Physiology and Genetics of : Applications for

by Olena I. Rzhepishevska

ISBN 978-91-7264-509-7 Umeå University Umeå 2008

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TABLE OF CONTENTS

ABSTRACT 5 ABBREVIATIONS AND CHEMICAL COMPOUNDS 6 PAPERS IN THIS THESIS 7 1. INTRODUCTION 9 1.1 General characteristics of the Acidithiobacillus genus 9 1.1.1 Classification 9 1.1.2 Natural habitats and growth requirements 9 1.1.3 and RISCs in natural and environments 10 1.1.4 The sulphur cycle and acidithiobacilli 11 1.1.5 Genetic manipulations in Acidithiobacillus spp. 13 1.1.6 Importance in industry and other applications 14 1.2 Acidithiobacillus spp. RISC and iron oxidation 15 1.2.1 RISC oxidation by Acidithiobacillus spp. 15 1.2.2 A. ferrooxidans iron oxidation and regulation 23 1.3 Mineral sulphide oxidation 26 1.4 Acid mine and rock drainage 30 1.5 Biomining 36 1.5.1 Biomining as an industrial process 36 1.5.2 Acidophilic microorganisms in industrial 38 2. AIMS OF THE STUDY 41 3. RESULTS AND DISCUSSION 43 4. CONCLUSIONS 51 5. ACKNOWLEDGMENTS 53 6. REFERENCES 55 7. PAPERS 75

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ABSTRACT

Bacteria from the genus Acidithiobacillus are often associated with biomining and . Biomining utilises acidophilic, sulphur and iron oxidising microorganisms for recovery of metals from sulphidic low grade and concentrates. Acid mine drainage results in acidification and contamination with metals of soil and water emanating from the dissolution of metal sulphides from deposits and mine waste storage. Acidophilic microorganisms play a central role in these processes by catalysing aerobic oxidation of sulphides. Acceleration of mineral solubilisation is a positive aspect in biomining whereas, in acid mine drainage it is undesirable and accordingly, microbial iron and sulphur oxidation is promoted in the first case and measures are taken to inhibit it in the second case. In this thesis, several approaches were taken in order to understand and increase oxidation efficiency in biomining and to gain an insight into the biochemical reactions taking place in these environments. A laboratory scale bioreactor was designed and tested allowing simulation of bioleaching in heaps of mine at different aeration, irrigation and particle size conditions (Paper I). A new psychrotolerant strain of Acidithiobacillus ferrooxidans was characterised that has an application in boreal heap bioleaching. Iron, reduced inorganic sulphur compound oxidation and bioleaching of various ores by this strain was studied as well as gene expression during oxidation of tetrathionate and/or ferrous iron (Papers III & IV). Expression and regulation of a tetrathionate hydrolase from , a key enzyme in reduced inorganic sulphur compound metabolism of this bacterium was investigated and the presence of this enzyme in a bioleaching mixed culture was shown. The gene cluster that harbours the gene coding for tetrathionate hydrolase (tetH) was described for the first time (Paper II).

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ABBREVIATIONS AND CHEMICAL COMPOUNDS

AMD - acid mine drainage ARD - acid rock drainage DGGE - denaturing gradient gel electrophoresis EPS- extracellular polymeric substance FISH - fluorescent in situ hybridisation GSH - glutathione GSSG - GSH disulphide GSSH - glutathione persulphide - GSSO3 - glutathione S- sulphonate HiPIP - high potential iron-sulphur protein

H2S - sulphide PQQ - pyrrolo-quinoline quinone RISCs - reduced inorganic sulphur compounds SFORase - sulphide:Fe(III) oxidoreductase SORa - sulphur oxygenase reductase SQR - sulphide quinone oxidoreductase StOR - sulphite:cytochrome c oxidoreductase 2- Sn - polysulphides S0 - elemental sulphur 2- S2O3 - thiosulphate 2- S4O6 - tetrathionate 2- S5O6 - pentathionate 2- SO3 - sulphite SRB - sulphate reducing TQO - thiosulphate:quinone oxidoreductase TQR - thiosulphate quinone oxidoreductase

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PAPERS IN THIS THESIS

I Olena I. Rzhepishevska, E. Börje Lindström, Olli H. Tuovinen & Mark Dopson (2005) Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor. Biotechnol Bioengin 92(5):559-567

II Olena I. Rzhepishevska, Jorge Valdes, Liucija Marcinkeviciene, Camelia Algora Gallardo, Rolandas Meskys, Violaine Bonnefoy, David S. Holmes & Mark Dopson (2007) Regulation of a novel Acidithiobacillus caldus gene cluster involved in metabolism of reduced inorganic compounds. Appl Environ Microbiol 73(22):7367-7372

III Daniel Kupka, Olena I. Rzhepishevska, Mark Dopson, E. Börje Lindström, Olia V. Karnachuk & Olli H. Tuovinen (2007) Bacterial oxidation of ferrous iron at low temperatures. Biotechnol Bioengin 97(6):1470-1478.

