 1 % Crude Oil 37°C after 24 hours. . . 74

7 Cell Cellular and Colony Morphologies of Isolate 1 70. . . . . . 1 02

8 Cell Cellular and Colony Morphologies of Isolate 1 81 . . . . . . 1 03

9 Cell Cellular and Colony Morphologies of Isolate 1 83. . . . .. 1 04

1 0 Cell Morphology of Isolate 224 after 20 hours (magnification 1 OOOx) . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 05

1 1 Cell Morphology of Isolate 21 7 after 20 hours (magnification 1 000x) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 06

xi

LIST OF ABBREVIATIONS

BHA Bushnell Hass agar

BH Bushnell Hass

BMA basal media agar

BM basal media

CFU colony forming unit

F lO flame ionization detector

FT-IR Fourier transform infra red

GC gas chromatograph

He hydrocarbons

nm nanometer

NA nutrient agar

NP nitrogen and phosphorus

00 optical density

rpm round per minute

TSB trypticase soy broth

xii

Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.

CRUDE OIL DEGRADING BACTERIA: ISOLATION, GROWTH AND BIODEGRADATION STUDIES

By

FARAG M. AU

April 1998

Chairman : Associate Professor Dr. Che Nyonya Abdul Razak

Faculty: Science and Environmental Studies

A total of 61 bacteria isolates vuere isolated from the four soil samples

during the primary screening using 0.1 % crude oil (v/v) as sole carbon

source by the direct plating and enrichment method. Hovuever, only 23

isolates gave good growth when grown in 1 % crude oil out of which 9 .

isolates were able to grow in crude oil up to 50% concentration.

Substrate specificity studies done on 5 of the 9 isolates, shovued that

all the 5 isolates ( 170, 1 81 , 1 83, 21 7 and 224) were able to grow on all the

1 0 different hydrocarbon substrates tested with varying preferences.

Optimum growth for a/l the 5 isolates vuere observed at 30°C, pH 7.5, with

1 % nitrogen and phosphorous supplements and shaken at 1 50 rpm. They

were able to grow in media containing up to 2.3 % NaCI concentration.

xiii

Among the 5 isolates, isolate 1 70 gave the highest 00540 reading (0.469)

and viability count ( 1 .9x1 07) after 5 days. Isolates 21 7 and 1 83 gave the

lowest growth with 00540 and viability count after 5 days, respectively.

Infrared spectrometry (IR) analysis showed that all the 5 isolates caused

some peaks reduction, disappearance, appearance and intensification after

7 days incubation.

Biochemical and morphological studies on the 5 isolates revealed

that isolate 1 70, 1 83 and 21 7 belong to Bacillus sp, while, isolates 1 81 and

224 are Pseudomonas sp. Detai led biochemical identification indicate that

isolate 1 70 is Bacillus subtilis and isolate 1 81 is Pseudomonas aeruginosa.

The bioremediation potential of the isolate 1 70 studied using liquid

media and soi l containing 0.5% crude oil showed that the isolate was able to

degrade more then 80% of the hydrocarbons compounds of crude oil. The

GC profi le on day th, 1 4th, 21 st and 30th day; showed that 26, 48, 78 and

81 % respectively, of the total hydrocarbon were degraded in liquid media. '

Simi larly with the soil sample, GC analysis showed that 21 , 57, 71 and 86%

reduction of total hydrocarbon in the samples occurred after 1 5th , 30th , 45th

and 60th day incubation. Most of the degradation occurred during the

exponential phase of the growth.

xiv

Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia bagi memenuhi keperluan Ijazah Master Sa ins.

BAKTERIA PENDEGRADASI MINYAK MENTAH: KAJIAN KE ATAS PEMENCILAN, PERTUMBUHAN DAN BIODEGRADASI

Oleh

FARAG M. ALI

April 1998

Pengerusi: Profesor Madya Dr. Che Nyonya Abdul Razak

Fakulti : Sains dan Pengajian Alam Sekitar

Sejumlah 61 pencil an bakteria telah dipencilkan daripada 4 sampel

tanah semasa penyaringan awal, menggunakan 0. 1 % minyak mentah ( iIi)

sebagai sumber karbon tunggal, melalui kaedah pemplatan secara terus

dan kaedah pengkayaan. Walaubagaimanapun, hanya 23 pencilan sahaja

yang memberikan pertumbuhan yang baik apabila ditumbuhkan didalam 1 %

minyak mentah. Oaripada jumlah tersebut, 9 pencilan berupaya untuk

tumbuh didalam minyak mentah sehingga kepekatan 50%.

