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Faculty of Resource Science and Technology

DISTRIBUTION OF HEAVY METALS, HYDROCARBONS AND HYDROCARBON-DEGRADING BACTERIA IN FRESHWATER SEDIMENTS

NUR AFIQAH BINTI MOHAMAD (24524)

Bachelor of Science with Honours (Biotechnology Resource) 2012

DISTRIBUTION OF HEAVY METALS, HYDROCARBONS AND HYDROCARBONDEGRADING BACTERIA IN FRESHWATER SEDIMENTS

by NUR AFIQAH BINTI MOHAMAD (Student Number: 24524)

Supervisors Prof Dr Zaini Assim Dr Azham Zulkarnain Assoc Prof Dr Awg Ahmad Sallehin Awg Hussaini

This project is submitted in partial fulfillment of the requirements for The degree of Bachelor of Science with Honours (Resource Biotechnology)

Program of Resource Biotechnology Department of Molecular Biology Faculty of Resource Science and Technology UNIVERSITY MALAYSIA SARAWAK

DECLARATION I hereby declare that no portion of this work referred to in dissertation has been submitted in support of an application for another degree or qualification to this university or any other institution of higher learning.

_________________________ (Nur Afiqah Binti Mohamad) Department of Molecular Biology Faculty of Resource Science and Technology University Malaysia Sarawak

i

ACKNOWLEDGEMENTS

Special thank is directed to my supervisor, Prof Dr Zaini Assim for giving me an opportunity to work on this project. I would like to thank him for giving me excellent guidance and knowledge throughout the making and conduction of this project. The special thank also goes to my cosupervisors, Assoc Prof Dr Awg Ahmad Sallehin Awg Hussaini and Dr Azham Zulkarnain for the supervision and support that they gave truly help the progression and the smoothness of the completion of the project. My thanks and appreciations also go to the laboratories assistant that have willingly helped me out with their abilities and for the wise idea throughout the project. Not forget, great appreciation go to my friends that help me from time to time along the way to finish my project. Last but not least, I would like to thank my parents Mr. Mohamad Bin Sait and Mdm. Hamiah Binti Hamdan, for the unconditional support they have always given me. Thank you.

ii

TABLE OF CONTENTS DECLARATION

i

ACKNOWMEDGEMENTS

ii

TABLE OF CONTENTS

iii

LIST OF FIGURES

vii

LIST OF TABLES

ix

LIST OF ABBREVIATION

xi

ABTRACT

xii

1

CHAPTER 1 INRODUCTION 1.1 General Introduction

1

1.2 Objectives of the Projects

3

CHAPTER 2 LITERATURE REVIEWS

4

2.1 Heavy metals in sediments

4

2.2 Hydrocarbons in sediments

4

2.3 Hydrocarbon-degrading bacteria in sediments

5

2.4 Importance of sediments studies

5

iii

CHAPTER 3 MATERIALS AND METHODS

7

3.1 Study Area and Sample Collection

7

3.2 Heavy Metals Analysis

9

3.4 Hydrocarbons Analysis

11

3.4.1 : Sample Extraction and Fractionation

11

3.4.2 Column chromatography

11

3.4.3 Gas chromatography (GC) analysis

12

3.5 Hydrocarbon-degrading bacteria Analysis

CHAPTER 4

13

3.5.1 Enrichment cultures and isolation of bacteria

13

3.5.2 Determination of bacterial cell number

14

RESULTS AND DISCUSSION

15

4.1 Calibration Analysis for Heavy Metal Standards on Atomic

15

Absorption Spectrophotometer (AAS) 4.2 Distribution of Heavy Metals from Freshwater Surface

18

Sediments 4.1.1 Nickel (Ni)

19

4.1.2 Copper (Cu)

20

iv

4.1.3 Manganese (Mn)

21

4.1.4 Zinc (Zn)

22

4.1.5 Chromium (Cr)

23

4.1.6 Silver (Ag)

24

4.1.7 Tin (Sn)

25

4.3 Sediment Quality Criteria and Environmental Status of

26

Sediment from Batang Ai Hydroelectric Dam based on Heavy metals Content 4.4 Distribution of Hydrocarbons in Freshwater Surface Sediments