IV Olena I. Rzhepishevska & Mark Dopson (2008) Sulphur Accumulation During Low Temperature Bioleaching (Manuscript in preparation)

Papers in this thesis have been reprinted with permission from Wiley (papers I & III) and ASM (paper II)

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1. INTRODUCTION

1.1 General characteristics of the Acidithiobacillus genus

1.1.1 Classification The Acidithiobacillus genus belongs to the γ- and comprises four species: Acidithiobacillus albertensis, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans (Kelly & Wood, 2000). The genus was formed when the former genus was split into the genera Acidithiobacillus, and . The members of the genus are acidophilic Gram-negative rods, motile by one or more flagella and comprise both mesophiles and moderate . All four species are capable of growth utilising inorganic compounds such as reduced inorganic sulphur compounds (RISCs) as sole energy substrate, whereas A. ferrooxidans is the only representative of the genus that can also oxidise Fe2+. A. ferrooxidans is represented by a number of strains with pronounced genetic variation (Bergamo et al., 2004; Karavaiko et al., 2003; Kelly & Wood, 2000; Paulino et al., 2001) which are predicted by Kelly and Wood (2000) to be reclassified into separate species. A. albertensis was thought to be lost from culture and its 16S rRNA sequence was previously unavailable, but in 2002 the 16S rRNA gene sequence of DSM strain 14366 was published (accession number AJ459804).

1.1.2 Natural habitats and growth requirements Acidithiobacilli use various RISCs or Fe2+ as electron donor and therefore, are often found in metal sulphide deposits all over the world (Karavaiko et al., 2003), fresh waters associated with sulphide deposits (Gonzalez-Toril et al., 2003) and seawater (Kamimura et al., 2003). A flexible chemolithoautotrophic metabolism allows A. ferrooxidans and A. caldus to grow on as the

9 electron donor which could potentially broaden their ecological niche (Drobner et al., 1990; Hallberg & Lindström, 1994). The final electron acceptor is usually oxygen (Kelly & Wood, 2000) however, Fe3+ and S0 were reported to be alternative electron acceptors for A. ferrooxidans under anaerobic conditions (Ohmura et al., 2002; Pronk et al., 1991a; Sugio et al., 1992a). Presence of Calvin cycle enzymes (Appia-Ayme et al., 2006; Kusano et al., 1991) and hence the ability to fix CO2 makes acidithiobacilli independent of an organic carbon source and allows them to thrive in mine waste ecosystems where the organic carbon concentration is extremely low (Dold et al., 2005). Despite the reports that organic compounds are toxic to A. ferrooxidans (Tuttle & Dugan, 1976; Tuttle et al., 1977), A. caldus was reported to reach higher growth yeilds with addition of yeast extract or and A. ferrooxidans was able to utilise glucose and formate (Hallberg & Lindström, 1994; Pronk et al., 1991b; Tabita & Lundgren, 1971). All members of the genus are with optimum pH from 2 to 4 but the upper limit for A. thiooxidans reaches 5.5. All of the acidithiobacilli are mesophiles with the exception of A. caldus which is a moderate with an optimum at 45°C (Kelly & Wood, 2000). When cultivated in laboratory conditions, acidithiobacilli generally grow much slower and give much lower yields compared to such as Escherichia coli (Eccleston & Kelly, 1978; Kamimura et al., 2003; Leduc et al., 1993).

1.1.3 Iron and RISCs in natural and mining environments Sulphur is one of the most abundant elements in minerals after oxygen and silicon; together with all its known compounds it constitutes 0.03 % of the Earth’s crust. Native sulphur (S0) is mainly found in volcanic, oil-bearing or sedimentary deposits. The biggest sedimentary deposits are located in Texas and Louisiana (USA) and Sicily (Italy). In the form of sulphate, sulphur is abundant in oceans and in the soil. Sulphur reacts with all metals except and platinum forming sulphides. The most known and exploited ores that contain

10 sulphides are chalcocite (Cu2S), (FeS2), chalcopyrite (CuFeS2), galena

(PbS), cinnabar (HgS), sphalerite (ZnS), argenite (Ag2S) and arsenopyrite (FeAsS) (Weller, 1960; In Encyclopaedia Britanica; http://search.eb.com/eb/article-9070248). Pyrite is the most widespread metal sulphide in the world and is often used for sulphur production and rich pyrite deposits are found in Spain, USA, Canada, Norway, Russia and Portugal. Galena is often found in association with sphalerite and argenite in USA, Australia and England, while chalcopyrite is present in almost every sulphide deposit. The estimated amount of iron in the Earth’s crust is 5 %; it occurs in hundreds of different compounds but the most common source of Fe2+ for iron oxidising acidophiles such as A. ferrooxidans are iron sulphides (Mottana, 1983; Weller, 1960). The interests of sulphur/iron oxidising microorganisms and humans collide at sulphide deposits as mining activities exposes sulphide sources buried under the ground and creates conditions (i.e. exposure to air and water) for the existence of acidithiobacilli and other acidophilic microorganisms.

