QR92.S8 As 5 blthr C.l G355582 University of Ghana http://ugspace.ug.edu.gh ISOLATION AND CHARACTERIZATION OF BIOOXIDIZING BACTERIA FROM THE OBUASI GOLD MINING SITE BY RICHARD HARRY ASMAH University of Ghana http://ugspace.ug.edu.gh ISOLATION AND CHARACTERIZATION OF BIOOXIDIZING BACTERIA FROM THE OBUASI GOLD MINING SITE A Thesis Presented to the The board o f Graduate studies University o f Ghana, Legon. Ghana. In Partial Fulfillment o f the Requirement for the Degree o f Master o f Philosopy (M.Phil.) in Biochemistry. By RICHARD HARRY ASMAH BSc. (Hons.) Department o f Biochemistry, Faculty o f Science, University o f Ghana, Legon, Accra, Ghana. September, 1998 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare lhat except for references to other people’s work which I have duly acknowledged, this exercise is a result o f my own research, and this thesis, either in whole, or in part has not been presented for another degree elsewhere. (Student) Dr. Y. D. OSEI (Supervisor) Dr. K. M. BOSOMPEM (Supervisor) University of Ghana http://ugspace.ug.edu.gh DEDICATION To my mother Beatrice Ellis and in memory o f my late father, Harry Benjamin Asmah and late aunties Victoria, Sarah and Christina Ellis. And to my brothers, sisters, cousins and entire family. Thank you for all your love and care. University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS I am glad to have this opportunity to express my deepest gratitude to my supervisors, Drs. Y. D. Osei and F. K. Rodrigues o f the Department o f Biochemistry and Dr. K. M. Bosompem o f the Noguchi Memorial Institute For Medical Research (NMIMR). This work was completed through their expert guidance, unfailing courtesy, help and co-operation. Their invaluable comments and critical evaluation enabled me to greatly improve the quality o f this work. I also wish to express my sincere appreciation to Prof. M. E. Addy, the former head o f Department o f Biochemistry, University o f Ghana, for suggesting to me the project area as well as helping me to obtain the samples used in the work. I am also appreciative o f her invaluable comments and help in shaping the work. My deepest gratitude goes to Mr. C. Clement for the invaluable assistance in initiation o f the project as well as for his continued interest and encouragement. I also express my appreciation to Dr. J. P. Adjimani and all the lecturers o f the Department o f Biochemistry for their help in difficult times. I wish to acknowledge my indebtedness to all individuals and institutions who in various ways, contributed to the formulation, execution and submission o f the work described in this thesis. My sincerest thanks go particularly to the Ashanti Goldfields Company Limited for permitting me to collect the samples used in project. I really appreciated the help and co-operation o f the technical staff, Mr. F. O. Bosompem, the chief technician, Mr. S. A. Y. Feyi, Mrs. Doris Amanor and Mrs. R. Nyarko, the administrative staff, Mr. S. Dramanu, Mrs. C. Nettey, and Mrs. H. Antwi-Boasiakoh, thank you all. I also appreciate the companionship and fruitful discussions with my mates Messrs J. Asiedu-Larbi, H. Asare-Anane, R. M. Yawson, D. Akakpo. To my roommate Mr. Kennedy Acquaah I say thank you for your support through tough times. I wish also to express my sincere thanks to Mr. Appawu, Dr. Armah, Dr. Wilson and Dr. Akanmori o f the NMIMR for their help and encouragement, and for allowing me to University of Ghana http://ugspace.ug.edu.gh use their laboratory facilities. I am extremely grateful to Mrs. Irene Ayi, Mr. William Anyan, Mrs. Bridgette Ogoe, Miss Anita Ofosu-Okyere, Mr. Mike Osei-Atweneboana o f the Parasitology Unit, Mr. Mike Ofori o f the Immunology Unit o f the NMIMR and Mr. Ismaela Abubakar o f the computer room for their helpful advice, encouragement and assistance during the work described in this thesis. To my friends at Campus Christian Family, Miss Juliana Dei-Frempong, Mr. Parker Yarney and Mr. Charles Brown I wish to express my appreciation for their encouragement and prayers. I finally acknowledge the help o f all those whose names I might have not mentioned but contributed in making this project a success. May God bountifully bless you. University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS D EC LA RA T IO N ..................................................................................................................................................................................................... 11 D ED IC A T IO N ........................................................................................................................................................................................................ m A CK N OW LED G EM EN TS ...............................................................................................................................................................................IV TABLE O F C O N T EN T S ....................................................................................................................................................................................^ L IST O F F IG U R E S ...........................................................................................................................................................................................V1 1 1 L IST S O F TABLES ...................................................................................................................................................... IX ABBREV IA T ION S ..................................................................................................................................................................................................X A B STRA C T ............................................................................................................................................................................................................. X I C H A PT ER 1 ............................................................................................................................................................................................................... 1 1.0 INTRODUCTION AND L ITERATURE R E V IEW ................................................................................................................ 1 1.1 Introduction....................................................................................................................................................................I 1.2 Objectives o f Study ........................................................................................................................................................ 5 1.3 Literature Review ..........................................................................................................................................................6 1.3.1 Biomining........................................................................................................................................................................................6 1.3.2 Biohydrometallurgy and Conventional Methods o f Low-Grade Ore Processing............................................................... 6 1.3.3 Bacterial Leaching of Mineral Ore..............................................................................................................................................7 1.3.4 Classification o f Biooxidizing Bacteria used in Mining Operations.....................................................................................8 (i) Ferrous Iron and Sulpho-oxidizing Bacteria.............................................................................................................................8 (ii) Bacterial Growth Temperatures..............................................................................................................................................8 1.3.5 Chemolithotrophic Bacteria as Catalytic Agents in M ining................................................................................................... 9 (i) Genus Thiobacillus........................................................................................................................................................................ 9 (a) Thiobacillus thiooxidans.................................................................................................................................................. 10 (b) Thiobacillus ferrooxidans................................................................................................................................................ 11 (ii) Genus Leptospirillum............................................................................................................................................................ 13 (a) Leptospirillum ferrooxidans.............................................................................................................................................13 (b) Leptospirillum thermoferrooxidans................................................................................................................................13 1.3.6 Mechanisms of Biooxidation.....................................................................................................................................................14 (i) Direct Contact Mechanism of Biooxidation............................................................................................................................ 14 (ii) Indirect Contact Mechanism of Biooxidation....................................................................................................................16 1.3.7 Isolation o f Biooxidizing Bacteria from Natural Sources.....................................................................................................16 (i) Important Factors Considered in Isolating Biooxidizing Bacteria...................................................................................... 17 (a) Sample source....................................................................................................................................................................17 (b) Energy substrate, pH and temperature........................................................................................................................... 18 (c) Liquid medium...................................................................................................................................................................19 (d) Solid medium.....................................................................................................................................................................20 (ii) Purification o f Isolates...........................................................................................................................................................21 (iii) Maintenance and Preservation o f Bacterial Cultures....................................................................................................... 22 1.3.8 Identification o f Biooxidizing Bacteria....................................................................................................................................23 (i) Morphological Characteristics.................................................................................................................................................. 23 (ii) Cultural Characteristics......................................................................................................................................................... 24 (iii) Physiological Characteristics............................................................................................................................................... 25 (iv) Biochemical Characteristics..................................................................................................................................................26 (a) Formation of Metabolites.................................................................................................................................................27 (b) Genetic Relatedness o f Biooxidizing Bacteria............................................................................................................ 27 (c) Chemotaxonomy...............................................................................................................................................................28 1.3.9 Comparative Characterization of Biooxidizing Bacteria...................................................................................................... 29 (i) Use of Antigenic Properties.....................................................................................................................................................29 (ii) Use of Mineral Ore Oxidation Rate, pH, Temperature and Substrate Concentration Profiles..................................30 (iii) Use o f Protein Profiles in Characterization.......................................................................................................................3 1 (iv) Use of DNA Profiles in Characterization........................................................................................................................... 32 CH A PTER 2 ............................................................................................................................................................................................................33 University of Ghana http://ugspace.ug.edu.gh 2.0 MATERIALS AND METHODS.................................................................................................................................. 33 2.1 Samples........................................................................................................................................................................ 33 2.2 Isolation, Purification and Maintenance o f Ferrous Iron Oxidizing Bacteria.....................................................33 (i) Isolation......................................................................................................................................................................................... 33 (ii) Pu rifica tion .............................................................................................................................................................................................. 37 (a) T. ferrooxidans-like Bacteria......................................................................................................................................... 37 (b) L. ferrooxidans-like Bacteria......................................................................................................................................... 38 (iii) Maintenance.............................................................................................................................................................................39 2.3 Isolation, Purification and Maintenance o f Sulpho-Oxidizing Bacteria...............................................................39 (i) Isolation......................................................................................................................................................................................... 39 (ii) Purification o f T. thiooxidans-like Bacteria........................................................................................................................ 39 (iii) Maintenance............................................................................................................................................................................ 40 2.4 Preservation o f Ferrous Iron and Sulpho-oxidizing Bacterial Isolates.................................................................40 2.5 Characterization o f Biooxidizing Bacteria............................................................................................................... 41 2.5.1 Staining of Bacterial Isolates..................................................................................................................................................... 41 (i) Basic Dye and Gram’s Staining................................................................................................................................................41 2.5.2 SDS-PAGE Analysis o f Bacterial Cell Lysates...................................................................................................................... 42 (i) Preparation o f Crude Bacterial Cell Lysates for SDS-PAGE and Electrophoretic ru n ...................................................42 2.5.3 Preparation and Restriction Endonuclease Cleavage o f Biooxidizing Bacterial Genomic DNA ............43 (i) Preparation o f Genomic DNA................................................................................................................................................... 43 (ii) Cleavage o f Genomic DNA with Restriction Endonucleases..........................................................................................45 CHAPTER 3 ...................................................................................................................................................................................46 3.0 RESULTS........................................................................................................................................................................46 3.1 Bacterial Isolates obtained from the Different Samples..........................................................................................46 3.1.1 Ferrous Iron Oxidizing Bacterial Isolates................................................................................................................................46 3.1.2 Sulpho-oxidizing Bacterial Isolates.......................................................................................................................................... 46 3.2 Designations o f Bacterial Isolates.............................................................................................................................48 3.3 Identification o f Purified Bacterial Isolates.............................................................................................................48 3.3.1 Ferrous Iron Oxidizing Bacteria................................................................................................................................................48 3.3.2 Sulpho-oxidizing Bacteria..........................................................................................................................................................49 3.4 Physiological, Cultural, Morphological and Biochemical Characteristics o f Selected Bacterial Isolates 60 3.5 Viability o f Stored Bacterial Isolates......................................................................................................................... 