Kajian kespesifikan substrat yang dijalankan keatas 5 daripada 9

pencilan, menunjukkan bahawa kelima-lima pencilan tersebut (1 70, 1 80,

1 83, 21 7 dan 224) berupaya tumbuh didalam kesemua sepuluh substrat

xv

hidrokarbon yang diuj i dengan kecenderungan yang berbeza. Pertumbuhan

optima untuk kelima-lima pencil an dicerap pada 30°C, pH 7.5, dengan

pertambahan 1 % nitrogen dan phosforus dan digoncang pada 1 50ppm.

Mereka berupaya tumbuh dalam media yang mengandungi kepekatan NaCI

hingga 2.3%. Diantara kel ima-lima pencilan, pencilan 1 70 memberikan

bacaan OD5<CO (0.469) dan bilangan viabi l iti ( 1 .9x1 0\ tertinggi selepas 5

hari. Manakala penci lan 2 17 dan 1 83 memberikan pertumbuhan yang

terendah dengan bacaan OD5<CO dan bilangan viabiliti, masing-masing.

Analisis spektrometri inframerah ( IR) menunjukkan bahawa kel ima-lima

pencilan menyebabkan berlaku penyusutan, kehilangan, kemunculan dan

peningkatan keamatan keatas beberapa puncak selepas 7 hari

pengeraman.

Kajian biokimia dan morphologi keatas kel ima-lima pencilan

menunjukkan bahawa pencilan 1 70, 1 83 dan 21 7 adalah dari kumpulan

Bacillus sp. , manakala pencilan 1 81 dan 224 adalah Pseudomonas sp.

Identifikasi biokimia yang terperinci menunjukkan bahawa pencilan 1 70'

adalah Bacillus subtilis dan pencilan 1 81 adaJah Pseudomonas aeruginosa.

Potensi bioremediasi oleh pencilan 1 70 yang dikaji menggunakan

media cecair dan tanah yang mengandungi 0.5% minyak mentah,

menunjukkan bahawa pencil an tersebut berupaya mengdegradasikan lebih

80% dari sebatian hidrokarbon dalam minyak mentah. Profil GC pada hari 7,

14, 21 dan 30, menunjukkan bahawa 26, 48, 78 dan 81 %, masing-masing

daripada jumlah hidrokarbon telah didegradasikan, dalam media cecair.

xvi

Keputusan yang sama diperolehi dengan sampel tanah d imana analisis GC

menunjukkan lebih 21 , 57, 71 dan 86% penurunan jumlah hidrokarbon

didalam sampel berlaku selepas 1 5, 30, 45 dan 60 hari eraman.

Kebanyakkan degradasi berlaku semasa fasa eksponen pertumbuhan.

xvii

CHAPTER I

INTRODUCTION

Oil pol lution is probably one of the most important subjects in any

discussion on environmental pollution. The air vve breathe, the water we

drink and bathe in, the soi l in which our crops are grown, and the

environments in which animals and plants grow are continual ly

contaminated by a variety of synthetic chemicals. The environmental

contamination by petroleum and its derivatives is a problem of increasing

magnitude with obvious ecological and economic implications. In fact,

considerable amounts of oil and hydrocarbon materials have always found

their way into the ecosystem. Hydrocarbon contamination in water and soi l

are for example a consequence of spi l ls from oi l tankers and activities

carried out in oil-dri l l ing sites, petroleum refineries and storage facil ities.

The elimination of these pollutants occurs in part through the activity

of some microorganisms which have the capacity to utilise different

hydrocarbons as carbon and energy source (Heitkamp and Cerniglia,

1 988). The role of microorganisms in the degradation of organic rT:Iaterials is

vvell established. Certain microorganisms had been shown to be able to use

petroleum and its derivatives as the sole source of carbon and energy

1

2

(Bossert and Burtha, 1 984; Heitkamp and Cemigila, 1 988). Thus,

biodegradation of hydrocarbon by microorganisms represents one of the

primary mechanisms through vvhich hydrocarbons pollutants could be

el iminated from the environment (Leahy and Colvvell, 1 990).

Practical appl ications for a controlled microbiological process of fossi l

hydrocarbon conversions are numerous, ranging from product formation to

removal of harmful materials. Biodegradation of oils is by no means a simple

subject since, in nature, hydrocarbon decomposition involves interactions of

complex physical, chemical and biological processes, the rates of, vvhich are

interdependent. Thus, it is understandable that the enzymatic systems and

responsible organisms involved in the complete biodegradation of crude oil

are iII-defined. Moreover, since crude oils vary in constituency from vvell to

vvell, the complexity of developing a microbial system for decomposing fossil

hydrocarbons becomes even more difficult. Evidence suggests the need to

adequately supplement crude oil-enriched waters with proper nutrients (Le. ,

nitrogen and phosphate) to stimulate and maintain the biodegradation'

process (Ahearn and Meyers, 1 973).