1.1.4 The sulphur cycle and acidithiobacilli Sulphur is one of the key elements in proteins that are present in every living cell and at the same time sulphur compounds can be used as electron donors or electron acceptors by various microorganisms. Such a tight dependence of all forms of life on sulphur creates its turnover in nature, termed the sulphur cycle. In this process, sulphur atoms are present at different oxidation states and are incorporated into amino acids of proteins as well as mineralised to inorganic compounds. The main reactions of the sulphur cycle are assimilatory sulphate reduction, mineralisation (desulphurisation), dissimilatory oxidation of hydrogen sulphide and sulphur, and dissimilatory sulphate and sulphur reduction. A number of groups of microorganisms are involved in dissimilatory oxidation of RISCs. Among those are the acidithiobacilli, green and purple

11 phototrophic bacteria (e.g. Clorobium and Allochromatium), some thermophilic such as Acidianus and , the facultatively lithoautotrphic neutrophile Parracoccus pantotrophus and bacteria that live in association with oceanic sulphurous hot springs from the genera Beggiatoa and Thioploca (Friedrich et al., 2005; Madigan & Martinko, 2006). Acidithiobacillus spp. are active in several steps of the sulphur cycle (Fig. 1) but their most important role in sulphur mobilisation is to catalyse metal sulphide oxidation at low pH (see section 1.2 for details), thereby releasing sulphate into solution (Schrenk et al., 1998).

Organic compounds

Assimilatory sulphate R-SH Mineralization reduction

Biological aerobic oxidation

2- 2- S4O6 , S2O3

Dissimilatory sulphate reduction S2- -Metal SO 2- H S 4 sulphides 2

Biological aerobic and anaerobic oxidation; spontaneous oxidation 2- SO3 2- Sn

Dissimilatory Biological aerobic and 0 S0 anaerobic oxidation S sulphur reduction

Fig. 1 The sulphur cycle - modified from Robertson & Kuenen (1992). Steps that include specific activities by acidithiobacilli are underlined. Oxidation steps are solid lines and dashed lines are reductive.

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Assimilatory sulphate reduction occurs in all animals, plants and all microorganisms. Desulphurisation is also a general process which involves decomposition of proteins through decay and excretion. Dissimilatory sulphate and sulphur reduction is carried out by bacteria generally termed sulphate reducers (e.g. the genera Desulfovibrio and Desulfotomaculum) and dissimilatory sulphur-reducing bacteria (Desulfuromonas). Sulphate reducers use sulphate as an anaerobic terminal electron acceptor, while dissimilatory sulphur-reducing bacteria couple anaerobic oxidation of organic compounds to reduction of S0 to hydrogen sulphide. Certain facultatively aerobic heterotrophs like spp. and Salmonella spp. are able to reduce sulphur and other sulphur compounds (Madigan & Martinko, 2006; Robertson & Kuenen, 1992).

1.1.5 Genetic manipulations in Acidithiobacillus spp. Genetic manipulations such as gene mutagenesis and cloning, mutation complementation and protein expression provide excellent approaches to study gene and protein function in different organisms. Unfortunately, many of these techniques are inefficient in Acidithiobacillus spp. mainly due to that the outer membrane and periplasmic space of these bacteria are adapted to acidophilic conditions, while most of the methods generally used in molecular biology work optimally at neutral pH (Holmes & Bonnefoy, 2007). One of the main problems is the absence of an efficient method to import DNA into acidithiobacilli cells. Though plasmid transfer through conjugation with E. coli was achieved for all species except A. albertensis, the frequency of transconjugates was only 10-5-10- 7 transconjugates per recipient (Jin et al., 1992; Liu et al., 2007; Peng et al., 1994; van Zyl et al., 2003). Electroporation was also possible in A. ferrooxidans but it was apparently only successful for certain strains (Kusano et al., 1992). Heterologous protein expression in E. coli or other well studied microorganisms is one way to confirm its function and an efficient option to produce sufficient protein for further studies. Indeed, some A. ferrooxidans and

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A. caldus cytoplasmic and inner membrane proteins were successfully expressed in E. coli (de Groot et al., 2003; Zeng et al., 2007) but outer membrane and periplasmic proteins often fail to fold correctly at neutral pH (Bengrine et al., 1998; Bruscella et al., 2005). Expression of heterogeneous phosphofructokinase in A. thiooxidans was also reported and resulted in glucose consumption by this chemolithothroph (Tian et al., 2003). A few mutants achieved both by direct and random mutagenesis have been reported for A. ferrooxidans. One of them was due to insertion of ISAfe1 (an insertion sequence element) and the other was constructed using a suicide plasmid with a selectable marker (Holmes et al., 2001; Liu et al., 2000). Recently, a successful mutant construction protocol involving a suicide plasmid was developed for A. caldus (D. Rawlings personal communication) however; this protocol is un-published and not available for general use. As a result, molecular genetic studies in acidithiobacilli is a challenging and time consuming task specially considering their long generation times, low yields in liquid media and poor growth on solid media.

1.1.6 Importance in industry and other applications The importance of acidithiobacilli in industry developed as a function of their ability to catalyse solubilisation of metal sulphides and to produce sulphuric acid (see chapter 1.4 for details). In some cases their presence is favourable for industrial processes and sometimes it is undesirable. Acidithiobacilli are employed for efficient extraction of many valuable metals from sulphidic minerals, an approach generally termed ‘biomining’ (Rawlings, 2002; Rawlings, 2005). On the other hand, their activity often causes an environmentally hazardous process called acid mine drainage (AMD) and acid rock drainage (ARD; for more information see chapter 1.3) (Canovas et al., 2007; Lefebvre et al., 2001a; Lefebvre et al., 2001b; Olias et al., 2004; Vigneault et al., 2001). Moreover, A. thiooxidans and A. ferrooxidans speed up concrete corrosion

14 processes (Maeda et al., 1999; Sand, 1987) and A. thiooxidans is reported to dissolve human urinary stones in in vivo experiments (Nishio et al., 2003). Lately, promising experiments were carried out showing the possibility of A. ferrooxidans usage in a microbial fuel cell running on Fe2+ (Ter Heijne et al., 2007).