63 3.6 Comparison o f Bacterial Isolates using SDS-PA GE ............................................................................................... 63 3.7 Comparison o f Bacterial Isolates by Genomic DNA Analysis using RFLP ......................................................... 69 CHAPTER 4 .................................................................................................................................................................................. 71 4.0 DISCUSSION AND CONCLUSIONS....................................................................................................................... 71 RECOMMENDATIONS............................................................................................................................................................. 75 REFERENCES.............................................................................................................................................................................. 76 APPENDICES .91 University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES F ig u re 1 P ho tog ra ph sh ow in g t h e su lph id e t r ea tm en t pla n t (S T P ) a t O b u a s i . A = r e a c t o r ta n k , B = SETTLING TANK.................................................................................................................................................................................................. 34 F ig ure 2 P ho tog ra ph sh ow in g su r fa c e m in in g s it e a t O bua s i w h er e t h e su r fa c e g o ld o r e sam pl e (SO ) w a s c o l l e c t e d .....................................................................................................................................................................................35 F ig u r e 3 P h o tog ra ph s sh ow in g u n d erg r o u n d m in in g s it e s w h er e th e u n d erg r o u n d g o ld o re sam pl e (U O ) w as co l l e c t ed ....................................................................................................................................................................................36 F ig u re 4 P ho tog ra ph sh ow in g sta g es o f g row th o f th e fer ro u s ir o n o x id iz in g ba c t e r ia ......................................4 7 F ig u r e 5 Iso la te B T 1 -F e2+ g row n on fer rou s a g a r o se sh ow in g o ra n g e , sm a l l p in po in t co lo n ie s ‘F ’.............52 F ig u r e 6 S m a l l , p in p o in t , r e d d i s h b r o w n ( i r o n e n c r u s t e d ) c o l o n i e s ‘G ’ o f i s o l a t e B T1 -F e 2+ g r o w n fer rou s a g a r ................................................................................................................................................................................................... 52 F ig u r e 7 L arg e a n d c ir c u la r co lo n y ‘A ’ o f iso la t e B T 1 -F e2+ g row n o n fer r o u s a g a r ..............................................53 F ig u r e 8 C h a r a c t e r i s t i c “ f r o s t y ” c o l o n i e s ‘B ’ o f i s o l a t e B T 1 -F e 2+ g r o w n o n s o d iu m t h i o s u l p h a t e AGAR........................................................................................................................................................................................................................53 F ig u r e 9 L a rg e , s pr ea d in g co lon ie s ‘E ’ o f iso la t e B T 1 -F e2+ g row n on fer r o u s / so d ium th io su l ph a te AGAR. ‘D ’ SHOWS IRON ENCRUSTED CENTER AND ’G ’ SMALL, PINPOINT COLONIES...................................................... 54 F ig u r e 10 L a rg e , s pr ea d in g co lo n y ‘E ’ o f iso la t e U W 1 -F e2+ g row n on fer r o u s /s o d ium th io su l ph a te AGAR....................................................................................................................................................................................................................... 54 F ig u r e 11 P ha se -co n tra st ph o tom ic r o g ra ph (m a g . X 1000) sh ow in g s tr a ig h t ro d c e l l s ‘N ’ o f iso la t e B T 1 -F e2+...............................................................................................................................................................................................................55 F ig u r e 12 P ha se -co n tra st ph o tom ic r o g ra ph (m a g . X 1000) sh ow in g str a ig h t rod c e l l s ‘M ’ o f iso la te UW 1 -F e2+.............................................................................................................................................................................................................55 F ig u r e 13 P ha se -co n tra st ph o tom ic r o g ra ph (m a g . X 1000) sh ow in g c l u st e r e d str a ig h t rod c ell s ‘O ’ iso la t e S 0 1 -F e2+............................................................................................................................................................................................ 56 F ig u r e 14 P ha se -co n tra st ph o tom ic r o g ra ph (m a g . X 1000) sh ow in g cu r v ed o r com m a rod cell s ‘P ’ of iso la te BT1 -F e2+. S l en d er a rrow sh ow s sp ir a l sh a ped c e l l s ....................................................................................57 F ig u r e 15 P u n c t i f o rm o r m in u t e c o l o n i e s ‘R ’ o f i s o l a t e B T 3 -S 0 g r o w n o n s o d iu m t h i o s u l p h a t e a g a r . In ser t sh ow s en la rg ed co lon ies in box w ith th e a rrow po in t in g to a s in g l e co lo n y tha t a ppea r s as a w h it e s p o t .............................................. 58 F ig u r e 16 P ha se -co n tra st ph o tom icr o g ra ph (m a g . X 1000) sh ow in g str a ig h t rod cell s ‘X ’ o f iso la t e B T 3 -S 0...................................................................................................................................................................................................................59 F ig u r e 17 C om pa r iso n o f ba c te r ia l c e l l ly sa te s o f iso la t es by S D S -PA G E .........................................................................65 F ig u r e 18 SD S -PA G E o f b a c t e r i a l c e l l l y s a t e s o f T. f e r ro o x id a n s f r o m b i o o x i d a t i o n t a n k .................................66 F ig u r e 19 SD S -PA G E of ba c ter ia l ce l l ly sa te s o f L . fer ro ox id a n s from b io o x id a t io n t a n k ............................... 67 F ig u r e 2 0 SD S -PA G E o f b a c t e r i a l c e l l l y s a t e s o f T. th io o x id a n s i s o l a t e s f r o m t h e b i o o x i d a t i o n t a n k .......................................................................................................................................................................................................................68 F ig u r e 21 A g a ro se g e l e lec tr o ph o r e s is o f un d ig e st ed b io o x id iz in g ba c ter ia D N A ...................................................... 70 F ig u r e 2 2 R e str ic t io n e n d o n u c lea se (H in d II I) d ig e st ed D N A o f b io o x id iz in g ba c te r ia l iso la t es from b io o x id a t io n t a n k .......................................................................................................................................................................................70 University of Ghana http://ugspace.ug.edu.gh LISTS OF TABLES T a b l e 1 S e lec ted b io o x id iz in g ba c te r ia l iso la t e s a n d t h e ir d e s ig n a t ed c o d e s ............................................................... 50 T a b l e 2 P h y s io lo g ic a l c h a ra c t er is t ic s o f b io o x id iz in g ba c te r ia l is o l a t e s ....................................................................... 51 T a b l e 3 C u l t u r a l a n d m o r p h o l o g i c a l c h a r a c t e r i s t i c s o f b a c t e r i a l i s o l a t e s o n a g a r a n d a g a r o se m e d ia ................................................................................................................................................................................................ 61 T a b l e 4 C u l tu r a l , m o r ph o lo g ic a l and b io c h em ic a l ch a ra c t er is t ic s o f b io o x id iz in g ba c ter ia l iso la t es in 9 k l iq u id m e d ia ....................................................................................................................................................................62 T a b l e 5 V i a b i l i t y o f s t o r e d b a c t e r i a l i s o l a t e s ........................................................................................................................................64 ix University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS APS -Ammonium per sulphate 2-ME -2-mercaptoethanol NMIMR -Noguchi Memorial Institute for Medical Research SDS-PAGE -Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis TEMED -N, N, N ’- N-tetramethylethylenediamine RFLP -Restriction Fragment Length Polymorphism PMSF -Phenyl methyl sulfonyl fluoride TPCK -Tosyl phenyl alanine chloromethyl ketone TLCK -Tosyl lysine alanine chloromethyl ketone University of Ghana http://ugspace.ug.edu.gh ABSTRACT Processing o f gold arsenopyrite and sulphide ores is currently done with biooxidizing bacteria. This procedure is preferred because o f its environmental friendliness and efficiency o f gold recovery from ores, compared to the conventional methods. The efficiency o f biomining is, however, largely influenced by the origin o f biooxidizing bacteria used and local organisms have been found to be better adapted to extracting gold from the ore from which they were isolated. The work reported in this thesis was conducted with the objective o f isolating and characterizing local acidophilic bioleaching bacteria from surface arsenopyrite and underground sulphide gold ores and underground mine water at the Ashanti Goldfields Company (AGC) in Ghana, one o f the world’s richest gold mines. Biooxidizing bacteria were also isolated from slurry from the commercial bioleaching tank at AGC and characterized for comparative purposes. Local biooxidizing bacteria were isolated from surface arsenopyrite and underground sulphide gold ores and underground mine water and identified using cultural, physiological, morphological and biochemical criteria. In all, eleven bacterial isolates were obtained from the samples collected. Their cell morphology showed that seven isolates were straight rods while the others were curved rods. All the bacterial isolates were physiologically either ferrous iron (Fe2+) oxidizers or sulpho (S0)- oxidizers, Gram negative, mesophiles as well as aerobes. Three representative pure biooxidizing bacteria isolates were obtained from the surface ore (SO). These were, S01-Fe2+ and S02-Fe2+ both o f which were ferrous iron oxidizers and S03-S0 which was a sulpho oxidizer. Similarly, three pure isolates were obtained from the underground ore (UOl- Fe2+, U02-Fe2+ and U 03-S0). However, only two representative ferrous iron oxidizers were purified from underground mine water (UWl-Fe2+ and UW2-Fe2+). The ferrous iron bacterial isolate 2 (S02- / xi University of Ghana http://ugspace.ug.edu.gh Fe2+, U02-Fe2+ and UW2-Fe2+) from each o f the samples failed to grow on solid media whilst, the ferrous isolate l(S01-F e2+, U01-Fe2+. UW l-Fe2+) as well as the sulpho oxidizers (S03-S0 and U 03 - S°) grew on solid media. Biooxidizing bacterial isolates (BT1-Fe2+, BT2-Fe2+and BT3-S0) obtained from the commercial bioleaching tanks exhibited similar characteristics. Characterization o f the purified bacterial isolates using cultural, physiological, morphological and biochemical criteria revealed that the ferrous iron oxidizers, isolate 1 (BT1-Fe2+, S01-Fe2+, U 01-Fe2+ and UW l-Fe2+) were T. ferrooxidans and the second isolates (BT2-Fe2+, S02-Fe2+, U 02-Fe2+ and UW2-Fe2+) were L. ferrooxidans. The sulpho oxidizers (BT3-S0, S03-S0 and U 03-S0) were identified as T. thiooxidans. In addition to the absence o f sulpho-oxidizing bacterium (T. thiooxidans) in the underground mine water, the iron oxidation rates o f T. ferrooxidans and L. ferrooxidans isolates obtained from the sample were slower compared to isolates o f the same bacteria from the other samples. Furthermore, T. ferrooxidans isolated from underground mine water did not exhibit pleomorphism and had a unique colony size (2-5mm) as compared to the other isolates (0.5-5mm) thus suggesting that it could be o f a different strain. Attempts to characterize the bacterial isolates and detect strain differences by restriction fragment length polymorphism o f their DNA, however, proved difficult because o f smearing o f bands. Soluble proteins from crude bacterial cell lysates o f all the purified isolates (BT1-Fe2+, S01-Fe2+, U01-Fe2+, UW l-Fe2+, BT3-S0, S03-S0 U 03-S0 BT2-Fe2+, S02-Fe2+, U02-Fe2+ UW2-Fe2+) were electrophoresed on 15-20 % acrylamide gradient gels, using the SDS-tris-glycine discontinuous buffer system, and the protein profiles analyzed. This electrophoretic analysis showed that the T. ferrooxidans isolates (BT1-Fe2+, S01-Fe2+, U01-Fe2+ and UW l-Fe2+) had similar protein profiles. Similarly, T. thiooxidans isolates (BT3-S0, S03-S0 and U 03-S0) and L. ferrooxidans isolates (BT2- Fe2+, S02-Fe2+ and U02-Fe2+) had unique protein profiles. The protein profiles clearly differentiated University of Ghana http://ugspace.ug.edu.gh the bacterial isolates at the species level. This study has therefore demostrated the presence o f local isolates o f the 3 important biooxidizing bacteria utilized in biomining and appears to suggest that some o f the local isolates are different strains that may be used for more effficent gold extraction at the AGC. xiii University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.0 INTRODUCTION AND LITERATURE REVIEW 1.1 Introduction Trading in precious metals especially gold has for centuries been an important economic activity in Ghana. Today, mining, processing and export o f mineral ores including gold, bauxite, manganese and diamond constitute a sizeable percentage o f the total export earnings o f Ghana. Presently, gold, the largest income generating export commodity in Ghana accounts for over 50% o f the national foreign exchange earnings (AGC Annual report, 1997). This contributes to the financing o f health, education and infrastructural development amongst other developmental priorities o f the nation. With civilization and technological advances, the consumption o f diverse metal products has increased immensely. Unfortunately, just like in other parts o f the world, there is an apparent exhaustion o f the supply o f high grade mineral ore in Ghana due to the successive mining o f such mineral deposits (Rawlings and Woods, 1995). There are, however abundant quantities o f low-grade ore but this is difficult to process using conventional technology or traditional methods. The conventional technology used in processing this type o f ore (roasting at high temperatures and pressure oxidation) cannot efficiently handle extraction o f the metal. These methods are known to be inefficient, expensive and environmentally degrading (Ehrlich and Holmes, 1985; Barrett et al., 1993). It is therefore, necessary to shift to the use o f more efficient extraction methods such as biotechnological applications, i f the industry is to survive into the next century (Barrett et al., 1993; Morin, 1995; Pizzaro et al., 1996). The applicability o f biotechnological methods such as processing mineral ore with microbes (biomining) in some o f the major gold mining industries o f the world was communicated by Rawlings and Silver (1995). Microbial mining (biohydrometallurgy) began in 1963 when results o f laboratory studies confirmed the involvement o f bacteria in the solubilization o f copper from sulphidic ore (Beck, 1967). Subsequently, various sulpho-oxidizing and ferrous l University of Ghana http://ugspace.ug.edu.gh oxidizing bacteria were reported to enhance the extraction o f gold, uranium and copper from their respective low grade ores (Rawlings and Silver, 1995). Today, biomining is known to be easy to implement, cost effective and has minimal effects on the environment (Barrett et al., 1993). Chemolithotrophic bacteria o f the genera Thiobacilli and Leptospirilli are used in the extraction o f gold from refractory ore. In refractory ore, gold is trapped in a compound matrix with sulphur, ferrous iron and arsenic (Barrett et al., 1993). This process which is known as bioleaching, gives 95 to 99% efficiency in the recovery o f gold as compared to conventional methods o f roasting and cyanide treatment which give 30 to 50% recovery (Norman and Snyman, 1988; Morin, 1995). A synergistic mixture o f Leptospirillium ferrooxidans, Thiobacillus ferrooxidans and Thiobacillus thiooxidans are extensively used in the gold mining industries o f the world (Morin, 1995; Jerez et al., 1995). These bacteria obtain energy by oxidizing sulphide and ferrous iron components o f the ore thereby releasing chemically trapped gold particles. Other products o f these reactions are sulphuric acid and ferric compounds. The importance o f biomining at one o f the richest gold mines in the world, the Ashanti Goldfields Company Limited (AGC) in Ghana cannot be over-emphasized. AGC has proven reserves o f about 21 million ounces o f gold and it currently operates the world's largest bioleaching plant for refractory gold ore (Osae et al., 1995; AGC Annual Report, 1996). The plant processes about 800 tons o f ore per day and provides about 30 to 40% o f the total gold ore processed by the company (AGC Annual Report, 1996). Microbial mining activity at AGC mining site is likely to increase in the future because o f the progressive depletion o f high grade oxide ore in the presence o f large quantities o f low grade arsenopyrite and sulphide ores (AGC Annual Report, 1994). It is, therefore important to ensure that the efficiency o f the bioleaching plant is maintained at a high level. One way to ensure such efficiency is to use local strains o f bioleaching organisms (Rawlings and Woods, 1995). This is 2 University of Ghana http://ugspace.ug.edu.gh because efficiency in biooxidization depends on the bacterial strains found in the particular environment. It has also been observed that every ore deposit is unique with respect to its mineralogy and chemical composition (Rawlings and Woods, 1995) thus bacterial populations that rapidly oxidize one ore deposit may not be very active on another deposit which has higher concentrations o f heavy metals such as arsenic and silver which may be toxic to the bacteria (Pakniker and Agate, 1987). Interestingly, bacterial strains used by the AGC in the sulphide treatment plant (STP) were obtained from Gencor Company in South Africa and they are being used to process surface arsenopyrite and underground sulphide ores that have different characteristics. These biooxidizing bacteria are known to exist in soils containing sulphur compounds (Waksman and Joffe, 1922), refractory gold ore (Livesey-Goldblatt et al., 1983; Livesey-Goldblatt, 1986), acid mine drainage and underground mine water (Colmer et al., 1950; Beck, 1960; Rawlings and Woods, 1995). It is therefore possible to obtain local isolates o f biooxidizing bacteria from the AGC (Obuasi) mining site, which may be more efficient than the strains currently in use at the sulphide treatment plant. It is equally important to characterize any local isolates that might be found since efficiency in biooxidation depends on the bacterial strain found in a particular environment (Rawlings and Woods, 1995). It was based on these observations that the work described in this thesis was conducted to isolate and characterize biooxidizing bacteria from different sources at the Obuasi mining site. 3 University of Ghana http://ugspace.ug.edu.gh JUSTIFICATION FOR THE WORK: The isolation and characterization o f local biooxidizing bacteria from the Obuasi mining site (Ghana) would provide valuable information on local biooxidizing organisms from a part o f the world that had previously not been explored. Furthermore, the characterization o f local biooxidizing bacteria may lead to the identification o f bacterial strains which are better suited for more efficient extraction o f gold from surface arsenopyrite and underground sulphide ores o f different characteristics. 