UNIVERSITI PUTRA MALAYSIA

BIOLEACHING AND BIOSORPTION OF HEAVY METALS FROM GOLD MINE TAILINGS BY ASPERGILLUS FUMIGATUS IN PENJOM,

MALAYSIA

BAHI JALILI SEH BARDAN

FP 2012 2

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BIOLEACHING AND BIOSORPTION OF HEAVY METALS FROM GOLD MINE TAILINGS BY ASPERGILLUS

FUMIGATUS IN PENJOM, MALAYSIA

By

BAHI JALILI SEH BARDAN

Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree

of Doctor of Philosophy

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DEDICATION

This work is dedicated to:

My husband, Fardin, for his endless patience and encouragement.

My father, Amir, who is a true scholar, my favorite storyteller, and always

encourages my interests in science.

My mother, Ameneh, whose love, nurturing, and motivation during my

graduate career were invaluable, and who I always admire for her endless

energy.

My sisters, brothers, sister-in-law and brothers-in-law for their understanding

and love that has supported me throughout the various challenging and

rewarding experiences of my life.

My wonderful nephew, Ahura, who changed my life when he was born.

My best friend, Mahtab, for her sisterly moral support.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

BIOLEACHING AND BIOSORPTION OF HEAVY METALS FROM GOLD MINE TAILINGS BY ASPERGILLUS FUMIGATUS IN PENJOM,

MALAYSIA

By

BAHI JALILI SHE BARDAN

January 2012

Chairman: Radziah Othman, PhD

Faculty: Agriculture

Mine tailings is an important source of heavy metal contamination in the

environment. Decontamination of the mine tailings is necessary for the

protection of the environment. In recent years, bioleaching process has

gained increasing attention for extraction of metals from solid substrate, since

it is simple, environmentally friendly and economical. The objectives of this

study were (i) to characterize some physicochemical properties of gold mine

tailings and to isolate and identify indigenous fungi from the tailings, (ii) to

evaluate the ability of the indigenous fungus to bioleach heavy metals in step

bioleaching and column bioleaching processes using distributed and surface

applied techniques, and (iii) to assess the efficiency of A. fumigatus biomass

to biosorb and remove metal from synthetic solutions and from leachate

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derived from a gold mine. Bioleaching experiments were conducted for the

removal of heavy metals from mine tailings using a fungal strain isolated from

the gold mine tailings and it was identified as A. fumigatus based on its 18S

rDNA analysis. The bioleaching processes were carried out in one-step and

spent medium leaching at 1%, 2%, 4% and 8% (w/v) tailing concentrations.

Column bioleaching experiments were carried out to compare the

effectiveness of the fungus to bioleach heavy metals from the tailings using

distribution technique (DT) and surfaced applied technique (SAT). The ability

of A. fumigatus to remove As, Fe, Mn, Pb and Zn from synthetic solutions

and from leachate derived from gold mine during bioleaching was assessed.

Batch sorption experiments were carried out to characterize the capability of

fungal biomass (FB) and iron-coated fungal biomass (ICFB) to remove metal

ions in single and multi-solute systems. Results showed that in the one step

bioleaching process, production of oxalic acid was the highest among the

other organic acids; while in the spent medium leaching citric acid was

dominant. The removal of As, Fe, Mn and Zn was higher in the one step than

the spent medium leaching but the reverse was observed for Pb. Heavy

metals removal efficiency decreased with increasing concentration of tailings

in both bioleaching processes. The column bioleaching study showed that DT

produced higher oxalic acid than the SAT, therefore more As, Fe, Mn, and Zn

were removed by the DT method compared to the SAT. However, Pb

removal was low by DT than the SAT probably due to the precipitation of Pb

as its oxalates. The DT was a more promising method for removal of metal

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ions from the mine tailings because distributing the fungus throughout the