1.2 Acidithiobacillus spp. RISC and iron oxidation

1.2.1 RISC oxidation by Acidithiobacillus spp. Chemolithotrophic RISC oxidation occurs in various groups of microorganisms and several types of enzyme systems are known that can carry out this process. The well studied Sox complex from α-proteobacterium P. pantotrophus is important for oxidation of thiosulphate to sulphate and also occurs in many other bacterial species (Friedrich et al., 2005). RISC oxidation by Archaea has been studied in Acidianus ambivalens where sulphur oxygenase reductase (SORa) and thiosulphate:quinone oxidoreductase (TQO) have been discovered. SORa catalyses the disproportionation of sulphur to sulphite, thiosulphate and hydrogen sulphide and TQO couples the reduction of caldariella quinone to thiosulphate oxidation with tetrathionate as a product (Muller et al., 2004; Urich et al., 2004). Interestingly, neither sox nor sor genes have been found in the A. ferrooxidansT genome (ATCC 23270), but instead acidithiobacilli have their own unique enzymes dedicated to RISC oxidation. Though many of these enzymes have been purified and characterised, the whole picture is far from being clear. Another unsolved question is how S0 is transported into the periplasm of acidithiobacilli. It is proposed that it is mobilised by thiol groups of putative outer-membrane proteins and transported to the periplasm in form of persulphide sulphur (Rohwerder & Sand, 2003; Silver & Lundgren, 1968b). Acidithiobacillus thiooxidans. A. thiooxidans was the first bacterium of the genus to be discovered (Kelly & Wood, 2000; Waksman, 1922). It was shown

15 not to oxidise Fe2+ but was able to metabolise various RISCs such as S0, 2- 2- 2- thiosulphate (S2O3 ), tetrathionate (S4O6 ) and sulphite (SO3 ) with different pH optimums for certain compounds (Masau et al., 2001; Suzuki, 1965a; Suzuki, 1965b; Suzuki et al., 1992). Thus, the pH optimum for sulphur oxidation is 2.3 but pH 5 for tetrathionate, thiosulphate and sulphite (Masau et al., 2001). The enzyme responsible for oxidation of S0 was purified in 1965 (Suzuki, 1965b) and participation of glutathione (GSH) in the reaction was reported even earlier (Suzuki & Werkman, 1959). Rohwerder and Sand (2003) reported that GSH activates sulphur through a non-enzymatic reaction forming glutathione persulphide (GSSH), which in its turn yields sulphite in an enzymatic reaction catalysed by sulphur dioxygenase. Sulphite was further oxidised in a non- enzymatic reaction to sulphate, thiosulphate and glutathione S- sulphonate - (GSSO3 ). Oxidation of H2S occurred only in the presence of GSH disulphide (GSSG). Oxidation of thiosulphate by A. thiooxidans follows two different pathways depending on the pH (Masau et al., 2001). At pH 5, the intermediate products are sulphite and sulphur and at pH 2.3 they are tetrathionate and sulphur. In both cases sulphur and sulphite are finally oxidised to sulphate. Thiosulphate dehydrogenase containing cytochrome c was purified from A. thiooxidans JCM 7814 (Nakamura et al., 2001) and tetrathionate decomposing enzyme was purified from A. thiooxidans ON 107 (Tano et al., 1996). The main product of tetrathionate metabolism in A. thiooxidans ON 107 was thiosulphate. Sulphite oxidation in A. thiooxidans JCM 7814 was studied by Nakamura et al. (Nakamura et al., 1992; Nakamura et al., 1995) and proven to be catalysed by an adenosine 5-monophosphate independent membrane bound sulphite:cytochrome c oxidoreductase (sulphite dehydrogenase). Sugio et al. (2006) purified an aa3- type ubiquinol oxidase and demonstrated the existence of sulphite:ubiquinone oxidoreductase activity in A. thiooxidans BN1-3. These findings allowed them

16 to propose aa3- type ubiquinol oxidase as a terminal oxidase in sulphite and consequently the A. thiooxidans sulphur oxidation pathway. Acidithiobacillus caldus. Only three studies have been dedicated to sulphur metabolism in A. caldus (Bugaytsova & Lindström, 2004; Dopson et al., 2002; Hallberg et al., 1996). It was shown that A. caldus could metabolise S2-, S0, 2- 2- 2- S2O3 , S4O6 and SO3 and an increase in phosphorylation potential was observed during oxidation of all these compounds (Dopson et al., 2002).

Concentration (mM)

0 4 8 12 16 20 24 minutes

Fig. 2 Oxidation of thiosulphate (■) to tetrathionate (●) by A. caldus KU in the absence (A) and in the presence (B) of 200 μM 2,4-dinitrophenol. Re-printed from Hallberg et al. (1996).