4 University of Ghana http://ugspace.ug.edu.gh 1.2 Objectives of Study (a) To isolate and identify the iron and sulpho-oxidizing bacteria (T. ferrooxidans, T. thiooxidans and L. ferrooxidans) from the Sulphide Treatment Plant (STP) reactor tanks at the Obuasi mining site. (b) To isolate and identify local strains o f these organisms from the surface arsenopyrite and underground sulphide ores, and from underground mine water at Obuasi. (c) To establish pH, temperature and substrate concentration profiles for optimum growth o f the local biooxidizing bacterial isolates. (d) To determine possible similarities and differences in protein profiles between local biooxidizing bacterial isolates and those present at the STP using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). (e) To determine possible similarities and differences in DNA fingerprints between local biooxidizing bacterial isolates and those present at the STP using Restriction Fragment Length Polymorphism (RFLP) o f genomic DNA. 5 University of Ghana http://ugspace.ug.edu.gh 1.3 Literature Review 1.3.1 Biomining Micro-organisms have been used as biotechnological tools for many purposes. Biomining is the process o f metal extraction which uses micro-organisms such as bacteria in commercial mining operations (reviewed by Brierley, 1978; Lundgren and Silver, 1980). The bacteria attack and help dissolve mineral ore, by converting insoluble metal deposits such as metal sulphides or oxides o f gold, copper and uranium through metabolic processes to soluble metal sulphates. According to Rawlings and Silver (1995) and Barrett et al. (1993) this exploitation o f micro-organisms simplifies metal extraction from low grade mineral ore. The involvement o f bacteria in metal leaching is reported to be physiological and is as a consequence o f the bacteria’s mode o f metabolism which results in their growth and survival in the mining environment (Lundgren and Silver, 1980). 1.3.2 Biohvdrometallurgy and Conventional Methods o f Low-Grade Ore Processing Hydrometallurgy is the process o f dissolving metals from mineral ore and the recovery o f the desired metals. The involvement o f bacteria in this process is called biohydrometallurgy. Two main conventional methods were developed for commercial extraction o f gold from low grade ore. These were (1) pressure oxidation, involving digestion o f the ore with acid in a pressure cooker in an oxygen enriched atmosphere and (2) roasting the ore in a furnace at 700°C in the presence o f oxygen (Barrett et al., 1993; Morin, 1995). The application o f these mining methods were found necessary because o f several reasons. For example, the depletion o f high grade ore deposits forced mining companies to work low grade ores and also to develop more efficient methods o f recovering small quantities o f metals left after physical processing o f richer ore materials (Rawlings and Woods, 1995). Nevertheless, the use o f these conventional mining methods led to other related problems such as high cost o f metal extraction compared to value o f metal recovered. Also there was the need for highly skilled operators to run processing plants (Morin, 1995) and environmental pollutants were 6 University of Ghana http://ugspace.ug.edu.gh created (Rawlings and Silver, 1995). Mining companies therefore had to look for alternative methods that were preferably more cost effective and environmentally friendly for recovering valuable metals from low grade ores (Livesey-Goldblatt et al., 1983; Ehrlich and Holmes). The most suitable alternative to conventional mining techniques so far introduced is bioleaching (Morin, 1995). Bioleaching is a hydrometallurgical process in which mineral ore is differentially or collectively solubilized (Barrett et al., 1993). By this process insoluble metal sulphides and oxides are made soluble (leached from ores) through the natural action o f autotrophic bacteria under suitable growth conditions. These bacteria called biooxidizers, carry out oxidation o f the mineral ores as a means o f generating energy for cell growth, cell division and other cellular metabolic processes. Most commercial bioleaching is however, carried out with a consortium o f chemolithotrophic bacteria which efficiently oxidize ferrous compounds and reduced sulphur compounds present in mineral ores. However, heterotrophic bacteria and other micro-organisms such as yeast, fungi, algae and mould are believed to play significant but undetermined roles which enhance the biooxidation o f mineral ore. According to Barrett et al. (1993) the overall significance o f the heterotrophic species in metal leaching systems is not yet clear because o f lack o f suitable data. 1.3.3 Bacterial Leaching o f Mineral Ore Some inorganic ion oxidizing chemolithotrophic bacteria have the ability to oxidize insoluble metal sulphides and solubilize a wide range o f metals including copper, uranium and more recently gold bearing arsenopyrite ores in great tonnages. Other ore types processed using bacterial leaching include sulphides o f zinc, lead, cobalt, nickel, bismuth, molybdenum and manganese (Brierley, 1978; Brierley, 1982; Barrett et al., 1993; Rawlings and Silver, 1995). It is reported that bacterial leaching activity can achieve 100% recovery o f metal from ores containing as low as 0.03% to 0.3% metal (Rawlings and Silver, 1995). Most strains o f bacteria being used in bioleaching operations have been 7 University of Ghana http://ugspace.ug.edu.gh isolated from sites where natural leaching takes place (Lundgren and Silver, 1980; Hamson, 1984) and the bacteria are therefore classified on the basis o f the ore they oxidize. Commercial bioleaching processes include dump, in situ, heap, vat, and agitated tank leaching methods (Barrett et al., 1993). In bioleaching o f refractory gold ore, the agitated or stirred reactor method is mostly used (Brierley, 1978; Barrett et al., 1993; Rawlings and Silver, 1995). 1.3.4 Classification o f Biooxidizing Bacteria used in Mining Operations (i) Ferrous Iron and Sulpho-oxidizing Bacteria Ferrous iron bacteria oxidize iron in the +2 oxidation state (Fe2+) to its +3 state (Fe3+). Those used in mining operations belong to the genera Thiobacillus and Leptospirillum. The ore types oxidized by these bacteria include arsenopyrite (FeAsS), pyrite (FeS2), mascasite (Fe2S), pyrrhotite (FeS) and chalcopyrite (FeCuS2) (Lundgren and Silver, 1980; Barrett et al., 1993; Rawlings and Silver, 1995). Unlike ferrous iron oxidizers, sulpho-oxidizing bacteria oxidize elemental sulphur as well as sulphur in other oxidation states (reduced sulphur and partially reduced sulphur as found in sulphidic ore) to +4 oxidation state to form sulphuric acid. Sulpho-oxidizers used in the mining industry belong mainly to the genus Thiobacillus (Rawlings and Silver, 1995). (ii) Bacterial Growth Temperatures The optimum growth temperature o f biooxidizing bacteria is one o f the criteria used in the classification o f the microbes utilized in biohydrometallurgical processes. The important groups based on temperature are: (a) mesophiles o f the genera Thiobacillus and Leptospirillum which grow at temperatures from 4 to 40 °C, (b) moderate thermophiles o f the genus Sulfobacillus together with a number o f unidentified strains, which grow at temperatures from 40 to 55°C, (c) extreme thermophiles o f the genera Sulfolobus, Acidanus, Metallosphaera and Sulfurococcus which grow at 8 University of Ghana http://ugspace.ug.edu.gh temperatures greater than 55°C and (d) heterotrophic micro-organisms o f the genera Acidophilum and Acetobacter among others with growth temperatures o f 59°C and 5 to 42 °C, respectively. Some of the heterotrophic bacteria have commensal relationship with the mesophilic biooxidizing bacteria (Brierley, 1978; Lundgren and Silver, 1980; Harrison, 1984). However, most bacteria presently used in commercial mining are mesophiles o f the genera Thiobacillus and Leptospirillum, (Morin, 1995) even though, the other types o f micro-organisms mentioned above are also believed to play important roles in the biooxidation o f mineral ore (Brierley, 1978; Rawlings and Silver, 1995). 1.3.5 Chemolithotrophic Bacteria as Catalytic Agents in Mining Chemolithotrophic bacteria make use o f inorganic salts in deriving energy for their life processes. They thus act as biological catalysts in mineral ore processing by helping in the oxidation o f the ore leading to the release o f metals. Most bacteria used in catalytic oxidation o f mineral ore are mixed cultures which share synergistic benefits, making the oxidation o f low grade ore more efficient than the conventional methods used (Barrett et al., 1993; Rawlings and Silver, 1995). Presently, the most extensively (commercially) exploited bacterial species which efficiently and rapidly attack mineral ore are T. thiooxidans and T. ferrooxidans from the genus Thiobacillus and L. ferrooxidans from the genus Leptospirillum (Rawlings and Woods, 1995). (i) Genus Thiobacillus Sulphur oxidizing bacteria were discovered in 1902 by Beijerinck (cited by Vishnac, 1975). Since then at least 14 species have been reported. These microbes utilize elemental sulphur, sulphur compounds and sulphuric ore as substrates for energy metabolism (Vishnac, 1975; Barrett et al., 1993). The most studied o f the Thiobacillus associated with metal extraction are T. thiooxidans and 9 University of Ghana http://ugspace.ug.edu.gh T. ferrooxidans. Although there is evidence that most o f the remaining 12 species may participate in biooxidation o f mineral ore, only the two species mentioned are important in biomining (Rawlings and Silver, 1995). An important characteristic o f the two species is that they can better tolerate ions o f heavy metals like copper, nickel, zinc, uranium and arsenic. The high resistance o f these bacterial strains to metal ions cannot be explained by only physiological and genetic properties o f the organisms (Barrett et al., 1993). It is reported that this property may just be a pseudo-resistance which depends on the state o f the metallic ions in the different environments such as: (1) complexing o f the ions with dead bacterial cells or organic metabolites, (2) precipitation o f the metal ions, (3) low pH o f the media which makes the bacteria surface binding sites less available for metals and (4) presence o f other substances that compete with the toxic or metalloid species (Barrett et al., 1993; Rawlings and Kusano 1994). Nevertheless, it is known that other heavy metals such as silver, mercury, antimony, cadmium, chromium and molybdenum may inhibit T. thiooxidans and T. ferrooxidans (Brierley, 1978; Barrett et al., 1993; Rawlings and Silver, 1995). (a) Thiobacillus thiooxidans Thiobacillus thiooxidans was first isolated in 1922 (Waksman and Joffe, 1922) from compost soil. Three species types (1, II and III) are classified under genus Thiobacilli based on their cellular lipid composition (Vishnac, 1975). The T. thiooxidans utilized in biooxidation is placed under the type HI species. Their growth temperature ranges from 2 to 40 °C, with the optimum between 25 to 35 °C (Barrett et al., 1993). The organisms are reported to grow at pH ranging from pH 0.5 to 6 (Vishnac, 1975; Konishi et al., 1995) with an optimum from pH 1.5 to 2.0 (Rawlings and Silver, 1995). Thus, T. thiooxidans is able to grow under very high acidic conditions (pH <1) mostly because o f its ability to rapidly oxidize elemental sulphur or partially reduced sulphur compounds, such as sodium thiosulphate (Na,S20 3) to sulphuric acid (Vishnac, 1975). 10 University of Ghana http://ugspace.ug.edu.gh T. thiooxidans organisms are strict autotrophs and aerobes. Strains o f the bacteria have been isolated from acid mine waters, corroding steel and concrete, as well as from sulphidic ore (Vishnac, 1975; Lane et a l, 1992; Padival et a l, 1995). The organisms are not able to withstand environmental temperatures above 45°C due to their low resistance to desiccation (Starkey, 1925; Hamson, 1982; Barrett et a l, 1993). In the laboratory, ammonium sulphate serves as their nitrogen source during cultivation, though urea and nitrates can also be used (Newburgh, 1954; Brierley and Brierley, 1968; Vishnac, 1975; Brierley, 1978; Harrison, 1984). The identification o f T. thiooxidans is based partly on the mole % o f guanine and cytosine (G+C) content o f their DNA which is reported to range from 50 to 65 % depending on the type o f isolate (Harrison, 1984; Lane et a l , 1992). T. thiooxidans strains have been found to exhibit various morphological forms and behaviour in liquid medium supplemented with sulphur. (b) Thiobacillus ferrooxidans T. ferrooxidans was first isolated in 1949 (Colmer et al., 1950) and characterized in 1951 (Temple and Colmer, 1951). The bacterium has the ability to oxidize ferrous iron, elemental sulphur, as well as partially reduced sulphur compounds to derive energy for metabolism (Silverman and Lundgren, 1959; Unz and Lundgren, 1961; Harrison, 1984; Rawlings and Silver, 1995). Leathen et a l (1956) isolated an iron oxidizing bacterium which they named Ferrobacillus ferrooxidans based on its inability to utilize sulphur as energy substrate. Kinsel (1960) also isolated a biooxidizing bacterium which he named Ferrobacillus sulfooxidans based on its ability to utilize ferrous iron and sulphur but not sodium thiosulphate. However, careful work by Kelly and Tuovinen (1972) showed that both Ferrobacillus ferrooxidans (Leathen et a l, 1956) and Ferrobacillus sulfooxidans (Kinsel, 1960) could indeed utilize ferrous iron, as well as sulphur and sodium thiosulphate, thus invalidating the different names given and confirming the organisms to be T. ferrooxidans. Interestingly, many 11 University of Ghana http://ugspace.ug.edu.gh strains o f T. ferrooxidans have also been found to fix nitrogen (Temple and Colmer, 1951; Mackintosh, 1978; Harrison, 1984; Rawlings and Kusano, 1994). Many strains o f T. ferrooxidans have been isolated from different geographical areas (Lane et al., 1992). The bacteria are mainly autotrophs that preferentially utilize ferrous iron in the presence o f both ferrous iron and sulphur compounds. Nevertheless, in the presence o f large amounts o f sulphide ore it oxidizes sulphur rather than ferrous iron (Margalith et al., 1966; Harrison, 1984; Shrihari et al., 1991). For T. ferrooxidans, the amount o f dissolved oxygen available for growth plays a critical factor in its oxidation o f mineral ore (Lui et al., 1988; Barrett et al., 1993), thus most strains o f T. ferrooxidans isolated are aerobes but some are anaerobes (Rawlings and Kusano, 1994; Das and Mishra, 1996). T. ferrooxidans has been shown to be involved in the oxidation o f pyrite, marcasite and coal seams to ferric sulphate and sulphuric acid (Colmer and Hinkle, 1947; Das and Mishra, 1996; Nyavor et al., 1996). The bacterium is considered to play a principal role in bioleaching operations because it oxidizes both ferrous and sulphuric ore, and is found in most mining sites where a form o f leaching occurs. T. ferrooxidans is also useful in desulphurization o f coal (Ehrlich and Holmes, 1985; Harrison, 1986; Rawlings and Silver, 1995; Gaylarde and Videla, 1995; Hallberg et al., 1996). Its growth temperature ranges from 2 to 40 °C (Barrett et al., 1993) with an optimum from 25 to 35 °C (Ferroni et al., 1986; Ahonen and Tuovinen, 1991). Its range o f pH adaptability is similar to that o f T. thiooxidans (pH 0.5 to 6) with an optimum from pH 1.5 to 2.5 (Harrison, 1984; Barrett et al., 1993; Rawlings and Silver, 1995). 12 University of Ghana http://ugspace.ug.edu.gh («) Genus Leptospirillum The genus Leptospirillum is classified under the family Spirillaceae. The genus includes mesophilic species and moderate thermophiles o f which L. ferrooxidans and L. thermoferroxidans are, respectively the most important in biooxidation. Members o f the genus Leptospirillum have been isolated from mining environment (Harrison, 1986). L. ferrooxidans are acidophiles which utilize ferrous iron, pyrite and some types o f mineral ore as energy substrate (Harrison, 1984; Harrison and Norris, 1985; Sand et al., 1992). (a) Leptospirillum ferrooxidans L. ferrooxidans was first isolated by Markosyan in an Armenian copper deposit (Markosyan, 1972) and characterized in 1974 (Balashova et al., 1974). The bacterium oxidizes ferrous iron slowly, however, in the presence o f cysteine in its growth medium the bacterium rapidly oxidize Fe2+ to Fe3+ (Harrison, 1984). It grows between a temperature range o f 4 to 45 °C with an optimum from 25 to 35 °C. It also grows at a pH range o f pH 1 to 4 with an optimum pH between pH 1.5 to 2.0 (Barrett et al., 1993; Rawlings and Silver, 1995). Most strains o f L. ferrooxidans are reported to have a mole % (G+C) above 50% (Lane et al., 1992). (b) Leptospirillum thermoferrooxidans L. thermoferrooxidans is an obligate autotroph. It is a moderate thermophile (Barrett et al., 1993) with optimum growth temperature ranging from 45 to 50 °C and optimum pH from pH 1.65 to 1.9. Even though this bacterium plays an important role in commercial bioleaching o f gold ore it is not used extensively because o f its thermophilic properties (Barrett et al., 1993; Rawlings and Silver, 1995). L. thermoferrooxidans is a strict aerobe that obtains energy solely by oxidizing Fe2+ in aqueous solution (Lane et al., 1992; Sand et al., 1992). Its mole % (G+C) is 65%. 