Each species generally metabolizes only a narrow spectrum of

homologous hydrocarbons. Certain species produce constitutive enzymes,

but in a good many species the enzymes vvhich catalyze the oxidation of

hydrocarbons are adaptive. Quite commonly pure cultures of various

species have been induced to attack hydrocarbons by cultivating them in

3

appropriate heterotrophic media enriched with one or more hydrocarbons

(Ahearn and Meyers, 1 973).

Bioremediation is the use of microorganisms to mitigate hazards of

soils or groundwater contaminated with organic waste materials. The

technology has been receiving increased attention over the past 6-8 years

due to the fol lowing reasons, it is cheaper compared to physical and

chemical methods since it involve minimum amount of equipment and space,

it can be carried out in situ and does not have toxic effect, thus

environmental friendly and has long term effect.

Biological degradation of organic wastes can and usually does, result

in complete destruction of the contaminants and elimination. Bioremediation

has become more popular and subsequently more widely used. However, as

the popularity of using biological treatment increase, the pressures for

performance beyond developed capabil ities also increase. It has long been

realised that microorganisms are capable of metabolising naturally occurring ·

organic compounds. However, many problematic environmental sites also

have xenobiotic or synthetic compounds, which may, or may not be

biodegradable. This latter phase has been the focus of much microbiology

research in the laboratory for the past few years. However, field

developments have primarily focused on the application of naturally

occurring organic compounds, such as petroleum waste products.

4

Bioremediation of petroieum in soil using bacteria can be carried out

by two methods. Firstly, through addition of nutrients into soil to stimulate

the endogenous bacteria already present in the soil . These bacteria are

stimulated to grow by introducing nutrients into the soil and thereby

enhancing the biodegradation. The second method is through addition of

exogenous bacteria with petroleum degradabil ity to the contaminated site.

However, there are confl icting conclusions in the l iterature concerning

the abil ity of individual isolates to degrade both the aliphatic and aromatic

components in crude oil, Bushnell and Haas (1 940) reviewed the earlier

l iterature on this subject which was supportive of the idea that there were

isolates that could use al iphatic but not aromatic compounds for growth.

Similarly, Austin et a/. , (1 977) showed that there was some degree of

specificity in the types of hydrocarbons degraded by given bacterial species.

It is known that both extrinsic factors, that is, the physico-chemical

environment, and intrinsic microbial factors influence the rate of

biodegradation of a specific hydrocarbon compound. The environmental

constraints on degradation of petroleum hydrocarbons were extensively

reviewed by Bartha and Atlas (1 977) and Atlas ( 1 981 ). The more important

environmental factors include availabi l ity of oxygen, presence of nutrients

(nitrogen and phosphorus mainly), temperature, presence of .particulate

matter, creation of stable emulsion, cometabolising microbial community,

and previous exposure to petroleum hydrocarbons. These factors will

influence the values of 'hydrocarbon degradation activity'.

Pf<:�VU"'TAl(Af� UNI\lT�lSl�l Vii fHA MALAY�

Therefore, the specific objectives of the research are:

1. To screen and isolate hydrocarbon degrading bacteria.

2. To determine the substrate specificity of the potential isolates.

3. To determine the growth parameters affecting the growth of selected

isolates in 1 % crude oil.

4. To identify the potential isolates to genus or species level.

5. To study laboratory scale bioremediation process using water and soi l

contaminated with crude oil by a potential isolate.

CHAPTER II

UTERATURE REVIEW

Oil Toxicity

Oil pollution, whether due to spilling crude oil or refined product, may

damage the environment in many different ways. Many biological processes

which are important for the survival of marine organisms are affected by the

presence of low concentration of petroleum hydrocarbons in the seawater

(National Academy of Science, 1 985). Oil toxicity is caused by the

soluble fraction, which consists of low boiling point aromatic hydrocarbon

which include benzene, toluene, xylene, naphthalene and phenanthrene.

These substances can cause organism damage and mortality (Bishop,

1 983) and the following damages to;

a) Marine Plants

i) Inhibition of Photosynthesis

Little is known of the mechanisms by which petroleum affects either

photosynthesis or metabolism in marine plants. However, studies with

6