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No substrate level phosphorylation was detected and it was concluded that all ATP is synthesised by membrane bound ATPase driven by a proton gradient. Thiosulphate was oxidised to tetrathionate by A. caldus cells and in presence of 2,4-dinitrophenol tetrathionate was accumulated (Fig. 2). Inhibition of oxygen consumption by cyanide had a biphasic character that suggested the presence of more than one terminal oxidase. It was proposed that thiosulphate is oxidised to tetrathionate in the periplasm which agreed with the localisation of thiosulfate oxidising enzyme in other acidithiobacilli. The products of tetrathionate hydrolysis were thiosulphate, pentathionate and sulphate (Bugaytsova & Lindström, 2004). Based on inhibition by various uncouplers, it was suggested that S2- and S0 oxidation yields sulphite and enzymes for these reactions were located in the cytoplasm (Hallberg et al., 1996) which disagrees with proposed localisation of sulphur dioxygenase in other acidithiobacilli (Rohwerder & Sand, 2003). Based on subsequent data (Bugaytsova & Lindström, 2004) it is probable that the inhibition of S2- and S0 oxidation by uncouplers is due to their uptake to the periplasm rather than the cytoplasm and data in the Hallberg et al. (1996) study was misinterpreted. Acidithiobacillus albertensis. A. albertensis is the least studied species of all acidithiobacilli. DSM 14366 and BY-05 are the only two strains known and 0 2- 2- both were demonstrated to metabolise S , S2O3 , S4O6 and ZnS (Kelly, 1989; Xia et al., 2007). The details of S0 oxidation in this bacterium are unknown. A. albertenis was shown to colonise elemental sulphur particles by micro colonies and it was suggested that glycocalyx plays an important role in the attachment (Bryant et al., 1984). Acidithiobacillus ferrooxidans. A. ferrooxidans is the best studied representative of all acidithiobacilli. A. ferrooxidans can oxidise RISCs both aerobically and anaerobically and all known aerobic pathways of RISC oxidation were summarised (Fig. 3) by Holmes and Bonnefoy (2007). The S0 oxidising protein was purified by Silver and Lundgren in 1968 but the actual

18 mechanism of its activity was only explained in 2003 (Rohwerder & Sand, 2003; Silver & Lundgren, 1968b). As in the case of A. thiooxidans, prior activation of sulphur by glutathione is required and the enzymatic reaction yields sulphite. Sulphite is then oxidised to sulphate by sulphite oxidoreductase (Vestal & Lundgren, 1971). The thhiosulphate oxidising enzyme was also purified and a high thiosulphate:quinone reductase activity is present in A. ferrooxidans ATCC19859 cells grown on S0 (Brasseur et al., 2004a; Brasseur et al., 2004b; Silver & Lundgren, 1968a). Other important RISC enzymes include sulphide:quinone oxidoreductase (Wakai et al., 2007), tetrathionate hydrolase (de Jong et al., 1997; Kanao et al., 2007) and rhodanese (Tabita et al., 1969).

Tetrathionate 2- Sulphide S4O6 S2- Thiosulphate quinone oxidoreductase Tetrathionate hydrolase Sulphide:quinone oxidoreductase

Thiosulphate 0 Sulphur S O 2- S 2 3 Rhodanese ? Sulphate 2- (S) Trithionate SO4 hydrolase Sulphur dioxygenase Sulphite 2- oxidoreductase 2- S3O6 SO3 Trithionate Sulphite

Fig. 3 Proposed enzymatic steps involved in RISC oxidation by A. ferrooxidans. Re-drawn from Holmes & Bonnefoy (2007).

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A. ferrooxidans has a number of terminal oxidases and electron flow from various substrates to oxygen is quite complicated. The most recent findings suggest that electrons arising from growth on RISCs enter the respiratory chain through sulphide quinone oxidoreductase (SQR), TQO or sulphite oxidoreductase (StOR) and are transferred to bd- or bo3-type oxidases. Those electrons which are directed to the bo3-type oxidase pass through a bc1-complex (Fig. 4).

Operon petII Operon cyo Operon cyd Operon doxII sqr3

cycA2,petA2,petC, cyoB, cyoC, cydB, cydA doxDA, rod sdrA2, petB2, hip cyoA,cyoD

? SO SOR

2- 2- GSSG SO3 SO4 Hip 0 2- 2- CycA2 H2SS S2O3 S4O6 ( c4 )

bd quinol SQR TQR bc1 complex bo3 quinol oxidase oxidase UQ (PetABC2)

O2 H2OO2 H2O

Fig. 4 Model proposed for RISC energetic metabolism of A. ferrooxidans. The transcriptional units and the corresponding proteins located in the inner membrane are presented in the same color. Operons cyo, cyd and doxII are up regulated on S0; operons petII and sqr3 are expressed to the same or higher level on S0 comparing to Fe2+. TQR: thiosulphate quinol reductase; SQR: quinone reductase; Hip: High Potential Iron-sulphur Protein. Re-drawn from Quatrini et al. (2006).