13 University of Ghana http://ugspace.ug.edu.gh 1.3.6 Mechanisms o f Biooxidation Two main mechanisms o f oxidation o f iron and sulphur by biooxidizing bacteria have been reported (Silverman, 1967; Brierley, 1978; Lundgren and Silver, 1980; Barrett et al., 1993). These are: (1) the direct contact mechanism and (2) the indirect contact mechanism. Both processes o f oxidation have oxygen as the terminal electron acceptor that is reduced to water. 0 2 + 4H+ + 4e' 2H20 (i) Direct Contact Mechanism o f Biooxidation In the direct contact mechanism o f biooxidation there is an intimate physical contact between the bacteria and the sulphidic mineral ore. Under aerobic conditions bacterial leaching occurs through the action o f bacterial enzymes on the components o f the mineral ore that are susceptible to oxidation. This process o f solubilization o f the metal sulphate had been confirmed by observing the rate o f bacteria acceleration o f the oxidation o f pyrite (FeS2), chalcocite (CuS), covelite (CuS) as well as other types o f mineral ore (Razzell and Trussell, 1963; Silverman, 1967; Mustin et al., 1992; Sand et al., 1992). Scanning electron micrographs have also revealed that numerous bacteria attach themselves to the surface o f sulphide minerals in solutions supplemented with nutrients (Bennett and Tributsch, 1978; Brierley, 1982). The direct contact mechanism is, however, difficult to demonstrate using iron containing minerals such as arsenopyrite (2FeAsS), chalcopyrite (CuFeS2) and bomite (Cu3FeS4) owing to the release o f soluble iron during oxidation and probable occurrence o f the indirect contact mechanism at the same time (Silverman, 1967). Metals in these ores are thus released through oxidative metabolism o f micro-organisms or solubilized indirectly by chemical oxidant produced as metabolic products o f the micro-organisms (Lundgren et al., 1985). 14 University of Ghana http://ugspace.ug.edu.gh T. ferrooxidans and L. ferrooxidans are reported to utilize the direct contact mechanism o f biooxidation during leaching o f mineral ore (Beck and Brown, 1968; Pinches, 1975; Brierley, 1978; Sand et al., 1992; Rawlings and Silver, 1995). However, an investigation by Silverman (1967) revealed that oxidation o f pyrite by T. ferrooxidans involves not only the direct attack o f the ore by the bacteria but also chemical oxidation o f the ore by ferric iron produced by the bacteria and released in the leacheate. Similarly, T. ferrooxidans selectively attacks sulphide containing minerals and converts the insoluble metal compounds o f copper, lead, zinc, nickel, cadmium and cobalt to their soluble metal sulphates (Brierley, 1978; Rawlings and Kusano, 1994). In this type o f reaction ferric iron present in the ore acts as a primary oxidant in the oxidation o f elemental sulphur by T. ferrooxidans. It has also been reported that T. ferrooxidans is selective in choosing sites in mineral ores that are favourable for energy extraction depending upon availability o f substrate (Bennett and Tributsch, 1978; Lundgren and Silver, 1980; Rodriguez-Leiva and Tributsch, 1988). T. thiooxidans also utilizes the direct contact mechanism in its oxidation o f the sulphur component o f sulphide ore (Konishi et al., 1995). The bacteria play an indispensable role in oxidizing sulphur to sulphuric acid thus exposing the constituent metals for further leaching (Brierley, 1982; Mustin et al., 1992; Curutchet et al., 1995). It does this by attaching itself to the sulphur particle before oxidizing it (Starkey, 1937; Harrison, 1984; Rawlings and Silver, 1995). It was reported that most o f the oxidation processes involving sulphide minerals occur through the direct contact mechanism as they involve attachment o f the various kinds o f bacteria to the mineral lattice and subsequent oxidation o f the iron and the sulphur components (Sakaguchi et al., 1976; Barrett et al., 1993). The involvement o f the bacterial pili in their attachment to the sulphide mineral surface has been demonstrated by electron microscopy (Lizama and Suzuki, 1988). Generally, however, all the processes involved in the direct contact mechanism are not fully understood (Bennett and Tributsch, 1978; Mustin et al., 1992). 15 University of Ghana http://ugspace.ug.edu.gh (ii) Indirect Contact Mechanism o f Biooxidation In the indirect contact mechanism o f biooxidation, the bacteria generate ferric iron (a powerful oxidizing agent) by oxidizing soluble ferrous iron. The ferric iron in turn reacts with other metals in the sulphidic mineral ore transforming them into soluble oxidized forms in an acidic (sulphuric acid) solution, and is itself reduced to ferrous iron. The biooxidizing bacteria then oxidize the ferrous iron to the ferric state thereby regenerating the primary oxidant. This mechanism is also referred to as bacterial assisted leaching (Brierley, 1982). It is, however, difficult to determine the roles o f the direct and indirect leaching mechanisms quantitatively because most mineral ores contain some iron that could initiate indirect leaching alongside the direct process (Brierley, 1982). Hence, in practice, leaching o f metals by bacteria is far more complex than the theory may suggest. This is especially so as there are many other minor processes in addition to the direct and indirect contact mechanisms described above (Barrett et al., 1993). Some o f the minor processes lead to the formation o f secondary compounds and elemental sulphur from sulphide ore which can inactivate the reactive surfaces o f the ore where bacterial leaching is taking place, thereby inhibiting the process. Nevertheless, as reported by Lundgren et al. (1985) and Rawlings and Silver (1995), the oxidation o f pyrite (4FeS2) and arsenopyrite (4FeAsS) could be summarized in the following reactions: (a) 4FeS2 + 150, + 2H20 — -----► 2Fe2(S04)3 + 2H2S04 (b) 4FeAsS + 1402 + 4HzO — ---- * 4FeAs04 + 4H2S04 1.3.7 Isolation o f Biooxidizing Bacteria from Natural Sources Micro-organisms can be selectively obtained from natural habitats such as soil or water either by direct or by enrichment isolation. Direct isolation involves using a selective medium solidified with 16 University of Ghana http://ugspace.ug.edu.gh a gelling agent and allowing colonies to develop. On the other hand enrichment isolation involves the use o f an inoculum in a liquid selective medium which selects for the micro-organism o f interest (Stainer et al., 1992). Isolation o f bacterial cultures for use in biomining is carried out by inoculating appropriate selective nutrient media with bacteria containing samples such as mineral ore, acid mine water and underground mine water. The medium consists essentially o f nutrients that support growth o f the organisms that are expected in the samples. Inhibitory media could also be used to help select for the organisms o f interest (Cowan, 1985). The need for isolation o f a single or mixed culture determines the choice o f medium. However, for the purpose o f identification and characterization, it is essential that pure isolates be obtained. Several workers have communicated specific procedures for isolation o f specified types o f organisms (Salle, 1967; Pelczar and Chan, 1977; Joklik et al., 1980; Cowan, 1985; Collin and Lyne, 1989). The most important factors to consider in an isolation protocol include source o f sample, energy source, optimum temperature, pH and the suitability o f liquid or solid media. (i) Important Factors Considered in Isolating Biooxidizing Bacteria (a) Sample source The initial process o f isolation o f a microorganism, must take into consideration the possible habitats o f the organisms o f interest. For biooxidizing bacteria the environment o f interest is normally a leaching environment, a habitat in which oxidizable forms o f metals and sulphur occur. For example, Thiobacilli are widespread in ore deposits, sulphur springs and compost soils (Harrison, 1986; Said and Johnson, 1988; Barrett et al., 1993), while T. ferrooxidans and other acidophiles produce large quantities o f sulphuric acid in leaching environments which makes their ecological niche inhospitable to most organisms. The local temperature and pH o f the micro-environment also influence the character as well as composition o f bacterial species in any habitat. 17 University of Ghana http://ugspace.ug.edu.gh Most strains o f T. ferrooxidans have been isolated from acid mine drainage, coal spoils, copper deposits, mine effluents, uranium mines and gold mining sites (Livesey-Goldblatt et al., 1983; Murayama et al., 1985; Harrison, 1986; Lane et al., 1992; Rawlings and Woods, 1995). T. thiooxidans strains have been isolated from compost o f soil (Waksman and Joffe, 1922), acidic sulphate soil (Lane et al., 1992), sulphur springs, acid mine waters, corroding steel or concrete and most uranium, copper and gold mining sites (Vishnac, 1975; Harrison 1982; Harrison, 1986; Goebel and Stackebrandt, 1994). Similarly, L. ferrooxidans have been isolated in copper deposits, coal dumps, uranium mines and generally mine ore samples but rarely mine waters (Sand et al., 1992; Barrett et al., 1993; Goebel and Stackebrandt, 1994). (b) Energy substrate, pH and temperature The type o f energy substrate serves as one o f the most effective selective factors in isolating biooxidizing bacteria. Biooxidizing bacteria have a wide range o f specific energy requirements for growth and cellular metabolic processes. For example, the acidophiles oxidize inorganic materials to obtain energy, while the autotrophic bacteria use the energy generated out o f the oxidation process to fix carbon from atmospheric C 02 in their cellular matter. At the species level important biooxidizing bacterium like T. thiooxidans oxidizes elemental sulphur and reduced sulphur compounds such as sodium thiosulphate to produce sulphuric acid. Other nutrients needed for growth are obtained from inorganic salts (Unz and Lundgren, 1961; Otero et al., 1995). On the other hand, T. ferrooxidans uses ferrous iron compounds either in aqueous form or the iron (II) content o f pyrite ore as well as the oxidation o f sulphur even though ferrous iron utilisation is reported to be preferred to sulphur when both substrates are present (Beck, 1960; Harrison, 1984). All strains o f the other ferrous iron oxidizer, L ferrooxidans, described up to date use only iron (II) in aqueous solution or the ferrous iron content o f mineral ore as their energy substrate (Sand et al., 1992; Barrett et al., 1993; Rawlings 18 University of Ghana http://ugspace.ug.edu.gh and Woods, 1995). Since the energy sources used by these microbes are vital to their survival, their inclusion in liquid or solid media enable the cultivation and isolation o f biooxidizing bacteria. Biooxidizing bacteria are known to grow within defined temperature ranges with an optimum between 25 to 35 °C (Vishnac, 1975: Barrett et al., 1993). It is therefore convenient to isolate these bacteria at ambient temperatures (Ahonen and Tuovinen, 1991; Rawlings and Silver, 1995). Most biooxidizing organisms are extreme acidophiles growing best between pH 1 to 3.5 (Vishnac, 1975; Barrett et al., 1993; Battaglia et al., 1994; Rawlings and Silver, 1995). This pH range is highly selective because most micro-organisms are known to grow around pH 7 (Vishnac, 1975; Joklik et al., 1980). The required acidic condition therefore greatly reduces or in some cases eliminates the need for special sterilization methods, thus making biomining economical. (c) Liquid medium Whilst all biooxidizing bacteria grow in liquid media some are unable to grow on solid media (Harrison, 1984). Most isolates o f T. thiooxidans have been obtained in liquid medium with elemental sulphur or sodium thiosulphate as the energy source. However, high concentrations o f sodium thiosulphate is reported to inhibit the growth o f the organism in liquid medium (Starkey, 1925; Unz and Lundgren, 1961), however, while some strains o f T. thiooxidans do not utilize sodium thiosulphate at all (Waksman and Joffe, 1922; Adair, 1966; Harrison, 1982; Harrison, 1984). The pH o f the medium is initially lowered (pH <1) to eliminate contaminating micro-organisms (Harrison, 1982; Harrison, 1984; Sand et al., 1992) even though it may result in a decrease in the rate o f oxidation (Starkey, 1925). The culture medium used in growing T. thiooxidans is reported to develop a characteristic greyish colour which intensifies as growth proceeds (Starkey, 1925; Konisihi eta l., 1995). 19 University of Ghana http://ugspace.ug.edu.gh Similar to T. thiooxidans, most isolates o f T. ferrooxidans and L. ferrooxidans have been obtained using liquid medium but with ferrous iron as the energy source although elemental sulphur and sodium thiosulphate have also been used for T. ferrooxidans (Colmer et al., 1950; Temple and Colmer, 1951; Kinsel, 1960; Colmer, 1962; Harrison, 1984; Schrader and Holmes, 1988; Sand et al., 1992; Barrett et al., 1993). However, the propagation o f L. ferrooxidans in liquid medium requires the addition o f cysteine to enhance their growth (Harrison, 1984). In growing T. ferrooxidans the conversion o f Fe2+ to Fe3+ changes the initial colour o f the medium from colourless or pale blue, depending on pH, through amber to reddish brown. This colour change and the depletion o f iron (II) may be used as growth indicators. Other methods used include: (1) polarographic assay, (2) chemostat reactor measurements, (3) determination o f bacterial nitrogen, (4) rate o f carbon dioxide fixation, (5) manometric measurements involving the measurement o f small amounts o f oxygen uptake using a respirometer or an oxygen electrode and (6) most probable number (MPN) o f bacterial cells. The oxygen uptake has been reported to correlate directly with the oxidation o f Fe2+ to Fe3+ and is generally applicable, but the MPN is not suitable in situations where the bacteria are attached to solid substrates or entrapped in ferric iron precipitates (Silverman and Lundgren, 1959; Beck, 1960; Dugan and Lundgren, 1965; Smith et al., 1972; Brierley, 1978; Harrison, 1982; Kulpa et al, 1985; Barron and Leuking, 1990; Skoog et al, 1992; Mustin et al., 1992; Barrett et al, 1993; Hallberg et al., 1996). General microbiological methods such as the determination o f the turbidity o f cultures and direct counting o f the bacteria are not used for determining the growth o f biooxidizing bacteria because their energy substrates are inorganic salts that result in the formation o f inorganic precipitates (Brierley, 1978). (d) Solid medium The common method used for obtaining a pure isolate o f an organism from culture is to get it to grow as a colony in or on solid medium (Veldkamp, 1970). Solid media prepared using the same 20 University of Ghana http://ugspace.ug.edu.gh constituents as for liquid media and solidified with gelling agents such as purified agar, silica, silicic acid, agarose or polyacrylamide (Harrison, 1984; Barrett et al., 1993) have been used to isolate biooxidizing bacteria (Harrison, 1982; Barrett et al., 1993; Peng et al., 1994). Some types o f conventional agar are however, known to be inhibitory to some strains o f T. thiooxidans and T. ferrooxidans (Harrison, 1984). Nevertheless, Vishnac (1975), Harrison (1982, 1986) reported the isolation o f T. thiooxidans on sodium thiosulphate agar. Agarose has been reported to be a good gelling agent for isolating single colonies o f T. ferrooxidans (Vishnac, 1975; Harrison, 1984; Barron and Leuking, 1990). Yeast extract and tryptone soya are added to solid media where the aim is to isolate heterotrophic organisms (Harrison, 1984). L. ferrooxidans has also been isolated using this particular type o f medium (Johnson, 1995). (ii) Purification o f Isolates Pure cultures are needed for identification and characterization o f bacterial isolates. Methods used to obtain pure cultures include plating, selection by acid and use o f statistical dilution, single cell isolation using a micro-manipulator and selection with heavy metals. Plating is normally used for strains that produce colonies in or on solid medium. A single colony is usually selected and cultivated in liquid medium. The plating procedure is then repeated and a single colony is eventually selected for cultivation as a pure culture. This procedure is, however, not applicable to some strains o f biooxidizing bacteria that do not form well defined colonies (Harrison, 1984). Such bacteria may be purified by the acid selection method. This technique is based on the observation that biooxidizing bacteria (autotrophs) o f interest have the ability to grow at pH below 2 whilst many heterotrophs which are likely contaminants in the isolation procedure are unlikely to grow at such low pH. Hence rapid serial cultivation o f the bacteria in 9K liquid medium (pH below 2) enriches the isolation o f iron and sulpho-oxidizers o f interest (Silverman and Lundgren, 1959; Harrison, 1986; Sand etal., 1992). 