27

4.4.1 Distribution of Aliphatic Hydrocarbons

27

4.4.2 Environmental Status of Sediment from Batang Ai

30

Hydroelectric Dam based on Aliphatic Hydrocarbons Content 4.4.3 Distribution of Polycyclic Aromatic Hydrocarbons (PAHs)

34

4.4.4 Environmental Status of Sediment from Batang Ai

39

Hydroelectric

Dam

based

on

Polycyclic

Aromatic

Hydrocarbons Content 4.3

Hydrocarbon-degrading bacteria analysis

v

40

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

42

5.1 Conclusions

42

5.2 Recommendations

43 44

REFERENCES

vi

LIST OF FIGURES Figure

Figure 3.1

Page

Location of sampling sites at Batang Ai Hydroelectric Dam at

7

Lubuk Antu District Figure 4.1

Calibration curves for Ni and Cu analyzed on AAS

15

Figure 4.2

Calibration curves for Mn, Zn, Cr, Ag, Sn and Pb analyzed on AAS

16

Figure 4.3

Calibration graphs for Cd, As and Bi analyzed on AAS

17

Figure 4.4

Concentration (µg/g) of Nickel in freshwater lake, Batang Ai Dam

19

Figure 4.5

Concentration (µg/g) of Copper in freshwater lake, Batang Ai Dam

20

Figure 4.6

Concentration (µg/g) of Manganese in freshwater sediment of

21

Batang Ai Dam Figure 4.7

Concentration (µg/g) of Zinc in freshwater lake, Batang Ai Dam

22

Figure 4.8

Concentration (µg/g) of Chromium in freshwater lake, Batang Dam

23

Figure 4.9

Concentration (µg/g) of Silver in freshwater lake, Batang Ai Dam

24

Figure 4.10

Concentration (µg/g) of Tin in freshwater lake, Batang Ai Dam

25

Figure 4.11

Gas chromatogram of aliphatic fraction from St1

27

vii

Figure 4.12

Gas chromatogram of aliphatic fraction from St2

27

Figure 4.13

Gas chromatogram of aliphatic fraction from St3

27

Figure 4.14

Gas chromatogram of aliphatic fraction from St4

28

Figure 4.15

Gas chromatogram of aliphatic fraction from St5

28

Figure 4.16

Gas chromatogram of aliphatic fraction from St6

28

Figure 4.17

Gas chromatogram of aliphatic fraction from St7

29

Figure 4.18

Gas chromatogram of polycyclic aromatic fraction from St1

34

Figure 4.19

Gas chromatogram of polycyclic aromatic fraction from St2

34

Figure 4.20

Gas chromatogram of polycyclic aromatic fraction from St3

34

Figure 4.21

Gas chromatogram of polycyclic aromatic fraction from St4

35

Figure 4.22

Gas chromatogram of polycyclic aromatic fraction from St5

35

Figure 4.23

Gas chromatogram of polycyclic aromatic fraction from St6

35

Figure 4.24

Gas chromatogram of polycyclic aromatic fraction from St7

36

viii

LIST OF TABLES Tables

Page

Table 3.1

Description of each sampling sites

8

Table 3.2

Concentration of heavy metal standards in calibration analysis on AAS

10

Table 3.3

Chemical composition of SBM

13

Table 4.1

Concentration (µg/g) of selected heavy metal in freshwater sediment of

18

Batang Ai Dam Table 4.2

Concentration (µg/g) of heavy metal in freshwater sediment of Batang

26

Ai Dam in comparison with USEPA guideline classification values for sediment metal concentration (µg/g). Table 4.3