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Electrons resulting from sulphide and thiosulphate oxidation join the respiratory chain at the level of the quinol pool and those coming from sulphite oxidation enter at the level of cytochrome c4 or high potential iron-sulphur protein (HiPIP) protein (Fig. 4) (Holmes & Bonnefoy, 2007; Quatrini et al., 2006). Unusually, the electron transport chain in A. ferrooxidans uses two differentially expressed bc1 complexes (Appia-Ayme et al., 1999; Brasseur et al., 2002; Bruscella et al., 2007). The first is coded by the petI operon and is expressed during growth on Fe2+. The other is coded by the petII operon and 0 dominates in S grown A. ferrooxidans. It was shown that petII coded bc1 complex supports downhill electron transport to bo3-type oxidase, but the bc1 coded by petI works in the opposite direction and supplies electrons for NAD(H) reduction (for more details, see chapter 1.2.2). This theory was built using data obtained by microarray analysis of differential gene expression in A. ferrooxidans grown on Fe2+ and S0 and verified by quantitative PCR and supported by measurements of enzyme activity. Higher transcription levels were found for genes from petII, cyo, cyd and doxII operons from S0 grown A. ferrooxidansT ATCC 23270 compared to cells grown on Fe2+ (Brasseur et al., 2004b; Bruscella et al., 2007; Quatrini et al., 2006). Surprisingly, similar experiments in A. ferrooxidans strain NASF-1 showed the same level of cydA gene expression both in S0 and iron grown cells (Wakai et al., 2004). Moreover, coxB which was down regulated in S0 grown A. ferrooxidans ATCC 23270 (Quatrini et al., 2006) was equally expressed in A. ferrooxidans NASF-1. The same authors reported that a thiosulphate:ubiquinone oxidoreductase activity was not detected in NASF-1 strain which is contradictory to findings in A. ferrooxidans BRGM strain (Brasseur et al., 2002; Wakai et al., 2004). These contradictions might indicate that there could be other pathways of electron transport during aerobic RISC oxidation in different strains within the A. ferrooxidans species.

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A. ferrooxidansT was shown to grow in anaerobic conditions using S0 as electron donor and Fe3+ as electron acceptor (Pronk et al., 1992). Strain 21834 was capable of glycine uptake under the same conditions which was seen as a proof of energy transduction during Fe3+ respiration because glycine was not taken up by starving cells (Pronk et al., 1991a). Interestingly, A. ferrooxidans strain JCM 7811 expressed a specific cytochrome when grown on sulphur and respiring Fe3+. The cytochrome had a molecular mass of 28-kD, was stable at acidic pH and able to reduce Fe3+ (Ohmura et al., 2002). The observation that S0 reduction coupled to Fe3+ oxidation takes place even in the presence of oxygen gave rise to a new theory proposing an alternative mechanism for S0 oxidation in A. ferrooxidans strain AP19-3 under aerobic conditions. A soluble sulphur:Fe(III) oxidoreductase and a membrane bound sulfite:Fe(III) oxidoreductase were purified from strain AP19-3 (Sugio et al., 1992a; Sugio et al., 1992b). The first enzyme requires GSH and oxidises S0 to sulphite and the second one oxidises sulphite to sulphate. It is suggested that Fe2+ resulting from hydrogen sulphide:Fe(III) oxidoreductase (SFORase) and sulphite:Fe(III) oxidoreductase reactions is further oxidised via iron oxidase (Sugio et al., 1985; Sugio et al., 1988b). Sugio et al. (1989) proposed hydrogen sulphide to be the actual substrate for sulphur:Fe(III) oxidoreductase using the argument that it was oxidising hydrogen sulphide in the presence of 8mM GSH. However, contrary to this, Rohwerder and Sand (2003) argue that GSH at such a high concentration forms GSSG which reacts with H2S producing GSSH, which in its turn is the real substrate for the oxidoreductase. Nevertheless, the enzyme is often referred to as a SFORase. SFORase oxidises S0 using not only Fe3+ but also Mo6+ and Cu2+ as electron acceptors (Sugio et al., 1988a; Sugio et al., 1990b). SFORase and iron oxidase activities were high even in the type strain grown for many generations on S0 and SFORase activities comparable to that of strain AP19-3 were found in eight different strains of A. ferrooxidans and five strains of Leptospirillum ferrooxidans (Sugio et al., 1992b; Sugio et al., 2007).

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Both these facts were interpreted by the authors as evidence favouring a wide distribution of the alternative pathway for S0 oxidation in iron oxidising microorganisms. Sulphur oxidation is strongly inhibited by Fe2+ in strain AP19-3 due to inhibition of sulphite:Fe(III) oxidoreductase by this ion. As a result, sulphite accumulates in the cells and damages SFORase and iron oxidase which halts cell growth (Sugio et al., 1990a). Such sensitivity of strain AP19-3 to sulphite suggests that this strain might lack sulphite oxidoreductase.

1.2.2 A. ferrooxidans iron oxidation and regulation A. ferrooxidans is the only species of acidithiobacilli able to oxidise Fe2+ and consequently these bacteria have both RISCs and Fe2+ for energy metabolism at their disposal. Identification of the primary protein that oxidises Fe2+ to Fe3+ in A. ferrooxidans has a long history. Among others, high potential iron-sulphur protein (HiPIP) from Fe-1 and BMGR strains, the 26 kDa cytochrome c4 (also known as Cyc1 or CYC41) and outer membrane cytochrome Cyc2 have all been suggested to serve as the iron oxidase (Appia-Ayme et al., 1999; Cavazza et al., 1995; Giudici-Orticoni et al., 2000; Yamanaka & Fukumori, 1995). Both latter authors agreed that electrons from Fe2+ were transferred by the iron oxidase to rusticyanin, a blue protein which constitutes up to 5 % of the total protein in iron grown A. ferrooxidans. Later it was demonstrated that the iro gene (coding for HiPIP in Fe-1 and BMGR strains) had hip gene as the only counterpart in three other strains. Hip shared 51% similarity with Iro and was up regulated in S0 grown cells which suggests that it did not act as an iron oxidase

(Bruscella et al., 2005). At the same time, 26 kDa cytochrome c4 was shown by spectroscopic analysis and site specific mutagenesis to mediate electron transport between rusticyanin and cytochrome oxidase (Malarte et al., 2005).