entire soil column improved the bioleaching efficacy of the heavy metals. The

biosorption study showed that sorption data for all systems fitted well the

Langmuir isotherm equation. The maximum sorption capacities of metals by

both FB and ICFB were higher in the single metal system than the multi-

solute system. It indicates that the presence of multiple metal ions in as

solution suppressed adsorption of the individual metal ions. Sorption capacity

of ICFB was higher than FB for all metal ions because the net surface charge

of ICFB was more negative than FB at the pH range of 6 to 9, therefore the

ICFB could adsorb more cationic metals. As for As adsorption, the As formed

complexes with the Fe oxide of the ICFB surface sites. The FT-IR analysis

showed that the functional groups of the fungal biomass were involved in the

metal ions sorption. Overall, the results suggest that bioremediation process

using A. fumigatus was effective for the leaching of heavy metals from mine

tailings especially in the one-step bioleaching process using the DT method.

The ICFB was found to be effective in adsorbing the metals present in the

leachate of the tailing bioleaching process.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk mendapat ijazah Doktor Falsafah

BIOLURUHLARUTAN DAN BIOJERAPAN LOGAM BERAT DARIPADA SISA LOMBONG EMAS OLEH ASPERGILLUS FUMIGATUS

DALAM PENJOM, MALAYSIA

Oleh

BAHI JALILI SEHBARDAN

Januari 2012

Pengerusi: Radziah Othman, PhD

Fakulti: Pertanian

Tahi lombong adalah punca penting pencemaran logam berat dalam

persekitaran. Dekontaminasi tahi lombong adalah perlu bagi perlindungan

alam sekitar. Pada tahun-tahun kebelakangan ini, proses larutlesap telah

mendapat perhatian yang semakin meningkat bagi pengekstrakan logam

daripada substrat pepejal, kerana ia mudah, mesra alam dan ekonomi.

Objektif kajian ini adalah (i) untuk mencirikan beberapa sifat tahi lombong

emas dan untuk mengasingkan dan mengenal pasti kulat asli dari tahi

lombong tersebut, (ii) untuk menilai kemampuan kulat asli A. fumigatus untuk

melarutlesapbio logam berat dalam proses larutlesapbio di dalam kolum

menggunalan teknik edaran dan teknik taburan pada permukaan, dan (iii)

untuk menilai keberkesanan biojisim A. fumigatus untuk menjerapbio dan

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mengeluarkan ion logam dalam air yang terhasil dari proses

larutlesapbio.Eksperimen larutlesapbio telah dijalankan untuk penyingkiran

logam berat dari tahi lombong dengan menggunakan strain kulat yang

diasingkan daripada tahi lombong emas dan ia telah dikenal pasti sebagai A.

fumigatus berdasarkan analisis 18S rDNA. Proses larutlesapbio telah

dijalankan dalam satu langkah, dengan kepekatan media tahi lombong pada

kepekatan 1%, 2%, 4% dan 8% (w / v).Eksperimen larutlesapbio lajur

dijalankan untuk membandingkan keberkesanan kulat untuk

menglarutlesapbiologam berat daripada tahi lombong menggunakan teknik

pengagihan (TP) dan teknik taburan permukaan (TTP). Keupayaan A.

fumigatus untuk menghapuskan As, Fe, Mn, Pb dan Zn daripada larutan

sintetik dan daripada hasil larutlesap yang berasal dari lombong emas

semasa larutlesapbio dinilai. Eksperimen jerapan sekumpulan telah

dijalankan untuk mencirikan keupayaan biojisim kulat (BK) dan biojisim ferum

bersalut kulat (BFBK) untuk mengeluarkan ion logam dalam sistem tunggal

dan pelbagai bahan larut.Hasil kajian menunjukkan bahawa dalam proses

larutlesapbio satu langkah, pengeluaran asid oksalik adalah yang tertinggi di

kalangan asid organik yang lain, sedangkan dalam medium hasil larut lesap,

asid sitrik adalah dominan. Penyingkiran As, Fe, Mn dan Zn adalah lebih

tinggi dalam proses langkah salah satu daripada medium hasil larut lesap

tetapi sebaliknya dilihat untuk Pb. Kecekapan penyingkiran logam berat

menurun dengan peningkatam kepekatan tahi lombong di kedua-dua proses

larutlesapbio. Kajian larutlesapbio kolum menunjukkan TP menghasilkan asid

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oksalik yang lebih tinggi daripada TTP, oleh itu, lebih banyak As, Fe, Mn, dan