Concentration of Aliphatic Hydrocarbon (n-alkanes) in Freshwater

30

Surface Sediments from Batang Ai at different Sampling Sites (St) Table 4.4

Concentration of Aliphatic Hydrocarbons in freshwater sediment of

33

Batang Ai Dam in comparison with concentration of Aliphatic Hydrocarbons in sediments from other areas Table 4.5

Concentration of Polycyclic Aromatic Hydrocarbon (PAHs) in Freshwater Surface Sediments from Batang Ai at different Sampling Sites (St)

ix

37

Table 4.6

Concentration of PAHs in freshwater surface sediment of Batang Ai

39

Dam in comparison with concentration of PAHs in sediments from other areas Table 4.7

Number of bacterial colonies (CFU/µl) in freshwater lake, Batang Ai Dam

x

40

LIST OF ABBREVIATIONS µm

Micrometer

mL

Milliliter

mm

Millimeter

cm

Centimeter

sp.

Species

GC

gas chromatography

AAS

atomic absorption spectrophotometer

PAHs

polycyclic aromatic hydrocarbons

DCM

Dichloromethane

TEL

total extractable lipids

TAHs

total aliphatic hydrocarbons

SBM

saline basal medium

NA

nutrient agar

EDTA

ethylenediaminetetraacetic acid

xi

ABSTRACT Freshwater sediments from the Batang Ai hydroelectric Dam at Lubok Antu, Sri Aman were analyzed for heavy metals, hydrocarbons and hydrocarbon-degrading bacteria content. A total of 7 sampling sites within vicinity of Batang Ai Hydroelectric Reservoir were selected for investigation. The heavy metals content in sediment was analysed using atomic absorption spectrophotometer (AAS), while the aliphatic and aromatic hydrocarbons in sediments were analyzed by gas chromatography-flame ionization detector (GC-FID). Furthermore, distribution of hydrocarbon-degrading bacteria in sediments was analyzed using molecular biological technique. Heavy metal content in sediments show varying concentrations of nickel (Ni), copper (Cu), manganese (Mn), zinc (Zn), chromium (Cr), argentum (Ag), stannum (Sn) and bismuth (Bi). The highest concentration of Ni, Cu, Mn, Zn and Cr were detected at St2, while the highest concentration of Ag and Sn wrere detected at St1 and St4, respectively. Bismuth (Bi) was only detected at St5 and St6 with concentration 4.79 and 5.28 µg/g, respectively. Lead (Pb), cadmium (Cd) and arsenic (As) was not detected in all sampling station. The aliphatic hydrocarbons were ranged from 2 - 421145 ng/g dry weights. St1 and St2 indicate the higher content of aliphatic hydrocarbons, while St3 showed the lowest n-alkanes content. The total PAHs concentration of sediments in Batang Ai Dam ranged 2 - 5958 ng/g dry weights. The PAH composition pattern in sediments of Batang Ai suggest dominance by medium to high molecular weight compounds, and the ratio of certain related PAHs indicate anthropogenic sources and natural processes. The highest number of bacterial colonies was detected at St7 with 9.9 × 105 CFU/ml. The number of bacterial colonies in sediment is higher near the jetty than other sites of Batang Ai Hydroelectric Dam. Other sediments show moderate bacterial number with the lowest bacteria count was at St3 (1.4 × 101 CFU/ml). Keywords: heavy metals, aliphatic hydrocarbons, aromatic hydrocarbons, hydrocarbondegrading bacteria, sediments