21 University of Ghana http://ugspace.ug.edu.gh In the statistical dilution method the cell density o f a culture is determined using microscopic assay and serial dilutions made using sterile 9K liquid medium to obtain ^ 1 cell per specified volume. After a period o f growth the culture is assayed as before using microscopy and the procedure repeated several times. This method makes it possible to even separate two strains o f the same species (Harrison, 1984). Single cell isolation has also been achieved using a micro-manipulator, even though the method is subject to some form o f contamination (Unz and Lundgren, 1961; Harrison, 1984). The method o f selection with heavy metals is designed to exploit the ability o f autotrophic biooxidizers to readily adapt to high concentrations o f heavy metals like copper, arsenic and uranium (Brierley, 1978; Harrison, 1984; Barrett et al., 1993). The presence o f such heavy metals therefore eliminates a broad range o f contaminating organisms. A combination o f the methods described above are however normally used in the purification o f biooxidizing bacteria. (iii) Maintenance and Preservation o f Bacterial Cultures Biooxidizing bacterial isolates have been maintained in the laboratory using the procedure o f subculturing in liquid medium (Harrison, 1984). A working stock o f these organisms may also be maintained at 4°C on agarose slant under mineral oil (Barron and Leuking, 1990; Vandepitte et al., 1991). Long term storage methods employed include mixing the organisms with sterile ore (Larpage et al., 1970; Gupta and Agate, 1986) and freezing aliquots o f the bacteria at 4°C, -20°C or -70°C (Norris and Ribbons, 1970; Barron and Leuking, 1990), or freeze-drying the organisms ( Norris and Ribbons, 1970; Murakami et al., 1986; Wakao et al., 1990). Freeze-drying have been a preferred method for long term storage o f biooxidizing bacteria (Wakao et al, 1990). The method maintains large numbers o f viable organisms over long periods o f time and allows for easy recovery. For instance, T. ferrooxidans has been stored in the freeze-dried state for periods up to 2 years and T. thiooxidans up to 6 years (Norris and Ribbon, 1970; Wakao et al, 1990). The method o f choice for 22 University of Ghana http://ugspace.ug.edu.gh long term preservation, however, depends on the nature o f the organism being preserved and the type o f storage medium used. 1.3.8 Identification o f Biooxidizing Bacteria To identify a micro-organism, it is important to determine which characteristics have the greatest differentiating capacity. Morphological, cultural, physiological, ecological features and biochemical utilization o f organic or inorganic substances have all been used in the identification o f biooxidizing bacteria (Goodfellow and Board, 1980 ; Stainer et al., 1992). Tang et al. (1997) in a review described such biological characteristics or profiles as biograms, and the process o f determination o f the relatedness o f different organisms on the basis o f their biological profiles as biotyping. (i) Morphological Characteristics Chemolithotrophic and acidophilic bacteria o f the genus Thiobacillus are all straight rods or ovoid in shape (Harrison, 1982; Harrison, 1984). Rod cells o f T. ferrooxidans have rounded ends and measure 0.3 to 0.5 |im X 1 to 1.7 (am in size (Barrett et al., 1993). They are non-sporing Gram negative organisms and their cell sizes depend on the type o f liquid medium in which they are cultured. Colmer et al. (1950) reported that in liquid medium where sodium thiosulphate was the energy source, T. ferrooxidans cells were larger compared to when ferrous iron sulphate was used. Some strains o f T. ferrooxidans move by means o f flagella (Barrett et al., 1993) and the cells occur mostly in singles and pairs, but rarely in short chains (Beck, 1960; Vishnac ,1975; Harrison, 1982; Barrett et al., 1993). On the other hand, T. thiooxidans cells are short rods measuring 0.5|am X 1 to 2(im in size and they occur in singles, pairs or short chains. In young cultures some strains o f T. thiooxidans are motile whilst others are not (Waksman and Joffe, 1922; Unz and Lundgren, 1961; Harrison, 1984). 23 University of Ghana http://ugspace.ug.edu.gh L. ferrooxidans cells are vibrio or comma shaped when young but as they grow older they join together becoming spiral like in appearance (Harrison, 1986; Sand et al., 1992) and by accumulating iron precipitates, the cells may assume coccoid-like appearance (Hamson, 1982; Hamson, 1986; Sand et al., 1992; Barrett et al., 1993). They form aggregates or clusters in medium made slimy by capsular excretes from their outer cell envelopes (Harrison and Norris, 1985; Hamson, 1986; Barrett et al., 1993) and they are highly motile organisms. L. ferrooxidans cells measure 0.3 to 0.6 jam in diameter and 1 to 3.5|am in length. Generally, each bacterium usually exhibits a characteristic morphology in young cultures and in media where conditions are favourable for growth (Salle, 1967). Consequently, variation o f their cell size and overall morphological characteristics depend to a large extent on parameters such as, temperature o f incubation, age o f culture, concentration o f substrate, composition o f medium and the presence o f waste products. According to Salle (1967), changes in these parameters lead to corresponding decrease in bacterial cell size. («) Cultural Characteristics The distinctive characteristics o f biooxidizing bacteria on or in solid medium depend on constituents such as the type o f gelling agent used in solid medium (Beck, 1960; Harrison, 1984). The important characteristics on solid media are the form, color, margin, surface texture and elevation o f colonies. Various colony forms o f biooxidizing bacteria including punctiform, circular, filamentous, irregular, rhizoid or spindle-like shapes have been observed on solid media prepared with different gelling agents and energy substrates (Harrison, 1984). On thiosulphate agar T. thiooxidans colonies are punctiform whilst T. ferrooxidans colonies appear as punctiform or filamentous (frosty) (Colmer et al., 1950; Colmer, 1962; Vishnac, 1975). However, on ferrous agarose, T. ferrooxidans colonies are irregular in form but filamentous on ferrous agar. The colony colour intensity has also been reported to vary with age and is influenced by the type and concentration o f energy substrate in solid media (Kinsel, 1960; Vishnac, 1975). For example, young colonies o f T. ferrooxidans on ferrous agar are 24 University of Ghana http://ugspace.ug.edu.gh creamish or whitish with a brown pigment in the center, but become reddish brown with age. However, on ferrous agarose T. ferrooxidans colonies are orange or yellow, while on ferrous silica the young colonies are brown and age to become reddish (Vishnac, 1975; Harrison, 1984; Kawarazaki et al., 1986). On sodium thiosulphate agar T. thiooxidans colonies are initially pale yellow but become transparent as the colonies age. Colony elevation o f bacteria may be flat, raised, convex, pulvinate or umbonate. Most colonies o f T. ferrooxidans are raised whilst those o f T. thiooxidans are flat. Margins o f bacteria colonies can be entire, undulate, lobate, erose, filamentous or curled. T. thiooxidans colonies have entire margins whilst, T. ferrooxidans colonies are either filamentous, entire or undulate (Vishnac, 1975; Harrison, 1984). In liquid media, biooxidizing organisms form pellicles, flocculent or flaky sediments with their substrates and waste products (Vishnac, 1975). T. ferrooxidans and L. ferrooxidans cells form pellicles with ferric compounds such as ferric hydroxysulphate when the pH o f the culture medium is > 1.9 (Harrison, 1984). (iii) Physiological Characteristics Bacterial physiology essentially constitutes bacterial cell functions that enable the interrelationship between the organisms and the environment through utilization o f various substances, such as, carbon, nitrogen, oxygen and energy used for metabolic processes. Other important bacterial physiological properties are determined by the influence o f different organic compounds and stimulants on bacterial growth, the ability to grow at certain temperatures and production o f certain metabolites (Waksman and Joffe, 1922; Harrison, 1984; Harrison and Norris, 1985; Barrett et al., 1993; Goebel and Stackebrandt, 1994; Rawlings and Kusano, 1994). In general, differences in bacterial physiological properties provide a basis for characterization, differentiation and identification o f bacterial genera and to some extent their species. 25 University of Ghana http://ugspace.ug.edu.gh It is the unique physiological characteristics o f biooxidizing bacteria that enable them to grow in mining environments. Pronk et al. (1991b), reported that, even though most strains o f T. ferrooxidans use atmospheric C 02 as their carbon source, some strains can utilize formic acid. T. ferrooxidans is also reported to diazotropic; possessing genes for nitrogen fixation (Pretorius et al., 1986) thus, it can fix atmospheric nitrogen (Mackintosh, 1978; Pretorius et al., 1986). The organism is also capable o f utilizing nitrates as a source o f nitrogen (Vishnac, 1975). T. ferrooxidans normally grows best in an aerobic environment where it uses oxygen as a terminal acceptor in metabolic oxidation. However, i f oxygen is lacking, reduced sulphur compounds or formate act as the electron donor and ferric iron becomes the terminal electron acceptor. It is this property that enables some strains o f T. ferrooxidans to live under anaerobic conditions (Pronk et al., 1991a; Pronk et al., 1992; Sugio et al., 1992; Das and Mishra , 1996). Drobner et al. (1990) and Fischer et al. (1996) have also reported that other substrates such as hydrogen can be used as energy source by some strains o f T. ferrooxidans although, their ability to do this is limited by the presence o f ferrous iron or sulphur under aerobic conditions. Despite the wide range o f energy sources, most strains o f T. ferrooxidans cannot utilize organic compounds for energy (Harrison, 1984; Rawlings and Kusano, 1994). Trace elements used by biooxidizing bacteria for metabolic purposes are usually present as impurities in water or in the ore. Biooxidizing bacteria, therefore, have modest nutritional requirements that can be supplied by water, air and oxidizable iron or sulphur. For this reason, the organism s can adapt to severe adverse conditions in their environment (Rawlings and Kusano, 1994; Morin, 1995). (iv) Biochem ical Characteristics Several biochemical characteristics o f biooxidizing bacteria have been used in their identification. These distinguishing characteristics include: formation o f metabolites, DNA base composition differences, gene sequencing, ribosomal RNA (rRNA) sequencing, susceptibility to antibiotics 26 University of Ghana http://ugspace.ug.edu.gh (chemotaxonomy) and presence o f certain types o f plasmids (Harrison, 1982; Hamson, 1986; Visca et al., 1988; Lane et al., 1992; Stoner et al., 1996, Pizarro et al., 1996). (a) Formation o f Metabolites During the oxidation o f sulphur and metal sulphides biooxidizing bacteria release organic substances into their culture medium. These are exometabolites and are high molecular mass substances such as lipids and phospholipids and low molecular mass substances (LMM) like acids o f the tricarboxylic acid cycle, amino acids and ethanolamine. Their secretion is reported to correlate with bacterial growth and reaches a maximum in the exponential phase o f their growth. The function o f these exometabolites have not yet been extensively studied (Adair, 1966; Harrison, 1984; Barrett et al., 1993). However, Schaeffer et al. (1963) and Harrison (1984) suggested that the lipids and phospholipids act as moisturizing agents and contribute to the oxidation o f sulphur by converting it to colloidal states that can easily be metabolized by the bacteria. LMM substances also participate as complexing agents for ionic species such as aqueous iron (III) and arsenic (III) (Harrison, 1984). The exometabolites contribute significantly to the adhesion o f the bacteria to the surface o f mineral ore (Barrett et al., 1993). Also, exometabolites are beneficial to the organisms as they form complexes with toxic metal ions, thereby reducing the bioavailability o f these metal ions (Barrett et al., 1993). Nevertheless, a build-up o f the metabolites is toxic and affects the growth o f the bacteria. (b) Genetic Relatedness o f Biooxidizing Bacteria The overall DNA base composition o f an organism has been used as a valuable preliminary tool for assessing the relatedness o f one strain o f a microbe to another. It is also used in assigning newly isolated species to their specific genera (Lane et al., 1992). Organisms that are closely related at the species level are reported to have similar or nearly similar DNA base composition (Harrison, 1982; 27 University of Ghana http://ugspace.ug.edu.gh Harrison, 1986). Harrison (1986) and Lane et al. (1992) demonstrated genomic diversity amongst micro-organisms using their DNA base composition. Other methods that have been used to study genetic relatedness o f biooxidizing bacteria include (1) DNA hybridization analysis (homology), (2) 5S rRNA assay and (3) determination o f mole % o f guanine and cytosine (G+C) (Marmur et al., 1963; Harrison, 1982; Harrison, 1986; Lane et al., 1992; Rawlings and Kusano, 1994). On the basis o f inter-strain DNA hybridization analysis T. ferrooxidans strains were divided into seven homologous groups (Harrison, 1982), and Stoner et al. (1996) used 5S rRNA assay to differentiate between strains o f T. ferrooxidans, T. thiooxidans, L. ferrooxidans and other acidophiles. Determination o f (G+C) in mole % has been used in the characterization o f different strains o f biooxidizing bacteria (Vishnac, 1975). Barrett et al. (1993) reported that the (G+C) in mole % varied depending on the bacterial strain. For example, the mole % (G+C) for T. ferrooxidans strains ranged from 55 to 65 %, whilst that for T. thiooxidans strains was 52 to 65 %, and 50 to 56 % for L. ferrooxidans. However, Brierley (1978) and Rawlings et al. (1991) reported that variations existed in DNA base composition o f T. ferrooxidans strains and other acidophiles grown on different energy substrates. Nevertheless, Lane et al. (1992) showed that there is no quantitative relationship between mole % (G+C) and genotype o f organisms. (c) Chemotaxonomy The differentiation o f bacterial strains using their susceptibility to various types o f antibiotics is termed chemotaxonomy. The identification profiles obtained by the method are called antibiograms (Tang et al., 1997). Interestingly, all mesophilic isolates o f biooxidizing bacteria described up to date areGram negatives and sensitive to antibiotics such as ampicillin, vancomycin and kanamycin (Huber et al., 1985; Barrett et al., 1993). 28 University of Ghana http://ugspace.ug.edu.gh 1.3.9 Comparative Characterization o f Biooxidizing Bacteria Morphological, cultural, physiological and biochemical characteristics (biograms) o f micro­ organisms are used normally in their identification. However, Tang et al. (1997) in a review reported that in some cases these characteristics are unstable and therefore not reliable for identification o f bacterial strains. This is mainly because the characteristics may be influenced by genetic regulation, technical manipulation and gain or loss o f plasmids by the microbes. Other properties are therefore usually used in conjunction with these biograms to accurately identify biooxidizing bacteria strains. These include their antigenic properties, mineral ore oxidation rates, the optimum pH, temperature and substrate concentration, and protein/ DNA profiles. (i) Use o f Antigenic Properties In vitro reactions between antigens and their homologous antibodies have been widely used in the identification and characterization o f micro-organisms (Harlow and Lane, 1988; Tang et al., 1997). Serological reactions are reported to reveal marked differences among bacterial cultures that appear to be similar on the basis o f morphology and physiology (Weir, 1986). This type o f analysis distinguishes bacterial species on the basis o f their antigenicity. For example, Apel et al. (1976) adapted the fluorescent antibody staining technique to identify T. ferrooxidans isolates using rabbit anti-7! ferrooxidans immunoglobulin G (IgG) against 23 bacterial isolates. They observed fluorescence with T. ferrooxidans isolates grown with both iron and sulphur as energy substrates but noted cross-reactivity for two other unrelated bacterial species. Their antibodies, however, failed to react with known T. thiooxidans isolates. Nevertheless, Brierley (1978) reported in a review that the application o f the fluorescent antibody staining technique in the field using mineral ore samples was not very successful because o f low populations o f biooxidizing bacteria in the ore. Muyer et al. (1987) also demonstrated that antiserum raised against whole cells o f T. ferrooxidans reacted with a variety o f acidophiles and non-acidophiles in an enzyme-linked immunoabsorbent assay. They also 29 University of Ghana http://ugspace.ug.edu.gh observed reactivity o f their antiserum with biooxidizing bacteria in another experiment where an indirect immunofluorescence assay was combined with a DNA-fluorescence staining technique (Muyer et al., 1987). The above experiments thus demonstrated that polyclonal antisera raised against biooxidizing bacteria were not specific at the species level (Muyzer et al., 1987; Arrendondo and Jerez, 1989). The success so far achieved with polyclonal antibodies in identification o f some biooxidizing bacteria may however suggest that the use o f specific monoclonal antibodies would lead to more accurate characterization. («) Use o f Mineral Ore Oxidation Rate, pH, Temperature and Substrate Concentration Profiles The rate o f mineral ore solubilization by biooxidizing bacteria had been used in their characterization because o f its economic importance in commercial mining. The ability o f biooxidizing bacteria to leach different types o f mineral ores efficiently varies because o f environmental conditions such as pH, temperature and the concentrations o f various substrates (Ahonen and Tuovinen, 1991; Barrett et al., 1993). Hence, a mixed culture o f T. ferrooxidans, T. thiooxidans and L. ferrooxidans is normally used in bioleaching o f mineral ore since these bacteria have a sort o f synergistic relationship which enhances the biooxidation process rather than pure individual cultures o f the organisms (Barrett et al., 1993; Rawlings and Silver, 1995; Morin, 1995). Even with the same species o f biooxidizing bacteria it has been observed that organisms isolated from a particular mineral ore oxidize that ore better than isolates from a different ore (Rawlings and Woods, 1995). According to Lundgren and Silver (1980), bioleaching o f mineral ore is influenced by the chemical nature o f both the aqueous and solid crystal phases o f the ore. Morin (1995) explained that this was so, since mineral ore deposits from different geographical sources were unique with respect to their mineralogy and chemical composition which in turn affects the morphology and surface features o f the ore (Agate and Khinvasara, 1985; Baldi et al., 1992; Barrett et al., 1993; Rawlings and Woods, 1995; Morin, 1995). In general, however, the characterization o f biooxidizing bacteria using the mineral ore 30 University of Ghana http://ugspace.ug.edu.gh oxidation rates is enhanced by comparative measurement o f pH, temperature and substrate concentration profiles. (iii) Use o f Protein Profiles in Characterization Shapes and functions o f cells are determined by their proteins (Wilson and Walker, 1995). Some o f the protein molecules (enzymes) catalyze reactions which synthesize cell membranes, pigments and drive the mechanisms o f energy production from substrates. Indeed, it is the differences in the structure and composition o f these proteins that provide the basis o f species and strain designation o f microorganisms. For this reason, several electrophoretic methods are available for analysis o f proteins made by micro-organisms. In this respect, zone electrophoresis o f a mixture o f bacterial cell proteins in well defined standardized conditions produce protein profiles which are considered as fingerprints o f the different strains under investigation (Kersters and De Ley, 1980; Copeland, 1994). On the other hand, similar strains o f bacteria grown under standardized culture conditions have the same set o f proteins (Kersters and De Ley, 1980). Electrophoresis o f total soluble or outer membrane proteins o f bacterial strains therefore yield complex polypeptide protein bands that are similar for the same species or strains o f organisms. The protein bands usually consist o f a number o f structurally different molecules with identical electrophoretic mobilities (Copeland, 1994; Wilson and Walker, 1995; Tang et al., 1997). Several workers have as a result used protein band analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to identify and differentiate between bacterial isolates (Swings et al., 1976; Huber et al., 1985; Chamorro et al., 1988; Arrendondo and Jerez, 1989; Deutscher, 1990; Hames and Rickwood, 1994; Teixaeira et al., 1995). The accuracy o f the method was shown by Rawlings et al. (1991) who differentiated between T. ferrooxidans grown with ferrous iron as energy substrate and the same organism grown with elemental sulphur. 