Zn telah dikeluarkan dengan kaedah DT berbanding SAT. Walau

bagaimanapun, penyingkiran Pb adalah rendah oleh TP berbanding TTP

mungkin disebabkan oleh pemendakan Pb sebagai oksalat. Kaedah TP

adalah lebih berpotensi untuk penyingkiran ion logam daripada tahi lombong

kerana agihan kulat sepanjang kolum tanah meningkatkan keberkesanan

larutlesapbio logam berat. Kajian biojerapan menunjukkan bahawa data

jerapan untuk semua sistem dilengkapi boleh disesuaikan dengan

persamaan isotherma Langmuir. Kapasiti maksima jerapan logam oleh

kedua-dua BK dan BFBK dalam sistem logam tunggal adalah lebih tinggi

daripada sistem pelbagai bahan larut. Ia menunjukkan bahawa kehadiran

pelbagai ion logam menekan penjerapan penjerapan ion logam secara

individu. Kapasiti jerapan BFBK adalah lebih tinggi daripada BK untuk semua

ion logam kerana caj bersih permukaan BFBK adalah lebih negatif daripada

BK pada julat pH 6-9, oleh itu BFBKB boleh menjerap logam yang lebih

kationik. Bagi penjerapan As, ia membentuk kompleks dengan oksida Fe

pada permukaan BFBK. Analisa FT-IR menunjukkan bahawa kumpulan

berfungsi biojisim kulat terlibat dalam jerapan ion logam.Secara keseluruhan,

keputusan menunjukkan bahawa proses bioremediasi menggunakan A.

fumigatus berkesan untuk larutlesap logam berat dari tahi lombong

terutamanya dalam proses larutlesapbio satu langkah menggunakan kaedah

TP. BFBK didapati berkesan dalam menjerap logam yang hadir dalam hasil

larutlesap proses larutlesapbio tahi lombong.

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ACKNOWLEDGEMENTS

I wish to express my deepest appreciation to Assoc. Prof. Dr. Radziah

Othman for providing valuable academic advices, critical support and

guidance during my graduate study. Her friendship and monitoring helped

guide me throughout my graduate program. I would also like to thank my

committee members, Dr. Samsuri Abd Wahid and Assoc. Prof. Dr. Aminuddin

Husin who provided valuable assistance and advice to ensure the quality of

my research.

I am grateful for the assistance and support of my husband, Dr. Fardin

Sadegh-Zadeh, who provided experimental advice.

I am thankful to the members of the Land Management Department

especially to Mr Ramley Sulaiman and Mr. Alias Tahar for providing me the

equipment to conduct the research.

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I certify that a Thesis Examination Committee has met on 5 January 2012 to conduct the final examination of Bahi Jalili Seh Bardan on his Doctor of Philosophy thesis entitled “Bioleaching and biosorption of heavy Metals from gold mine tailings in Penjom, Malaysia, by Aspergillus fumigatus” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U. (A) 106] 15th March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Shamsuddin Jusop, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Sariah Meon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Ahmad Ismail Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Yen Peng Ting Associate Professor Department of chemical and bimolecular engineering National University of Singapore (External Examiner)

________________________

Seow Heng Fong, PhD

Professor and Deputy Dean School of Graduate Studies

Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:

Radziah Othman, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Samsuri Abd Wahid, PhD Lecturer Faculty of Agriculture Universiti Putra Malaysia (Member) Aminuddin Husin, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

______________________________

BUJANG BIN KIM HUAT, PhD Professor and Dean

School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations

which have been duly acknowledged. I also declare that it has not been

previously and is not concurrently, submitted for any other degree at

Universiti Putra Malaysia or other institutions.