xii

ABSTRAK

Sedimen air tawar dari empangan hidroelektrik Batang Ai di Lubok Antu, Sri Aman telah dianalisis untuk logam berat, hidrokarbon dan kandungan bakteria pengurai hidrokarbon. Sebanyak 7 lokasi pensampelan di lingkungan empangan hidroelektrik Batang Ai telah dipilih untuk kajian. Kandungan logam berat dalam sedimen telah dianalisis dengan menggunakan spektrofotometer serapan atom (SSA), sementara hidrokarbon alifatik dan aromatik dalam sedimen yang diperolehi dianalisis dengan kromatografi gas pengesan pengionan nyala (KGPPN). Selain itu, taburan bakterai pengurai hidrokarbon dalam sedimen telah dianalisis menggunakan teknik biologi molekul. Kandungan logam berat dalam sedimen menunjukkan pelbagai kepekatan nikel (Ni), tembaga (Cu), mangan (Mn), zink (Zn), kromium (Cr), perak (Ag), timah (Sn) dan bismut (Bi). Kepekatan tertinggi Ni, Cu, Mn, Zn dan Cr didapati daripada St2, manakala kepekatan tertinggi Ag dan Sn telah dikesan pada St1 dan st4, masing-masingnya. Bi hanya boleh dikesan di St5 dan St6 dengan kepekatan 4.79 dan 5.28 μg/g, masing-masingnya. Plumbum (Pb), kadmium (Cd) dan arsenik (As) tidak dijumpai di semua lokasi pensampelan. Kepekatan hidrokarbon alifatik adalah dalam julat 2 - 421145 ng/g berat kering. St1 dan St2 menunjukkan jumlah hidrokarbon alifatik tinggi, manakala St3 menunjukkan jumlah hidrokarbon alifatik yang terendah. Kepekatan hidrokarbon aromatic polisiklik (HAP) dalam sedimen adalah dalam julat 2 - 5958 ng/g berat kering. Corak komposisi HAP dalam sedimen Batang Ai didominasi oleh sebatian berat molekul yang tinggi dan sederhana, dan nisbah HAP tertentu menunjukkan sumber antropogenik dan proses semulajadi. Bilangan tertinggi koloni bakteria dikesan di St7 dengan 9.9 × 105 CFU/ml. Bilangan koloni bakteria berhampiran jeti lebih tinggi berbanding lokasi lain di empangan hidroelektrik Batang Ai. Sedimen lain menunjukkan bilangan bakteria sederhana dengan hitungan bakteria terendah di St3 (1.4 × 101 CFU/ml). Kata kunci: logam berat, hidrokarbon alifatik, hidrokarbon aromatic polisiklik (HAP), bakteria pengurai hidrokarbon, sedimen

xiii

CHAPTER 1 INTRODUCTION

1.1

General Introduction Sediments refer to the depositional site of mineral and organic particles that are

transported from the catchment area, as well as particles that forms and settles from within the water body (Lopez and Lluch, 2000). Sediments efficiently sequester hydrophobic chemical pollutants that inflowing water bodies such as lakes (Harikumar et al., 2009). During the formation and absorption process of sediments in freshwater lakes, it accumulates various compounds such as heavy metals, hydrocarbons and hydrocarbon-degrading bacteria. Therefore, sediments can be good indicators to monitor level of contaminants and pollution in aquatic environment (Harikumar et al., 2009). Heavy metals and organic pollutants normally accumulate in the sediments linked with organic particles, clay surfaces, sulphides and iron manganese hydroxides (Leivuori et al., 2000). Heavy metals refer to metals of relatively high density, or of high relative atomic weight. Examples of heavy metals are cadmium, copper, iron, manganese, lead, zinc, and nickel. Moreover, some of heavy metals are known to be dangerous to health and to the environment. Hydrocarbons refer to compounds that consists only the elements carbon and hydrogen. Hydrocarbons are divided into two types: aliphatic hydrocarbons and aromatic hydrocarbons (Loudon, 2002). Due to the tendency of aliphatic and polycyclic aromatic hydrocarbons to accumulate in sediments, they are considered to be ubiquitous sedimentary contamination. The