31 University of Ghana http://ugspace.ug.edu.gh (iv) Use o f DNA Profiles in Characterization Rapid progress in the molecular genetics o f procaryotes has made it possible to differentiate between bacteria up to the strain level using polymorphism in their nucleic acids. The genome o f most Gram negative bacteria consists o f a single circular molecule o f DNA with size ranging from 6.0 X 105 to 7.8 X107 base-pairs (Cavalier-Smith, 1985; Li and Graur, 1991). Some bacteria also contain additional small circular DNA molecules called plasmids that encode for a variety o f cellular functions (Hames and Higgins, 1991; Watson et al., 1992). Differences in DNA o f organisms may however arise from point mutations (insertions and deletions) which alter the sequence o f nucleotides or genes comprising genomic DNA. These mutations produce variations in the number o f tandemly repeated DNA sequences and alters the length o f DNA fragment between two recognition sites for restriction endonucleases (Watson et al., 1992; Pingoud et al., 1993), thereby providing the basis for differentiation between strains o f bacteria by analysis o f the electrophoretic mobility o f nucleic acids (Patterson and Hyypia, 1985). Variations detected this way serve as extremely valuable genetic markers (Rothwell, 1988) and is termed Restriction Fragment Length Polymorphism (RFLP). The digestion o f genomic DNA with type II restriction enzymes have been used in the characterization o f biooxidizing bacteria (Southern, 1979; Yates et al., 1986; Tang et al., 1997). According to Tang et al. (1997) the advantage o f restriction endonuclease analysis is that it is highly reproducible and very accurate in determining the relatedness o f microbial strains. 32 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 2.0 MATERIALS AND METHODS 2.1 Samples The samples used in this study were collected from the mining site o f the Ashanti Goldfields Company Limited, Obuasi. These were; (1) slurry from the biooxidation reactor tanks (BT) (see Figure 1 A, IB), (2) gold arsenopyrite ore (S2) from a surface mining site (see Figure 2), (3) gold sulphide ore (S3) from an underground mining site (see Figure 3) and (4) underground mine water (UW). Gold ore samples (mainly crushed ore) and 50ml o f underground mine water or slurry were collected into polythene bags or sterile 50ml centrifuge tubes, respectively and transported to the laboratory within 24 hours. The samples were subsequently stored at 4°C until use. 2.2 Isolation, Purification and Maintenance of Ferrous Iron Oxidizing Bacteria (i) Isolation Five millilitres o f slurry from the biooxidation tank were pipetted into 100ml o f 9K enrichment medium (Appendix A) supplemented with ferrous iron sulphate (9K-Fe2+), pH <1.8 in a 500ml Erlenmeyer flask. The culture was then incubated at room temperature (25 to 30 °C) for 8 to 10 days with continuous agitation at a speed o f 100 rpm on a rotary shaker (Vishnac, 1975; Harrison, 1984; Goebel and Stackebrant, 1994). 33 University of Ghana http://ugspace.ug.edu.gh AFigure 1 Photograph showing the sulphide treatment plant (STP) at Obuasi. A - reactor tank, B= settling tank. Row diagram showing system of operation of S IP at Obuasi. B arsenopyrtte concentrate H2 O I Inorganic nutrients Cyanidation of bEotddized ore and gold recovery 2nd aerafnn f tank / (s tage two agitator! A V «B Settling tank f at anraiicfi Leach liquor's pH is ad­ just' £ fh " Schematic diagram o f the STP at Obuasi. Sample BT was collected from reactor tank ‘A ’. 34 University of Ghana http://ugspace.ug.edu.gh Figure 2 Photograph showing surface mining site at Obuasi where the surface gold ore sample (SO) was collected. 35 University of Ghana http://ugspace.ug.edu.gh Figure 3 Photographs showing underground mining sites where the underground gold ore sample (UO) was collected. 36 University of Ghana http://ugspace.ug.edu.gh Similarly, 10ml o f underground mine water were added to 30ml o f enrichment medium in a 250ml conical flask or 3 grams o f surface and underground gold ore were added separately to 100ml o f the medium in a 500ml Erlenmeyer flask and cultured as described above (Barrett et al., 1993). The underground mine water was cultured for 2 to 3 weeks instead o f 10 days. (ii) Purification Purification o f ferrous iron oxidizing bacterial isolates were done using the methods o f enrichment dilution and colony isolation (Unz and Lundgren, 1961; Beck, 1960; Barron and Leuking, 1990) in either solid or liquid media. (a) T. ferrooxidans-like Bacteria Primary isolation o f T. ferrooxidans-like bacteria was done by spreading 100fj.l o f bacteria suspension at the logarithmic phase o f growth as indicated by medium colour (deep amber) and pH o f the medium (pH >1.8) on ferrous agar (FA) or on ferrous agarose (FAR) in petri plates (Appendix B). The plates were incubated at 37°C for 4 to 6 days during which bacterial cell colonies appeared on the solid medium. Single colonies were then transferred with a sterile Pasteur pipette back into 2ml o f 9k-Fe2+ liquid medium in a 24 well tissue culture plate (Becton Dickinson and Company, USA). The culture plate was incubated at room temperature (25 to 30 °C) with continuous agitation on a rotary shaker for 1 to 3 weeks during which growth o f bacteria was observed. The above procedure was repeated 3 times to ensure the purity o f the isolates. Light microscopy was used (mag. X 1000, objective X I00) to ascertain purity o f the isolates by their morphological characteristics (Sand etal., 1992; Goebel and Stackebrant, 1994). 37 University of Ghana http://ugspace.ug.edu.gh (b) L. ferrooxidans-like Bacteria Ten millilitres o f inoculum from the stationary phase o f enrichment cultures prepared with samples (Section 2.2.1) were pipetted into 100ml o f 9k-Fe2+ liquid medium added with L-cysteine (0.01%) which selects for L. ferrooxidans in 500ml Erlenmeyer flasks separately (Harrison, 1984). The individual cultures were incubated at room temperature (25 to 30 °C) with continuous agitation on a rotary shaker at a speed o f 100 rpm. An aliquot (10ml) o f each culture was then pipetted into fresh liquid medium in a 500ml Erlenmeyer flask under aseptic conditions in a laminar flow cabinet at 2 weeks intervals. Sub-culturing was repeated at least 10 times. Light microscopy was used to ascertain purity o f the isolates by their morphological characteristics (Sand et al., 1992; Goebel and Stackebrandt, 1994). Further purification was achieved using the method o f statistical dilution (Harrison, 1984). Briefly, a suspension o f L. ferrooxidans in culture at the logarithmic phase o f growth was titrated using 9K- Fe2+ medium with L-cysteine from 2 X 10"2 to 2 X 10'20 at 50 fold dilution. One hundred microlitres volumes o f each dilution was then pipetted into the wells o f a sterile 96 well tissue culture plate. The plates were then incubated at room temperature (25 to 30 °C) under continous agitation at a speed o f 30 rpm on a rotary shaker (Thomas Kagaku, Co. Ltd, Japan). The micro-plates were inspected at weekly intervals for signs o f growth as indicated by medium colour change. After every 30 days 100(j.l o f fresh sterile 9k-Fe2+/L-cysteine medium were added under aseptic conditions to make up for evaporative losses. Light microscopy was used to ascertain purity o f the isolates by their morphological characteristics (Sand et al., 1992; Goebel and Stackebrandt, 1994). 38 University of Ghana http://ugspace.ug.edu.gh (iii) Maintenance T. ferrooxidans-like bacterial isolates were maintained using 9k-Fe2+ liquid medium. Fresh cultures o f the bacterial isolates were prepared every two weeks by sub-culturing 10ml o f an on going culture into 100ml o f 9k liquid medium in a 500ml Erlenmeyer flask. However, sub-culturing o f purified bacterial isolates from underground mine water were done monthly. The cultures were continuously agitated on a rotary shaker at a speed o f 100 rpm at room temperature (25 to 30 °C) and opened every 5 days to allow fresh supply o f air into the flasks. On the other hand, purified L. ferrooxidans-like bacteria were maintained in 9k liquid medium with 0.01% L-cysteine, by sub-culturing every 2 weeks. 2.3 Isolation, Purification and Maintenance of Sulpho-Oxidizing Bacteria (i) Isolation One hundred millilitres o f 9K medium (pH < 1) supplemented with elemental sulphur (9k-S°) in a 500ml Erlenmeyer flask was inoculated with 5ml o f sample BT or 3 grams o f surface and underground gold ores (Barrett et al., 1993). The resulting enrichment medium was then continuously agitated at 100 rpm on a rotary shaker (Eyela Tokyo Rikikai Co., Ltd., Tokyo, Japan) for 30 days at room temperature (25 to 30 °C). Growth o f the bacteria was monitored by medium colour change which developed from colourless to intense grey (Sand et al., 1992; Goebel and Stackebrandt, 1994). (ii) Purification o f T. thiooxidans-like Bacteria T. thiooxidans-like bacteria were purified by the method o f selection by acid described by Harrison (1984) and by colony isolation. Briefly, the acid selection method utilizes a low pH medium which enabled only T. thiooxidans-Mke bacteria to grow (Torma, 1985). An aliquot (100^1) o f the resulting 39 University of Ghana http://ugspace.ug.edu.gh culture was spread on sodium thiosulphate agar (Appendix B) and incubated for 8 to 10 days at 37°C. During this period bacteria cell colonies appeared on the plates. Single colonies were picked with the tip o f a sterile needle because o f the minute sizes o f the colonies that developed and put back in 9k-S° medium. The transfer o f single bacterial colonies into liquid phase was repeated 2 to 3 times. Light microscopy was used to ascertain purity o f the isolates by their morphological characteristics (Sand et al., 1992; Goebel and Stackebrandt, 1994). (iii) Maintenance Sub-culturing o f sulpho-oxidizing bacterial isolates was done every 30 days by pipetting 10ml o f the on going culture into 100ml o f fresh 9k-S° liquid medium in a 500ml Erlenmeyer flask. The culture flask was opened every 5 days to allow a fresh supply o f air in a sterilized laminar flow cabinet. 2.4 Preservation of Ferrous Iron and Sulpho-oxidizing.Bacterial Isolates Ferrous iron or sulpho-oxidizing bacteria in 100ml cultures at the stationary phase o f growth were harvested by centrifugation at 10,000g for 1 hour. The pellet obtained was re-suspended in 2ml o f 9k liquid medium pH 6.5 (Appendix A). One hundred microlitres (1 OOjj.1) o f re-suspended bacterial cells were then pipetted into 5ml glass vials (Nichiden-Rika-garasu Co. Ltd, Japan) and frozen at - 20°C before freeze-drying using a Yamato freeze drying machine (Yamato Co. Ltd, Japan). Freeze- drying was done at -105°C for one hour. The freeze-dried samples were stored at 4°C until use. The viability o f freeze-dried bacterial cells was tested as follows: immediately after freeze-drying some o f the samples were re-suspended in 1 OOjal o f 9k liquid medium supplemented with ferrous iron or elemental sulphur as energy substrate for ferrous and sulpho-oxidizing bacteria respectively. The entire 100( 1^ bacterial suspensions were then pipetted separately into 2ml o f the respective media and agitated continuously on a rotary shaker at a speed o f 100 rpm in a 24 well tissue culture plate 40 University of Ghana http://ugspace.ug.edu.gh (Becton Dickinson and Company, USA). One hundred microlitres o f bacterial cell suspensions stored at 4°C and -20°C transferred into culture plates similarly were used as positive controls whilst lOOjal o f 9k liquid medium without any bacteria cells served as negative control. Growth o f the prepared cultures were monitored by medium colour change over a period o f 3 months. 2.5 Characterization of Biooxidizing Bacteria 2.5.1 Staining o f Bacterial Isolates (i) Basic Dye and Gram's Staining Basic dye stains were used in staining biooxidizing bacteria (Salle, 1967; Cowan, 1985; Lillie, 1990). Ten microlitres (IOjj.1) o f bacterial culture were pipetted and smeared evenly on a cleaned (grease free) labelled slide. The smear was air dried and then heat fixed by passing the slide through a bunsen flame 5 to 6 times. Prepared slides were flooded separately with any o f the basic dye solutions for 1 to 3 minutes (Appendix C). The slides were then washed with distilled water to remove all excess stain and air dried at room temperature. The stained cells were observed under oil immersion using a light microscope at a magnification o f X 1000 (objective X I00, ocular X I0). Photomicrographs o f stained bacterial cells were taken using an Olympus model phase-contrast light microscope (Olympus Optical Co. Ltd., Tokyo, Japan) with a camera attached. Gram’s staining was performed as described by Collins and Lyne (1989) with modification. Briefly, a suspension o f bacteria (approximately 1.5ml) at logarithmic phase o f growth was pelleted by centrifuging in a microfuge for 1 hour. The supernatant was decanted and the pellet re-suspended in IOOjj.1 o f 9k liquid medium pH 6.5 (Appendix A). The bacterial cells were then fixed onto microscope slides as described above before flooding with crystal violet solution. The slides were washed with excess distilled water and Logol’s iodine applied by dropwise addition for 1 minute (Appendix C). This was followed by washing with acetone until colour ceased to come out o f the 41 University of Ghana http://ugspace.ug.edu.gh preparation. The slides were again washed with distilled water and the smear counter-stained with carbol fuschin for 3min. The excess stain was then washed with distilled and the slide air dried. Microscopic examination was performed as described earlier. 2.5.2 SDS-PAGE Analysis o f Bacterial Cell Lvsates Electrophoresis o f bacterial cell lysates were performed with an ATTO CORPORATION slab gel apparatus (Bunkyo-Ku, Tokyo, Japan) using the SDS-tris-glycine discontinuous buffer system (Laemmli, 1970). All bacterial samples were electrophoresed on 15 - 20% resolution acrylamide gradient gels with a 3% stacking gel. (i) Preparation o f Cnide Bacterial Cell Lysates fo r SDS-PAGE and Electrophoretic run Crude bacterial cell lysates were prepared from the isolates obtained from different samples. Bacterial cells were harvested from a 100ml culture at stationary phase o f growth by centrifugation at 2900 Xg for 60 mins at 4°C using a Hitachi CF7D2 Centrifuge (Hitachi Co., Tokyo, Japan). The cell pellet was re-suspended in 100(il o f sample buffer (62.5mM, Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% 2-Mercaptoethanol) in a 1.5ml Eppendorf tube. Protease inhibitors [2.5(j.l Leupeptin (lOmg/ml), 2.5|il E64 (lOmg/ml), 5ul mixture containing lOOmM PMSF, 20mM TPCK and 5mM TLCK] were then added. The tubes were rapidly frozen in liquid nitrogen and transferred immediately to a water-bath at 37°C to thaw before vortexing briefly for 2min to break up the cells. The freeze-thaw process was repeated 20 times to enhance disruption o f the cells. The prepared crude bacterial cell lysates were boiled for 15min after the addition o f loading buffer (Huber et al., 1985) at 100°C in a water-bath before loading onto acrylamide gels for electrophoresis. Crude bacterial cell lysates were loaded into the wells o f the polymerized stacking gel alongside standard low molecular weight markers (Sigma Co, Ltd, St Louis, MO, USA) prepared as described 42 University of Ghana http://ugspace.ug.edu.gh by the manufacturer. A constant current o f 20mA was supplied by an electrophoresis power supply pack ATTO AE-3121 (ATTO Corporation, Japan) until the tracking dye (bromophenol blue) had travelled to the interface between the stacking and resolution gels. The current was then increased to 30mA to improve the sharpness o f resolution o f the peptide bands in the separating gel. A vertical strip o f the gel containing the standard molecular weight markers and resolved sample proteins was cut using a surgical blade and transferred into a plastic tray containing staining solution [0.5% (w/v) Coomassie blue, 10% (v/v) acetic acid, 50% (v/v) methanol] for 15min with slow shaking on a rotary shaker. The staining solution was discarded and the gel rinsed with first destaining solution [50% (v/v) methanol, 10% (v/v) acetic acid] for lmin. Destaining was continued using a second solution [10% (w/v) methanol, 7% (v/v) acetic acid] until the stained protein bands were visible in the gel. The destained gel was then visualized under an ordinary light illuminator and stored in distilled water to prevent it from drying up. The destained gels were soaked in 25% glycerol for 2-5 min and transferred onto chromatographic filter papers 3mnr thick (Toyo Roshi Kaishi, Ltd, Tokyo, Japan) and overlaid with a cellophane membrane previously softened by immersion in distilled water. The gels were dried in an ATTO gel drying machine (ATTO Corporation, Tokyo, Japan) at 60°C for 14 hours. The molecular weights o f prominent protein bands observed in the resolved bacterial proteins were determined using a relative mobilty curve (Appendix H). 