Bahi Jalili Seh Bardan

Date: 5 January 2012

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TABLE OF CONTENTS

Page

DEDICATION II ABSTRACT III ABSTRAK VI ACKNOWLEDGEMENTS X APPROVAL XI DECLARATION XIII LIST OF TABLES XVII LIST OF FIGURES XVIII LIST OF ABBREVIATIONS XXI 1 INTRODUCTION 1

2 LITERATURE REVIEW 5 2.1 Heavy metals 5 2.2 Mine tailings material 6 2.1.1Definition and characteristics of mine tailings 6 2.1.2 Metals species in mine tailings 6 2.2 Remediation technology 9 2.2.2 Bioleaching process 9 2.3 Remediation of metal-contaminated leachate using fungal 28 2.3.1 Use of fungal biomass for biosorption 29 2.3.2 Fungal structure 29 2.3.3 Effect of pre-treatment on biosorption by fungal biomass 30 2.3.4 Ecology and biology of Aspergillus fumigatus 32

3 MICROBIAL AND PHYSICOCHEMICAL ANALYSES OF GOLD MINE TAILINGS 34

3.1 Introduction 34 3.2 Materials and Methods 36 3.2.1 Mine tailing site 36 3.2.2 Microbial isolates from mine tailings 38 3.2.3 Molecular identification of fungal isolate 40 3.2.4 Mine tailings characterization 41 3.3 Result and Discussion 46 3.3.1 Selection of microbial isolates for bioleaching 46 3.3.2 Mine tailings characterization 50 3.4 Conclusions 60

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4 BIOLEACHING OF HEAVY METALS FROM MINE TAILINGS

BY ASPERGILLUS FUMIGATUS USING ONE-STEP AND TWO-STEP PROCESS 61

4.1 Introduction 61 4.2 Materials and Methods 62 4.2.1 Microorganisms 62 4.2.2 Bioleaching experiments 63 4.2.3 Chemical leaching experiment 65 4.2.4 Determination ofpH, organic acids and heavy metals and

concentration 65 4.2.5 Bioleaching yield 67 4.2.6 Statistical analyses 67 4.3. Results and Discussion 67 4.3.1 Bioleaching experiments 67 4.3.2 Chemical leaching experiment 80 4.4. Conclusions 84

5 BIOLEACHING OF HEAVY METALS FROM GOLD MINE TAILINGS BY ASPERGILLUS FUMIGATUS USING COLUMN TECHNIQUE 85

5.1 Introduction 85 5.2 Materials and methods 86 5.2.1 Medium for fungus 86 5.2.2 Column bioleaching designs 87 5.2.3 Statistical analyses 89 5.2.4 determination of heavy metals and organic acis

concentration 90 5.3 Results and Discussion 90 5.3.2 Bioleaching experiments 90 5.4 Conclusions 106

6 BIOSORPTION OF HEAVY METALS FROM LEACHATE DERIVED FROM BIOLEACHING OF GOLD MINE TAILINGS USING ASPERGILLUS FUMIGATUS 107

6.1 Introduction 107 6.2 Materials and methods 109 6.2.1 Chemicals preparation 109 6.2.2 Preparation of A. fumigatus biomass 110 6.2.3 Surface charge of fungal biomass 111 6.2.4 Preparation of metal solutions 111 6.2.5 Biosorpation experiments in single-soulte system 112 6.2.6 Biosorption experiments in multi-solute system 115 6.2.7 Preparation of leachate 115

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6.2.8 Biosorption of metal ions from leachate samples using ICFB 115

6.2.9 Fourier transforms infrared (FTIR) spectroscopy of UFB and ICFB before and after biosorption 116

6.2.10 Scanning electron microscopy of fungal biomass 116 6.2.11 Statistical analyses 116

6.3 Results and discussion 117 6.3.1 surface charge of fungal biomass 117 6.3.2 Biosorpation experiments in single system 118 6.3.3 Biosorption experiments in multi-solute system 133 6.3.4 Removal of metal ions from leachate using ICFB 136 6.3.5 FT-IR analysis 138 6.3.6 Scanning electron microscopy 143

6.4 Conclusions 144

7 SUMMARY, GENERAL CONCLUSIONS 145

REFERENCES 150 BIODATA OF STUDENT 164 LIST OF PUBLICATIONS 165

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