1

aliphatic hydrocarbons can be divided into three hydrocarbon families: alkanes, alkenes and alkynes (Loudon, 2002). Aromatic hydrocarbons that consist of two or more fused rings are known as polycyclic aromatic hydrocarbons (Loudon, 2002). Polycyclic aromatic hydrocarbons (PAHs) known to be highly mutagenic and carcinogenic. PAHs are derived from incomplete combustion of organic material and known as environmental pollutants (Landvik et al., 2006). Example of PAHs is naphthalene which is the simplest of the polycyclic aromatic hydrocarbon (Loudon, 2002). The pathway of aliphatic hydrocarbon and polycyclic aromatic hydrocarbon in the freshwater environment is usually linked to anthropogenic sources. The ability to catalyze the degradation of hydrocarbons is exhibited by wide variety of bacterial and fungal genera (Leahy and Colwell, 1990). This microorganism is known as hydrocarbon-degrading bacteria. Hydrocarbon-degrading bacteria are widely distributed in freshwater lake and marine environments and normally degrade numerous contaminating petroleum hydrocarbons and cleansing the ocean of oil pollutants (Sutiknowati, 2007). Thus, hydrocarbon-degrading bacteria plays a significant role in accelerate the pollution degradation in aquatic ecosystem. In the study, the surface sediments were collected from freshwater lake (Batang Ai hydroelectric dam). Batang Ai hydroelectric dam is located at Lubuk Antu, of Sri Aman Division and built to generate hydroelectric power for Sarawak.

2

1.2

Objectives of the Project The objectives of this project are: a. to determine the distribution of heavy metals and hydrocarbons (aliphatic and aromatic) in freshwater sediments from Batang Ai Hydroelectric Dam, b. to evaluate the distribution of the hydrocarbon-degrading bacteria in freshwater sediments, c. to assess environmental status of Batang Ai Lake based on concentration of heavy metals, hydrocarbons and hydrocarbon-degrading bacteria in sediments.

3

CHAPTER 2 LITERATURE REVIEW

2.1

Heavy metals in sediments Heavy metals are usually accumulated and incorporated in the sediments, hence added to

a body of natural water (Harikumar et al., 2009). Heavy metals are one of natural components of the environment which are mainly occurring in the bedrock and soil. Heavy metals enter pristine lakes from various sources, especially by the weathering of bedrock in catchments and consequently transported to the lakes as one of the important components of the accumulating material as dissolved or particulated form through runoff, and as wind-blown soil dust particles (Lepane et al., 2007). Heavy metals is distinguishable from other toxic pollutants with its main properties which is they are not biodegradable. The concentration of heavy metal in water column can be relatively low compare to the concentrations of heavy metal in the core sediment which may be elevated (Kabarssi et al., 2005). The occurrence of elevated levels of heavy metals mainly originate in the sediments can be a sensitive indicator for monitoring contaminants and man induced pollution and high levels of heavy metals can frequently be attributed to anthropogenic sources, rather than natural enrichment of sediment by geological weathering (Kabarssi et al., 2005).

4

2.2

Hydrocarbons in sediments One of significant parts of the land-derived organic inputs towards coastal regions is the

hydrocarbons (Wu et al., 2001). Hydrocarbons which are known as sedimentary aliphatic hydrocarbons (AHCs) have both natural (including biogenic and petrogenic) and anthropogenic sources (Peng X. et al., 2008). n-Alkanes are originate mainly from a large range of organisms. The assemblage of n-alkanes in both aquatic biota and in the surface waxes of higher plants is differentiated (Wu et al., 2001). Polycyclic aromatic hydrocarbons (PAHs) have carcinogenic properties and normally known as group of environmental pollutants derived mainly from all sorts of incomplete combustion and consequently may be considered to be ubiquitous (Jacob, 1996). This is especially true for many urban coastal areas where there are high anthropogenic sources and various environmental pollutants sources are presence (Wang et al., 2006).

2.3

Hydrocarbon-degrading bacteria in sediments Hydrocarbons in the environment are mainly biodegraded by the bacteria and fungi

(Leahy and Colwell, 1990). These microorganisms rely on nutrients for survival and these nutrients will be used to synthesize enzymes for the degradation of hydrocarbons (Hamzah et al., 2010). Bacteria are normally considered to symbolize the predominant hydrocarbon degrading element of microbial community especially in the marine environment (Leahy and Colwell, 1990). In some environments, bacterial attack can be crucial process and specific strains have been investigated and identified to determine their abilities to degrade particular hydrocarbons (Cormack and Fraile, 1997). Hydrocarbon-degrading bacteria that are capable of utilizing n-

5

alkanes of chain length C10-C40 as a sole source of carbon is Acinetobacter sp. (Throne-Holst et al., 2007).