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Operon rus Operon petI

cyc2, orf, coxA, coxD, cyc1, cycA1, petA1, petC1, coxB, coxC, rus sdrA1, petB1

Fe(II) Fe(III)

OM ORF Cyc2

Rusticyanin Periplasm Cyc1 CycA1 (c ) 4 (c4) NADHI Cytochrome bc complex IM 1 UQ oxidase (PetABC1)

Cytoplasm O H O NAD(P) NAD(P)H 2 2

Fig. 5 Model proposed for direct and reverse electron transport in A. ferrooxidans grown on iron. Figure re-drawn from Quatrini et al. (2006).

However, evidence favouring cytochrome Cyc2 as being a potential iron:rusticyanin oxidoreductase resulted in the model in Fig. 5 (Quatrini et al., 2006; Yarzabal et al., 2002; Yarzabal et al., 2004). Cyc2 oxidises Fe2+ and reduces rusticyanin which is the branching point in this electron transport chain. From rusticyanin, electrons are directed either to cytochrome oxidase via Cyc1 or to the bc1 complex via CycA1. Cyc1 and cytochrome oxidase represent the “downhill” electron pathway resulting in ATP production, while electron flow through CycA1 and bc1 is “uphill” i.e. an energy consuming pathway that results in NAD(P)H production. The requirement for A. ferrooxidans cultured on Fe2+ to expend energy on uphill or reverse electron transport was proposed as early as

24

1982 (Ingledew, 1982). The requirement for uphill electron transport results from that the redox potential of the couple Fe2+/Fe3+ at pH 2 is more positive than for NAD(P)H/NAD(P)+ at pH 6.5 (Ingledew, 1982). NAD(P)H is necessary in the Calvin cycle for CO2 fixation and carbohydrate synthesis (Fig. 6). As already mentioned, A. ferrooxidans has two bc1 complexes, one of which is upregulated in S0 grown cells and the other one is upregulated in iron grown bacteria. The latter bc1 complex is coded by the pet1 operon which also contains cycA1 and sdrA1. Gene sdrA1 is similar to ribitol/glucose oxidoreductases. Oxidation of ribitol yields ribulose which can be further converted to ribulose-5- P and directed to the Calvin cycle. Thus, the suggested role of SdrA1 is to trigger the Calvin cycle to utilise NAD(P)H produced by reverse electron transport (Levican et al., 2002). It is also noteworthy that two isotypes of rusticyanin are found in several strains of A. ferrooxidans. They differ in signal

-0.4 NAD(P)+ -0.2

0 0.2 Calvin cycle Volt 0.4

0.6

2+/3+ 0.8 Fe ∆μH+ ATP/ADP 1.0 O 2

+ Fig. 6 Redox and metabolic relationships in A. ferrooxidans. The ∆μH+ (H - electrochemical gradient) is used to drive ATP synthesis or reverse electron transport. Re-drawn from Ingledew (1982).

25

sequence and DNA context of their genes (Sasaki et al., 2003) and it is probable that these strains would not exactly fit to the model in Fig. 5. Recently, it was shown by several authors that expression of different protein complexes involved in oxidation of RISCs and Fe2+ is regulated depending on the availability of the substrate (Bruscella et al., 2007; Quatrini et al., 2006; Ramirez et al., 2004; Yarzabal et al., 2004). Transcription of petII, cyo, cyd and doxII operons is upregulated during cultivation on S0, while the rus and petI operons are upregulated in iron grown bacteria. Intriguingly, closer investigation of this process revealed transient expression of rus and petII operons on S0 and iron, respectively. Genes from the rus operon are always expressed in the early exponential phase on S0 but by the mid-exponential phase mRNA and corresponding proteins levels were drastically decreased (Yarzabal et al., 2004). This observation probably explains the conflicting reports on rusticyanin expression during A. ferrooxidans cultivation on S0 (Ramirez et al., 2004; Suzuki et al., 1990; Yarzabal et al., 2003). The rus operon is regulated from three different promoters and post transcriptional processing of its mRNA is proposed. A similar pattern is observed for petII promoter which is upregulated and suggested to be involved in direct electron transport in S0 grown A. ferrooxidans, but transient expression of the operon was observed at the early stages of growth on iron (Bruscella et al., 2007). The regulatory proteins involved in this process have not been identified though existence of Fur regulated genes in A. ferrooxidans was shown (Quatrini et al., 2005) but the binding site for this transcription factor has not been found for rus, petI or petII.