2.5.3 Preparation and Restriction Endonuclease Cleavage of Biooxidizing Bacterial Genomic DNA (i) Preparation o f Genomic DNA e protocol described by Sambrook et al. (1989) and Flook et al. (1992) for extraction o f genomic DNA, was adapted with slight modifications. Bacterial cells (100ml cultures) were harvested at the 43 University of Ghana http://ugspace.ug.edu.gh stationary phase o f growth by centrifuging at 2900g for 1 hour in a centrifuge at 4°C. After centrifugation the cell pellet was rinsed 3 times with 1M Tris-HCl pH 7.6 and re-suspended in 200(j.l o f Blender buffer (0.1M NaCl, 0.2M sucrose, 0.1M Tris-HCl pH 7.5, 0.05M EDTA pH 9.1, 0.5% SDS stored at 4°C) in an Eppendorf tube. The homogenate was incubated at 65°C for 1 hour and spun in a microfuge for lOmins (Kubota 1120 Kubota Corporation, Bunkyo-Ku Tokyo, Japan). Thirty microlitres (30|il) o f pre-chilled 8M potassium acetate were added, mixed well by tapping and left to stand on ice for 45min before centrifuging at 12,000g for lOmin. The supernatant (230|il) was transferred into a fresh Eppendorf tube and 2X volume cold absolute ethanol added. The sample was then kept at -50°C for 30min to precipitate DNA. The DNA was recovered in a pellet following centrifugation at 12,000g for lOmin and air dried. The DNA pellet was then dissolved in 50|^1 o f Tris-EDTA (TE) buffer pH 7.6 with lOmg/ml, RNAse solution (Appendix E) and further purified by phenol chloroform extraction. To the sample was added an equal volume o f phenol-chloroform mixture (1:1 v/v) and the contents mixed till an emulsion formed. The mixture was centrifuged for 5mins in a microfuge to separate the organic and aqueous phases, and the aqueous phase containing the DNA transferred into a fresh tube. This phenol chloroform extraction procedure was repeated two more times. The procedure was again repeated using only chloroform. Purified genomic DNA was then precipitated by the addition o f 1/10 volume o f sodium acetate pH 5.2 and 2 X volume cold absolute ethanol to the aqueous phase and keeping the mixture at -20°C overnight. The DNA was recovered by centrifugation at 12,000 Xg for lOmin. The DNA pellet obtained was washed with 70% ethanol, air dried at room temperature then dissolved in 30|il TE buffer (ImM EDTA, 10 mM Tris-HCl pH, 7.6, defined in appendix E) and stored at -20°C. The purity and yield o f extracted DNA was determined as described by Rodriguez and Tait (1983). DNA samples (10jj. 1 each) were diluted with 1.9ml o f distilled water and the absorbance at 260, 280 44 University of Ghana http://ugspace.ug.edu.gh and 300 nm read using silica cuvettes with distilled water as blank in a double-beam spectrophotometer (Shimadzu UV 190 Double beam, Japan). From the absorbance readings obtained, the quantity and purity o f DNA samples were calculated. The absorbance at 260nm was used to calculate the concentration o f DNA (OD 0.2 = 10(j.g DNA/ml) and the absorbance ratio A 26( / A 280 provided an estimate o f the purity o f DNA (Sambrook et al., 1989). («) Cleavage o f Genomic DNA with Restriction Endonucleases Cleavage o f genomic DNA with restriction endonucleases was done using the method o f Sambrook et al. (1989) and modified following the manufacturer’s recommendations. DNA solutions (10^1 each) were placed in sterile tubes and mixed with 8(il o f sterile distilled water to give 18(xl DNA solution with concentration ranging from 5 to 10 |ig. To these tubes were added 2\i\ o f 10 X restriction digestion buffer (Appendix F) and the contents mixed by tapping gently. The appropriate enzyme (1 (al) (Appendix F) was added directly into the reaction tubes and incubated at 37°C overnight. The tubes were heated at 70°C to stop the enzyme reaction and 4j_il o f 6X gel loading buffer added to each reaction mixture. Digested DNA samples were stored at -20°C until use. Electrophoresis o f digested and undigested DNA was performed as described by Sambrook et al. (1989). Undigested and digested DNA samples with restriction endonuclease mixed with loading buffer (Appendix F) were carefully dispensed using a micropipette into the slots in a 0.8% agarose gel submerged in electrophoresis buffer [IX Tris acetate EDTA (TAE) buffer] (Appendix G). Electrophoresis was performed using a mini-gel system (Biorad Model 200/2.0). The gels which contained 0.5^g/ml ethidium bromide were run for about 1 hour at 80V and photographed using a UV trans-illuminator and a polariod camera fitted with an orange filter o f wavelength 302nm. The size o f genomic DNA and digested DNA fragments were determined using a standard calibration curve (Appendix H). 45 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 3.0 RESULTS 3.1 Bacterial Isolates obtained from the Different Samples 3.1.1 Ferrous Iron Oxidizing Bacterial Isolates Enrichment cultures containing ferrous iron (Fe2+) as energy substrate were used to isolate ferrous iron oxidizing bacteria. All four sample types (slurry from biooxidation tank, surface arsenopyrite ore, underground gold sulphide ore and underground mine water) were found to contain ferrous iron oxidizing bacteria. The growth o f these organisms was monitored by the characteristic change o f medium colour fiom pale green to deep amber or reddish brown within 4 to 10 days o f incubation (Figure 4). Ferrous iron oxidizing bacterial isolates in the slurry, surface arsenopyrite and underground sulphide ores produced more intense medium colour change. Phase-contrast microscopical examination o f the culture revealed a diverse population o f straight rods and curved or spiral shaped cells. 3.1.2 Sulpho-oxidizing Bacterial Isolates Enrichment cultures containing elemental sulphur as energy substrate were used to isolate sulpho- oxidizing bacterial isolates. Sulpho-oxidizing bacterial isolates were obtained from only 3 sample types namely, slurry from biooxidation tank, surface arsenopyrite ore and underground gold sulphide ore. The growth o f the sulpho-oxidizing organisms was indicated by the change o f medium colour from colourless to intense grey after 30 days incubation. Phase-contrast microscopy revealed a population o f straight rods cell. 46 University of Ghana http://ugspace.ug.edu.gh Figure 4 Photograph showing stages o f growth o f the ferrous iron oxidizing bacteria. Colouration o f the medium indicated the growth phase o f the organisms. C: lag phase (plain in colour). D: logarithm phase (amber in colour). E: stationary phase (reddish brown or deep amber in colour). 47 University of Ghana http://ugspace.ug.edu.gh 3.2 Designations of Bacterial Isolates Table 1 contains a list o f selected representative bacterial isolates, their sources and designated abbreviated names. 3.3 Identification of Purified Bacterial Isolates 3.3.1 Ferrous Iron Oxidizing Bacteria Purified isolates o f two main ferrous iron oxidizing bacteria were obtained from all the samples analyzed. One o f the ferrous iron oxidizing bacteria from each o f the sample sources could grow on ferrous agar or agarose whilst the other grew only in liquid medium (Table 2 and Figures 5-10). The bacteria that grew on solid media showed as pinpoint iron encrusted colonies (Figures 5 and 6) or larger spreading colonies with opaque iron encrusted centres and transparent edges (Figures 7 and 9). These bacteria also grew on sodium thiosulphate agar (Figure 8) or ferrous/sodium thiosulphate agar (Figures 9 and 10). Transfer o f single colonies o f the organisms back into fresh liquid medium resulted in the oxidation o f ferrous iron (Fe2+) to ferric iron (Fe3+). These isolates were also able to grow in 9K medium supplemented with elemental sulphur or sodium thiosulphate (Table 2). Microscopical examination o f the cells obtained from single colonies revealed a homogenous culture o f straight rods that appeared in singles, pairs and in clusters (Figures 11, 12 and 13). The isolates were Gram negative. Representative isolates selected from the various sample sources for further characterization were BT1-Fe2+, S01-Fe2+, U01-Fe2+, and UW l-Fe2+. Serial cultivation o f crude ferrous iron oxidizing bacteria in 9K-Fe2+ liquid medium with L-cysteine produced bacteria that did not grow on solid media (Table 2) but oxidized Fe2+ to Fe3+ as indicated by change o f medium colour. Microscopical examination o f cultures revealed a homogenous sample o f curved or comma shaped rod cells and occasional spiral shaped cells (Figure 14). As shown the cells appeared in singles, pairs and in clusters. These cells were also Gram negative (Table 4). 48 University of Ghana http://ugspace.ug.edu.gh Representative isolates selected from the various sample sources for further characterization were BT2-Fe2+, S02-Fe2+, U02-Fe2+ and UW2-Fe2+. 3.3.2 Sulpho-oxidizing Bacteria Purified isolates o f two different sulpho-oxidizing bacteria were obtained from three o f the sample sources namely, slurry, surface arsenopyrite and underground sulphide ore. After growth, it was confirmed that one group o f bacteria were identical to ferrous iron oxidizers (section 3.3.1) so were designated BT1-Fe2+, S01-Fe2+, U01-Fe2+. These isolates grew on sodium thiosulphate agar, as well as ferrous agar or agarose and ferrous/sodium thiosulphate agar (Figures 5, 6, 8 and 9). The other isolates grew only on sodium thiosulphate agar forming minute pale yellow colonies (Figure 15). Single colonies transferred back into fresh 9K-S0 liquid medium grew elemental sulphur as energy substrate. Microscopic examination o f the sulpho-oxidizing bacterial culture revealed a homogenous sample o f straight rods that appeared in singles, pairs and in chains (Figure 16). The organisms were Gram negative (Table 4). 49 University of Ghana http://ugspace.ug.edu.gh Table 1 Selected b iooxid iz ing bacteria l isolates and their designated codes Source o f sample Designated sample Selected biooxidizing bacterial isolates code 1“ 2b 3C Biooxidation reactor tank BT BT1-Fe2+ BT2-Fe2+ BT3-S0 Surface arsenopyrite ore SO S01-Fe2+ S02-Fe2+ S03-S° Underground gold sulphide ore UO U01-Fe2+ U02-Fe2+ U03-S° Underground mine water UW UWl-Fe2+ UW2-Fe2+ * “Rod shaped ferrous iron oxidizers. b Curved shaped ferrous iron oxidizers. cRod shaped sulpho-oxidizing bacteria. * There was no sulpho-oxidizing bacteria in the sample. 50 University of Ghana http://ugspace.ug.edu.gh T a b l e 2 P hy si ol og ic al c ha ra ct er is ti cs of bi oo xi di zi ng ba ct er ia l is ol at es o < £ H cn o — Ul , a c o £ T3 < £ H o C/3 6 co •= a U < o u , 5 5 > < > ti. P - 6 o a 2 _ > s — CO a & o o CO cj ^ t/J ^ _ 7 ° ,-r o O J" u V) . i r PQ + • + + ' + + i + + ' + + + ■ + + • + + i + + + < + • + ■ i + i + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + > + + ' + + • + + ■ + + ' + + ' + + ' + + 00 o o U . C/5 1 ) o U. C/5 — N n H t i m . K K H r - M O O O O O P C P f f l P Q M M M D P D ■ c s ° . 2 £ p *8 2es CD Qh S- Oh too '■5 c o = s E g E E E o o- ■■ r “ W> E »r> ° s 1 • U . 2 v-v o O o PJ w >> • TD *o C cO Of) s j w cd o c3 O E D P u T p -T ? 5O '-Z o > U 5 (D ° 5 ,J= o O = > ' -e _o c o c U O C dj OQ o o e i o £ oT cC u * Ph roo C/3 w •a g p w ■auU5 < m >•' o ' p i P h" c J u P^ o p w o D >> P h P h & ; j§ " 3 -a 1 63 ca n . E .tr t/l II § “ s■a I 3 ca II II h. u . University of Ghana http://ugspace.ug.edu.gh Ta bi c 4 C ul tu ra l, m or ph ol og ic al a nd bi oc hc m ic al c ha ra ct er is ti cs of bi oo xi di zi ng ba ct cr ia l is ol at es in 9k liq ui d m ed ia § . a ' S3 ca 1 ! " Oa P ft, O — o p B = 2 • = o - 5"S uo C r~ 1/1 o r “ n r ; + + + o o 0 SL, fL, ^ 11 NJI Mi A \ yll Ml \ | l a 3c/3 'C +D -OJ i ? 2 9 ‘ * CO O ■gA 3 3^ XOJ ^ 'n OJU. ,& a U- 3 CO cj 6n 'Ju- Uh — OJ GJ 00Uh UU Uh Uh + u + rl o — Uh 0 0 Uh U h CO JL Ol m _ « r-1 — H H o o O 03 P3 03 CO CO CO + ~ fl*c5 aM yB e* Ewu Oic - aos N T 3 2 D B 3 >< O C/3 o C C/5 s 3 00 a> o 5 CO oV )X) II * II ^ c q * | S 5 |U0 C feb K I •3 ^ S..E A 1 1 £ O O . a. - c •o g « 2 « ■o o 3 0 _ t e o i f t 15 -g eg £ i s ii§ *0 ♦, « H 4> A ii .a cc fc- u £ J to u.- o- 0 - 0 ^ w E JC ra .£p cx = t ^ ES ^ 5 I I u01 < II C/3 D □ , u CO ■ University of Ghana http://ugspace.ug.edu.gh 3.5 Viability of Stored Bacterial Isolates Viability o f stored bacteria was assessed using aliquots o f representative bacterial isolates in suspension kept at 4°C and -20°C, as well as freeze-dried samples stored at 4°C (Table 5). The results indicated that the organisms retained viability under the various storage conditions. 3.6 Comparison of Bacterial Isolates using SDS-PAGE Bacterial cells lysates o f the isolates were analyzed by SDS-PAGE to determine their protein patterns. As shown in Figure 17, the organisms had different polypeptide band profiles. Whereas the T. ferrooxidans sample showed prominent bands ranging from 28 to 68 kDa, the T. thiooxidans sample had prominent bands ranging from 34 to 66 kDa with a large polypeptide band at 36 kDa. L. ferrooxidans on the other hand had 3 major clusters o f polypeptide bands ranging from 10 to 24, 24 to 29 and 29 to 66 kDa (Figure 17). The protein profile in lane 2 (Figure 18) illustrates a typical pattern seen for all isolates o f T. ferrooxidans from the different samples. Figures 19 and 20 show typical polypeptide band patterns for the different isolates o fL. ferrooxidans and T. thiooxidans from the samples analyzed. 63 University of Ghana http://ugspace.ug.edu.gh Table 5 Viability of stored bacterial isolates Growth o f bacterial preparations stored at different S e l e c t e d _______________________temperatures and periods____________________ bacterial Bacteria in suspension Freeze dried specimen stored at 4°C isolate 3 mons (4°C) 3 mons 1 mon 2 mons 3 mons __________________________________ (-20°C)_______________________________________ BT1-Fe2+ + + + + + BT2-Fe2+ + + + + + BT3-S0 + + + + + S01-Fe2+ + + + + + S02-Fe2+ + + + + + S03-S0 + + + + + UO l-Fe2" + + + + + U02-Fe2+ + + + + + U 03-S0 + + + + + UW l-Fe2+ + + + + + UW2-Fe2* + + + + + mons = period o f storage o f samples in months. + = Biooxidizing bacterial cells were able to grow when put back in 9k liquid media. 64 University of Ghana http://ugspace.ug.edu.gh 1 KDa 6 6 - - » 4 5 - * » 36 *» 4 * 2 9- «.» 4 24- # 20 - m 1 4 *^ # Figure 17 Comparison o f bacterial cell lysates o f isolates by SDS-PAGE. Lane 1 shows the standard molecular weight marker, lanes 2, 3 and 4 show protein band profiles o f T. ferrooxidans, T. thiooxidans and L. ferrooxidans respectively, from the biooxidation tank. Arrowheads and brackets indicate prominent bands. 65 University of Ghana http://ugspace.ug.edu.gh Figure 18 SDS-PAGE of bacterial cell lysates o f T. ferrooxidans from biooxidation tank (lane 2). Lane 1 shows standard molecular weight marker and arrowheads indicate prominent polypeptide bands. 66 University of Ghana http://ugspace.ug.edu.gh Figure 20 SDS-PAGE of bacterial cell lysates o f T. thiooxidans isolates from the biooxidation tank. Lane 1 shows standard molecular weight marker and arrowheads indicate prominent polypeptide bands. Lane 2, T. thiooxidans isolate. 68 University of Ghana http://ugspace.ug.edu.gh 3.7 Comparison of Bacterial Isolates by Genomic DNA Analysis using RFLP DNA obtained from the bacterial isolates were analyzed using Restriction Fragment Length Polymorphism (RFLP). The yield and purity o f DNA for a 100ml culture (T. ferrooxidans, T. thiooxidans and L ferrooxidans isolates) were similar (5-10 P-g and purity approximately ) shown in Figure 21, the different bacterial isolates had similar DNA size (approximately 23 kb). Restriction endonuclease digestion o f the DNA gave mostly smears (Figure 22). 69 University of Ghana http://ugspace.ug.edu.gh Figure 21 Agarose gel electrophoresis o f undigested biooxidizing bacteria DNA. Lane M shows a Lambda Hind III digested DNA marker. Lanes 1-3 undigested DNA o f T. thiooxidans isolates lanes 4-6 undigested DNA o f T. ferrooxidans isolates and Lanes 7-9 undigested DNA o f L. ferrooxidans isolates from biooxidation tank, underground gold sulphide and surface arsenopyrite ores respectively. kb 23.13—» 9.42-> 6.56-> 4.36-> 2.32-> 2.03—» Figure 22 Restriction endonuclease (Hind III) digested DNA o f biooxidizing bacterial isolates from biooxidation tank. (M) Lambda Hind III digested DNA marker. (1) Undigested DNA o f L. ferrooxidans. (2) Digested DNA o f L. ferrooxidans. (4) Undigested DNA o f T. ferrooxidans. (5) Digested DNA o f T. ferrooxidans. 70 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 4.0 DISCUSSION AND CONCLUSIONS The main purpose o f the study described in this thesis was to isolate and characterize biooxidizing bacteria from different sites at the Obuasi gold mining concession using microbiological methods, SDS-PAGE analysis o f bacterial cell lysates and RFLP o f their genomic DNA using restriction endonuclease digestion. The biooxidizing bacterial isolates obtained in this study could be placed in three main groups based on morphological, biochemical, and physiological characteristics. The first group consisting o f isolates, BT1-Fe2+, S01-Fe2+, U01-Fe2+ and UW l-Fe2+ were initially identified as T. ferrooxidans-like organisms due to reported characteristics such as being straight rods, Gram negative, ability to grow on solid media and oxidize iron II to iron III (Vishnac, 1975; Harrison, 1984; Torma, 1985; Hamson, 1986; Goebel and Stackebrandt, 1994). The ability o f these T. ferrooxidans-hke bacteria to utilize both iron II and elemental sulphur as energy substrates and their pleomorphic behaviour on solid media indicated that they were indeed T. ferrooxidans (Vishnac, 1975; Colmer et al., 1950, Leathen et al., 1956; Kinsel, 1960; Colmer, 1962; Harrison, 1984; Wakao et a l, 1991; Junior, 1991; Goebel and Stackebrandt, 1994). According to Schrader and Holmes (1988) the pleomorphic property o f T. ferrooxidans is due to a phenotypic switching mechanism which enables the organism to survive adverse environmental conditions. It is indeed this property that enables T. ferrooxidans to grow on solid medium and utilize both ferrous iron or sodium th iosulphate as energy substrate (Rawlings et al., 1991). The second group o f isolates (BT2-Fe2+, S 02 -F e2+, U02-Fe2+ and UW2-Fe2+) which among other characteristics were, curved rods, Gram negative and oxidized iron II to iron III were primarily considered as L. ferrooxidans based on earlier observations (H am son and Norris, 1985; Harrison, 1986; Sand et al., 1992). 