2.4

Importance of sediments studies Sediments provide useful information about the events that occurred in the past period in

the lakes and its catchments area. The history of sediment reflects the contamination history of an area (Harikumar et al., 2009). The study of the chemical composition of sediments from lake helps researchers to understand many processes occurring within the total lake system including its groundwater drainage basin (Lopez and Lluch, 2000). Sediments can be good indicators for monitoring contaminants in aquatic environments (Harikumar et al., 2009). Many researchers have used sediments to study the contamination caused by hydrocarbon (Wang et al., 2006; Peng et al., 2008). There were also researchers that used sediment to study the pollution history of aquatic ecosystem (Karbassi et al., 2005; Lopez and Lluch, 2000; Mohamed, 2005).

6

CHAPTER 3 MATERIALS AND METHODS

3.1

Study Area and Sample Collection The study area is Batang Ai Hydroelectric Dam at Lubok Antu District of Sri Aman

Division, Sarawak. This area is a man-made freshwater lake. A total of seven (7) sampling sites were selected for sediments collection as shown in Figure 3.1. The description of each sampling sites is presented in Table 3.1

St4 St1 St3 St5 St2 St7

St6

Figure 3.1: Location of sampling sites at Batang Ai Hydroelectric Dam at Lubok Antu District

7

Table 3.1: Description of each sampling sites

Sampling sites

Description

ST1

Adjacent to natural forest

ST2

Near the overflow of the Dam

ST3

Middle of the Lake

ST4

Jetty of Hilton Resort

ST5

Near the flow of river

ST6

Near the agricultural areas (rubber tree plantation)

ST7

Near the main jetty of the lake

The sediment samples were collected using a stainless steel grab sampler. The surface sediments for heavy metals analysis were placed into plastic bag. The samples were brought to the laboratory using plastic bag and placed in the cooler box for transportation. Whereas, the surface sediment samples for hydrocarbon analysis were wrapped with aluminum foil and placed in the cooler box for transportation. Upon arriving UNIMAS, the samples were stored frozen (20 ºC) until analysis in the laboratory. Sediment samples were dried and the dried sediments were sieve (125 µm) to remove large particles and debris before extraction (Wang et al., 2006). For microbiological analysis, to avoid any possible contamination, stringent measures against contamination were maintained throughout the sample collection and microbial isolation. As soon as the sediment was brought on board, the samples were placed into a sterile plastic bag, stored frozen (-4ºC) and transported to the laboratory for analysis.

8

3.2

Heavy Metal Analysis The sediment sample preparation was carried out according to procedure described by

Binning and Baird (2001). The sediments were dried in petri-dishes at room temperature and then ground into a powder. Approximately 0.5 g of each sample was weighed and placed into the volumetric flask. Then, 20 ml Aqua Regia (1:3 cHNO3: cHCl) was added into the sample. The mixture was placed on hot plate and heated to near dryness for two hours. The volumetric flask was covered by beaker to avoid the evaporation. Then, the mixture was allowed to cool before 5 ml HNO3 solution and 20 ml of deionized water were added. The mixture was again placed on hot plate and heated to near dryness for one hour. The samples were cooled and then were filtered through filter paper. The filtrates were transferred to a 100 ml volumetric flask and made up to the mark with deionized water. The metal determinations of the solution were performed on AAS by using the calibration curve method. Concentrations of the following metals were determined: Cu, Mn, Ni, Cd, Pb, Zn, Cr, Ag, As, Bi, Sn and Ba. Model of AAS used was Thermo SCIENTIFIC iCE 3000 SERIES. The concentration of respective heavy metals standard used during calibration analysis is shown in Table 3.2.

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