71 University of Ghana http://ugspace.ug.edu.gh Their inability to grow on solid media confirmed the identification (Hamson 1984; Goebel and Stackebrandt, 1994). Goebel and Stackebrandt (1994) isolated Gram negative, straight rod bacterial cells that grew at pH < 1 and utilized elemental sulphur as energy substrate, and identified them as T. thiooxidans. The third group o f isolates (BT3-S0, S03-S0 and U 03-S0) identified in this study exhibited sim ilar characteristics as T. thiooxidans. The present isolates also shared other T. thiooxidans characteristics such as the formation o f pale yellow minute colonies on sodium thiosulphate agar reported by earlier workers (Waksman and Joffe, 1922; Vishnac, 1975; H arrison , 1986; K onishi et al., 1995). The absence o t su lpho-oxidizing bacteria in underground mine water was not surprising since most isolates, especially T. thiooxidans had been obtained from soil samples (Waksman and Joffe, 1922; Vishnac, 1975; Harrison, 1986; Konishi et al., 1995). Lundgren and Silver (1980) reported that biooxidizing bacteria were likely to be found mostly in samples where their energy substrate is readily available. The absence o f T. thiooxidans in underground mine water is therefore attributable to a deficiency o f the energy substrate (elemental sulphur and reduced sulphur compounds). Raw lings and Kusano (1994) explained that low concentrations o f iron in the sample m ay result in small populations o f ferrous iron oxidizing bacteria with slow iron oxidation rate. This m ay also explain the slow ferrous iron oxidation rates o f T. ferrooxidans and L. ferrooxidans isolates obtained from underground mine water as compared to isolates o f the same bacteria from the other samples. However, the difficulty o f the T. ferrooxidans isolate from underground m ine water to undergo pleomorphism and its unique colony size (2-5mm) as compared to the other isolates (0.5-5nnn) may suggest that the isolate was o f a different strain. The mineralogy and chemical composition o f ore has been reported to determine the population o f biooxidizing bacteria present and their efficiency o f mineral ore oxidation (Murayama et a l, 12 University of Ghana http://ugspace.ug.edu.gh 1987; Suzuki et al., 1990; Baldi et al., 1992; Morin, 1995; Rawlings and Silver, 1995). This may suggest that different strains o f biooxidizing bacteria could be isolated from the surface arsenopyrite and underground sulphide ores at Obuasi. Isolates o f biooxidizing bacteria obtained in this study were therefore characterized using SDS-PAGE analysis o f bacterial cell lysates and RFLP o f their genom ic DNA w ith the objective o f revealing possible strain similarities and differences w ith in the same species o f organism isolated from the different ecological niches. Raw lings et al. (1991) used the SDS-PAGE and noted its high degree o f resolution o f bacterial cell proteins. Also, Kersters and De Ley (1980) reported that this method was particularly suited for com paring cell envelopes and ribosomal proteins. In th is w ork it was possible to differentiate between T. ferrooxidans, T. thiooxidans and L. ferrooxidans using their protein profiles. Similar studies by Huber et al. (1985), Harrison and Norris (19S5) and Chamorro et al. (1987) revealed differences in the protein bands o f the three organisms. The protein profiles obtained showed prominent bands that differentiated one bacterium from the other. The bands observed were comparable to those obtained by Huber et al. (1985). Harrison and Norris (1985 ) also identified individual strains o f biooxidizing b. -ieria using the SDS-PAGE. However, no differences in protein profiles were observed between the various isolates o f T. ferrooxidans, T. thiooxidans and L. ferrooxidans analyzed in this study. Even though the surface and underground ores used in this work had different mineralogy and chemical composition, the lack o f detectable strain differences by SDS-PAGE was not surprising since d iileren t b iooxidizing bacterial strains identified earlier were obtained from comp.etely different m etal ores nam ely, copper and uranium ores(Huber et al., 1985; Harrison and Norris, 1985; Chamorro cl al., 1987). 73 University of Ghana http://ugspace.ug.edu.gh Previous workers have characterized biooxidizing bacteria by RFLP (Shiratori et al., 1989, Raw lings, 1995). Tang et al. (1997) pointed out that a large number o f fragments obtained in the DNA digests o f bacteria had small size differences that resulted in overlapping separation normally observed as a smear. This may explain the inability to differentiate between the bacterial isolates in this study because o f smearing o f digested DNA fragments. Rawlings (1995) overcame this problem by amplifying specific DNA sequences before analysis by RFLP. Earlier on, Shiratori et al. (1989) employed Southern hybridization o f genomic DNA to identify strains o f biooxid izing bacteria. It was however not possible to use any o f these improved meth )ds in this study due to resource constraints. Nevertheless, the similarities between T. ferrooxidans, T. thiooxidans and L. ferrooxidans isolates from the biooxidation tank and the local ores bas^u on cultural, physio logical and morphological characteristics as well as protein profiles may suggest that the organism s from the biooxidation tanks are not different from the local isolates. Thi is an interesting observation since there is always the possibility o f local biooxidizing bacterial strains overgrow ing and replacing seeded organisms in long term culture systems. Such a situation may lead to inefficient gold recovery since not all the local biooxidizing bacterial strains are efficient oxidizers o f gold ore. I f this observation were true, then there is an urgent need to chara -; ize and select efficient local strains for the biooxidation process at the sulphide treatment plant at Obuasi. In conclusion, this study confirms the presence o f biooxidizing bacteria currently exploited in biomining namely, T. ferroxidans F. thiooxidans and L. ferrooxidan in the biooxidation tank at AGC, Obuasi. Furthermore, local isolates o f the three organisms with potential use foi .ore efficient gold extraction from local ores were obtained from surface and underground go!,, res as well as underground mine water. 74 University of Ghana http://ugspace.ug.edu.gh R ECOMMENDATIONS 1) There is the need to conduct farther studies to determine the gold recovery efficie.xy o f the isolated local biooxidizing bacteria. 2) More work is needed to determ ine the taxonomic significance o f bacterial isolates .Vom the underground m ine water which had some unique morphological and physiol -ical properties. 3) Further characterization o f the local biooxidizing bacterial isolates should emphasize jNA analytical methods, such as; nucleic acid hybridization, ribotyping (probing restriction patterns w ith labelled bacterial ribosomal operons that code for 16s or 23s rRN/ , and amplification of known segments of genomic DNA. 75 University of Ghana http://ugspace.ug.edu.gh REFERENCES Adair, F. W. (1966). 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Williams and Wilkins Company, Baltimore, USA. pp. 456-462. Wakao, N., Konno, J„ Sakurai, Y. & Shiota, H. (1990). Survival o f freeze-dried Thiobacillus ferrooxidans NC1H 8455 after long-term storage/. Gen.. Appl. Microbiol., 36, 283-286. Wakao, N., Lanada, K., Takahashi, A., Sakurai, Y. & Shiota, H. (1991). Morphological, physiologic; > and cheniotaxonomical characteristics o f iron and sulfur oxidizing bacteria isolated from acid mine drainage waters. J. Gen. Appl. Microbiol., 37, 35-48. Waksman. S. A. & Ioffe. J. S. (1922). Microorganisms concerned in the oxidation o f sulfur in tliL so i l . ./. jLtcicn I., 7, _.'9-255. Watson, J. D . Gilman, IN’., W ikowski, J. & Zoller, M. (1992). Recombinant DNA. 2nd ed. SciRtiiic .America Books Ltd. New York. Weir, D. M. (I9S6). Application o f Immunological Methods in Biomedical sciences. 4th ed. BI ckw ell Sv icntific Pi,U|ical ons Oxford, UK. Wilson, K & \ alk_r J. M. (i 5). Practical Biochemistry: Principles and techniques. 4th ed. Ca ibrid e I ,’niver i t y 1 . ss, Cambridge, UK. Yates, J. \ . , L boi, J. It & liolmes, D S. (1986). The use o f genetic probes to detect mi :roo.‘gnnisms in bioi ' :n:ng operations. J. Ind. Microbiol., 1, 129-135. 90 University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix A Composition and preparation o f 9k liquid medium (after Silverman and Lundgren, 1959) The composition and preparation o f 9k liquid medium used in isolation and cultivation o f biooxidizing bacteria is presented below. Solution A Ammonium sulphate 3.0g Potassium chloride O.lg Dipotassium hydrogen ph "'sphale 0.5g Magnesium sulphate hept;.hydrate 0.5g Calcium nitrate O.Olg Distilled water 700ml Solution B1 (energy suhsi■~ate for rerroiis iron oxidizers') Iron (II) sulphate heptahydrate 14.74g Sulphuri; acid (5M) 400pl Distilled water 100ml Solution 2 fen " e . - L i b s 1 ate ii • llnhur oxidizing bacteria: U nz and Lundgren. 196H Element;i! sulphur l.Og Sulphuric acid ( 5Mj 1.0ml Distilled water 20ml 91 University of Ghana http://ugspace.ug.edu.gh Solution A was sterili -,ed by autoclaving for 15min at a temperature o f 121°C and a pressure o f 1.5 atmospheres It v as then fill:red through a Whatman number 1 filter paper into a sterilized 1 litre volu letric flask coaled to room temperature and stored at 4°C until required. Solution HI was prepared by 11 lter-sterilizing the dissolved constituents through a 0.45pm millipore i i Iter (Millipore Co., Lid, Bedford, Ireland) into a sterilized 100ml volumetric flask and stored at 4 C. To prepare 9k medium supplemented with ferrous iron (Fe2+), 4 parts o f solution A was added to 1 part solution B1 in a 500ml Erlenmeyer flask rinsed with 5M H2S 0 4 On the otherhand 9k medium supplemented with elemental sulphur (S°) was prepared by adding 4 parts o f solutio A lo 1 pari sc ution 1 ’ 2 in an acid washed 500ml Erlenmeyer flask and sterilized by heating to 105 C in a \ ater bath 1 >r 45min on two successive days (Harrison, 1984). 92 University of Ghana http://ugspace.ug.edu.gh Composition and preparation oj solid media To prepare sol d media the following solutions were used: Solution 1: Sodium thiosulphale 0.5g Distilled water 2.5ml Solution 2 : Iron(II) sulphate heptahydrate (F eS04-7H20 ) Distilled water 5M H,SO_ Appendix B Solution ? : Ammoniu i sulphate 1.125g Potassiuir chloride 0.04g Magnesiu ' sulphate heptahydrale (M gS04-7H20 ) 0.1875g Distilled v ater 125ml pH o f sok :on was adjusted to 3.0 at room temperature (25-30°C) using dilute H2S 0 4. Solution - miQc.laverl'): Agar 2.5g Distilled water 120ml (a) Ferror r< id ■". i l l o f ferrous agar (FA) plates The amoui t o i l ron (II) su Iphate heptahydrate and sulphuric acid in solution 2 above was doubled and solution 1 eliminated. The procedure for preparation was the same as described in (a). (c) Prenar. . j f sodjum 'liosu ' ihate agar (STA1 The amou ct sodium ti iosulj iate and distilled water in solution 1 above was doubled and solution 2 . ’ui mated. The procedure for preparation was the same as described (a). (d) P rem '- ;cn o f ferrous tg a n r e (FAR-): Manning’s Modified ISP 9k solid m ed ium (M ann ing 1975: R an . 1982 Solution 1 Ammoniu a sulphate Potassium chlcride Magne; iu; > 1 phatc hcpluhydra '; Calcium n a. Iron(II) su ha.’eh ep t liyd ate (FeS04.7H20) Distilled \ alcr 0.5g 0.02g 0.02g O.OOlg 3-5g 50ml 94 University of Ghana http://ugspace.ug.edu.gh Solution 1 (pH 2.5) was mixed and filter-sterilized through a 0.45pm millipore filter and then warmed to 45 ’C in a waterbalh. Solution 2 i ioclavcil'1 Agarose Distilled w ter Solution 2 autoclave,! for 15min at 121°C and cooled to 45°C and solution 1 (at the same temperature) a Jded. The esulting mixture was aseptically poured into 9cm diameter sterile petri dishes and icU o set in a 1 uninar flow cabinet and stored at 4°C until required. 0.5g 50ml 95 University of Ghana http://ugspace.ug.edu.gh Appendix C Prepayatit n o f r eagents for staining bacterial isolates; (after Salle, 1967 and Collin and Lyne, 1989) (1) Crystal ii lot solmior Two grammes o f crystal violet and 0.8g o f ammonium oxalate were dissolved in 20ml o f 95% ethanol an ! S ml o f distilled water respectively. The two solutions were mixed and left to stand for 24 hours before filtering into a dark bottle using Whatman number 1 filter paper and stored at room tempera1 ure. (2) Iodine jl i iou Two grair. nc o f p o u s s i ’im iodide and l.Og o f iodine were grounded together in a motar and dissolved ia 2 )ml o f distilled water. The solution was made up to 100ml with distilled water when the ^ . h r js ha\ e co: lpletely dissolved. (3~) Carbo1 u hin ( P an. 1 Solution / . Basic Fust.iin Ethanol (9r% ) This soluf n f -\) was kept at 37"C overnight Solution B Phenol 5g Distilled \> .itv. r 100ml 10g 100ml 96 University of Ghana http://ugspace.ug.edu.gh To prepare slock Caiboi liischin stain, 10m! o f solution A was added » 100ml o f solution B. Carbol fus-hin used in staining prepared by diluting one volume o f stock solution with two parts distil ed water. (4) Neutn, , I solnii inXi o llin and T.vne. 1989~); Neutral re' ^ .lg ) Mas dissolved m 100ml o f distilled water and 0.2ml o f l%(v/v) acetic acid added. Tl' - p epared slain w as kept at room temperature until use. (5 )S a fran __s )ck sol 'lion Safranin (- 5y i was i i s solved in 95'. 4 ethanol to make a stock solution which was stored at room tempciatu I dilute salra m solution used in staining was prepared by making 1 in 10 dilution o f the stock. 97 University of Ghana http://ugspace.ug.edu.gh Appendix D Preparation of reagents fo r St. dium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAi E); S o l u t i o n 1 (M o n o m e r S o l u l i o n -) AcrylamBe 30.Og N,N-methylereBis-acrylamide (Bis-aorylamide) ®.8g Distilled v atet 100ml. The soluti a w as filtered through a 0.22pm millipore filter membrane into a dark bottle. Solution - n . ' M Trisamiu'i inet1’ e. pH 8.8) Tris-amm. methane (Tris) 18.16g Distilled \\ atei 100ml The pH o f sol'ition 2 w hicii was ed in preparing the resolution gel was adjusted to pH 8.8 with dilute HC.. S o l u t i o n ^ r 0 . 5 M T r i s I IC" H 6 . P ) Tris-amin ineinane ( i i is) 4.56g Distilled ate i 100ml The p i l e ' sc .it 311 2 .. Iik. w a. sd in preparing the stacking gel buffer was adjusted to pH 6.8 with dilute HC1. S o l u t i o n • l ac i v l a m i d » c I p o l v n < i z a t i o n i n i t i a t o r ) Amm< nii n i ii'-nlph tct.M'S) ' I soluii mi ( I0%)was always prepared fresh before use. 98 University of Ghana http://ugspace.ug.edu.gh Solution j ('c?ialvsQ N, N ,N\-NP-telramethvletliylen .diamine (TEMED). 3.03g 14.4g lg [0.1%(w/v)] 1000ml Solution (. f n innina hnlTer'l r X I 0 stock pH 8.3 1 Tris-amin Mneihane (Tris) Glycine SDS Distilled water The pH o! Sc!lit: i 6 was adju: . J to pH 8.3 with dilute HC1 and a working stock prepared by diluting the X 1 0 y]ock to obtain .. X I solution. S o lu t io n - , I! os a Sod ' uni P \ lecv l Sulphate ('SDS')! SDS (20r) v is ci isso l\ ocl in 100ml distilled water in a 100ml bottle and stored at room temperatu j . Cons 'itue’ is ofresoluHon and st; eking gels Solution A: ( 5"' res Uniati g e P 30%(w v; auy lamidc 0.8%(w/vj bt:-aci lamide 4.25ml 1.5M Tris-HC'l pH 8.S 2.125ml 20%(w/v) jDS 42.5(il 10%Cv/V» \ ’ 33.5|il TEMC ) 11.3(o.l Distilled v atcr 2.055ml 99 University of Ghana http://ugspace.ug.edu.gh Total v o lir 'ic 8 .5m l So lu tion P i 2 )% ■ , -1111 ion gel) 30%(w/v) r ylam ide 0 .8% (w /v ) ,.s-ac! lam idc 5.667m l 1.5M T r is - I IC’ I pH S.S 2 .125m l 20% (w /v ) SDS A2 .5\A 10% (w /v ) A >S 33 .5j j.1 T E M E D 1 1.3 ( j1 D is t i l le d v _r 663 .7p.l T o ta l v o lu 8 .5m l Solut' on C g Lit - 30%(w/v) r\ lai J l 0 .8% (\v /v ) A - , hum do 0 .4m l 0 .5M T r is " pH 6 A lm l 20%( w /v ' " ) S 20-° ll1 10%( .v /v ) i 32-8 ll1 T E M E D 6 -6 ^ D ic i i l le d ' 2 .560m l T o ta l v o h 4 0 m l Standard i dii 1 e. ’H mar' :rs 100 University of Ghana http://ugspace.ug.edu.gh Low n r o l c c lm » eighl standard markers (S.gnra Chemical Comply , USA.) used were; Albnmm from bovine r 6 kD»V Albumin tom egg (45 kDa), G lyceraldehyde-3-phosphate Dehydrogenase from rabl ii muscle l-o kDa), Carbonic anhydrase from bovine erythrocytes <29 kDa), Tiypsinog ) I om bovine pancreas (24 kDa), Trypsin inhibitor from soybean (20 kDa) and - Lactalbuir. n iom bovine milk i 14.2 kDa). 101 University of Ghana http://ugspace.ug.edu.gh Preparation o f reagents fo r isolation and purification o f DNA (1) 0.5M Eh I \ stock solution A t 8.0 Disodium ell ylcne diamine tclra-acetate (9.306g) was added to 40ml o f distilled water and vigorously :i ired whilst ihe j II was adjusted to 8.0 by the addition o f NaOH crystals. The volume v is il-.cn made up lo 5 nl with distilled water and the solution aliquoted and sterilized by autoclavin j at 12 T'C for30n ins. (2) 10% (\v v~> --odium lodccvl ■ inhate fSDS) SDS (lOgt wps rapidly dissoh in 90ml of distilled water by heating to 68°C. The pH was adjusted to 7.1 jy ;hc additioi of a few drops o f concentrated HC1 and the volume made up to 100ml with d billed v. ..ter. (3) 5M Pi a>- i im acetate Potassium ac i e ( Z4 " 3 : * v , dissolved in 50ml o f distilled water and the solution kept at -20°C. (4) Pot" h ceia' 1 Xii 5M po ta ; . urn celaie oluiion 0ml) was mixed with 11.5ml glacial acetic acid and made up to 100ml \vi: 'id i let v. ale;. Th oiution was stored at 4°C. (5~) 1M T '':s - ' ' ' ~'l str ■ o l l11ioi- - Ii 7.6 Tris bas-c 112 j | y as d isH lvi in 70ml o f distilled water. The pH was adjusted to 7.6 by the addition c I,.-ml u cd ' ;C I . ; solution wa allowed to cool to room temperature before final Appendix E 102 University of Ghana http://ugspace.ug.edu.gh adjustment to 'he pH was mac! The volume was then made to 100ml with distilled water. The solution v .is dispensed i n t o alii lots and sterilised by autoclaving. ( 6 ) T E J v '~en )H ~f t MnmM , ris-Cl n H 7 .6 : ImM EDTA. pH 8.0) This bu [ Te r w h prepared b \ di Lung 1 0 m l o f t h e EDTA stock solution and 10ml o f 1M Tris-Cl pH 7.6 an,In. .!•.::,o o f the olution is then made up with distilled water, dispensed ill > ■ liqi.ot and slei 'ed by autoclaving. (8) Phi d 'J m An ecL an.i i u ■ i ph .nol :1 e! 1 rolbrm were mixed and equilibrated by extracting the mixture ve i l ii - wit 0 . ! '1 T (pH .6). This solution was under an equal volume o f O.OlMTr -C (pH V ') and sic d in a dark bottle at 4°C. (9) IQ l 1 NaOH i -1 ’) oh,. I., billed water, sterilzed by autoclaving then stored at room temper:, e. 103 University of Ghana http://ugspace.ug.edu.gh Composi (1) Resi r Restrict i Endonu Hind 111 Bain HI P s t l Eco R1 S a i l The enz; (2) Res1 The bu!' concenlr G I Gel - ion untl j'i I'j'oriiiii >II < f Us fo r restriction endonuclease digestion; l i e n u i z i v ' s i ' 1 ' s o ill i Appendix F .ea.\ I’ rulciryoiic source Recognition sequence din ip, iul •iyh< icjucU iens i cmnci Ii -p i u h . m G Activity (units/fil) 5 ’-A/AGCTT-3’ 10 5’-G/GATCC-3’ 10 5 ’-CTGCA G-3’ 12 5’-G/AATTC-3’ 10 5’-GTCGA/C-3’ 10 les \ ere >i ed 2u C .■ enzymes were obtained from Gibco BRL. Li on ~i\y_ -s I I c r s ve pi " nufactureis, each enzyme has its specific buffer and at ioi, that u'c I i! Micenli ation used. The buffers were stored at-20°C. !'> hiit'v:' (5 v ' 'n 104 University of Ghana http://ugspace.ug.edu.gh 20% (\vA ) l;i ;oll, . m.VI EDTA. : >% (w/v) orange G, stored at room temperature. Final concentra ion used i ON/ sairpi ; IX. 105 University of Ghana http://ugspace.ug.edu.gh Reagen ts fo r agarose gel el ecu (1) E lec tr - csis N i l I ci' E ' This s o lu u 'n c nisi: ’s o [' 2 42■ ■ T: adjusted i ■ 7 (wii i "laeial acc: (2) E th id1 •'"ony . , 1 Ui gf" Ethidium T on id e w d c i (('.!:■ stored in .. cc i.ainc . rapped ; (3) N u c je J I m,i- kcts | I an * The sizes ■ o f i1 : fray iv and 125. • is n: 5OX Tris acetate (TAE) i s c , 57.1 ml glacial acetic acid, 100ml 0.5ml EDTA, with pH id) in 1 litre water. Working solution was IX TAE. Appendix G j added to 10ml o f distilled water. The solution was then inium foil and stored at room temperature. I III Digest (Gibco BRLY1 follows; 23,130, 9,416, 6,557, 4,361, 2,322, 2,027, 564, 106 University of Ghana http://ugspace.ug.edu.gh M ol ec ul ai w ei gh t / KD a Appendix H Standard Protein calibration cu rve Relative mobility (Rf) Standard DNA calibration cu rve University of Ghana http://ugspace.ug.edu.gh