1.3 Mineral sulphide oxidation

It is generally accepted that acidithiobacilli are involved in release of metals from metal sulphides (Colmer & Hinkle, 1947; Singer & Stumm, 1970). However, a debate on the nature of the metal sulphide oxidation has occurred

26 since Silverman and Ehrlich proposed the direct and indirect mechanisms (Silverman & Ehrlich, 1964; Silverman, 1967). The indirect mechanism implies that the actual leaching agent is Fe3+ ion which extracts electrons from the mineral while being reduced to Fe2+. The role of the bacteria is to oxidise Fe2+ into Fe3+ thereby, regenerating the primary oxidant. The direct mechanism suggests that the microorganisms themselves enzymatically oxidise the mineral whilst in direct contact. A few workers still support this theory (Lilova & Karamanev, 2005) though, no enzymes are reported which could be involved in direct metal sulphide oxidation. On the other hand, a study of the complex chemical reactions behind the indirect mechanism resulted in an integral model for bioleaching (Sand et al., 2001; Schippers & Sand, 1999). An important pre-requisite for this model was a study showing that there are two types of sulphides and accordingly, two types of possible indirect leaching mechanisms (Tributsch & Bennett, 1981). Sulphides with valence bands derived from metal electron orbitals only have their electrons extracted by 3+ the attack of Fe (e.g. MoS2, FeS2 and WS2) while sulphides with valence bands derived from both metal and sulphur electron orbitals could also be oxidised via proton attack (e.g. CdS and ZnS). The valence band in the band theory of solids is an energy level occupied by valence electrons (i.e. electrons from the outer shell of an atom) and valence electrons are thought to determine the chemical properties of atoms in reactions (Atkins & de Paula, 2006). Metal sulphides from the first group (MoS2, FeS2 and WS2) are oxidised via the indirect thiosulphate pathway (Fig. 7) that results in the production of thiosulphate and Fe2+ as the primary leaching products (Schippers & Sand, 1999). Thiosulphate is further oxidised in a cyclic manner to sulphate with tetrathionate, disulphane- monosulphonic acid and trithionate as intermediate products (Schippers et al., 1996). It is speculated that the oxidation of these sulphur compounds is mainly of a chemical character but participation of such enzymes as tetrathionatehydrolase of acidithiobacilli is also suggested (Sand et al., 2001).

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Thiosulphate mechanism Polysulphide mechanism

MS MS H+ Fe3+ Fe3+ Af,Lf Af,Lf

Fe2+ Fe2+ S 2- + Me 2+ 2- 2+ n S2O3 + Me (Af, At, Ac)

(Af, At, Ac) S8 Af, At, Ac

2- + 2- SO4 + H SO4

Fig. 7 Scheme of thiosulphate and polysulphide mechanism of metal sulphide (bio)leaching (modified from Schippers & Sand (1999)). MS = metal sulphide, M 2+ 2- 2- = metal ion, S8 – elemental sulphur, Sn = polisulphides, S2O3 = thiosulfate. Af, At, Ac and Lf = enzymatic reactions by Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. (Af), (At) and (Ac) = possible enzymatic reactions by these microorganisms.

The second group of sulphides is leached via the polysulphide pathway whereby, the key intermediate polysulphides are oxidised to S0 and then to sulphate (Fig. 7 and 8). The polysulphide pathway of sulphide degradation also occurs in the absence of Fe3+ through proton attack which results in formation of sulphide. The oxidation of sulphide by Fe3+ via polysulphides to S0 was

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H+ H+ 2H+ M2+ MS + HS* S [H2S* H2Sn] 8 Fe3+ Fe2+ Fe3+ Fe2+

Fig. 8 Simplified scheme from the polysulphide mechanism. Re-drawn from Schippers & Sand (1999).

demonstrated (Steudel, 1996) but no information is available on the involvement of microorganisms in this step. Additionally, Tributsch (2001) claims that polysulphides may also cause dissolution of metal sulphides. It was proposed to distinguish contact and non-contact leaching within indirect oxidation of metal sulphides (Tributsch, 2001) as A. ferrooxidans is able to attach to mineral surfaces and create dents and pits (Escobar et al., 1997; Gehrke et al., 1998; Mangold et al., 2008; Sand & Gehrke, 2006). Attachment occurred with the help of an extracellular polymeric substance (EPS) layer which is proposed to facilitate mineral dissolution due to increased Fe3+ concentration. The iron oxidising bacterium L. ferrooxidans has also been shown to attach to pyrite and create a characteristic hole pattern on the mineral surface (Rojas-Chapana & Tributsch, 2004). Thus, leaching by Fe3+ ions concentrated within EPS of the attached bacteria is referred to as contact indirect bioleaching. Leaching catalysed by free floating microorganisms is termed non- contact indirect bioleaching.

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1.4 Acid mine and rock drainage

AMD or ARD are terms used to describe drastic acidification (down to pH –3.6) of surface and ground waters by products of metal sulphide (bio)leaching (Nordstrom et al., 2000). Acidification is accompanied by high concentrations of iron and other metals such as copper, , lead, chromium and . Opencast and underground abandoned mines as well as mining industry waste (tailings) are the source of the AMD/ARD. The mining industry is widespread over the world and hence AMD is a common phenomenon and the involvement of microorganisms in AMD was proved long ago (Colmer & Hinkle, 1947). Leaching of metal sulphides exposed to water and oxygen occurs via a purely chemical mechanism but the presence of S0 and iron oxidising microorganisms increases the chemical reaction rate by more than 106 fold (Singer & Stumm, 1970). Several factors such as temperature, age, chemical composition of the rocks, hydrological regime of the site and mining technique influences AMD development. Consequently, every AMD is unique but nevertheless, some general trends in chemical and microbial characteristics can be found. An optimal combination of AMD forming factors gave one of the biggest and most studied AMDs in the world at Iron Mountain, Shasta County, northwest California. The Iron mountain mining area comprises several mines that were operated from the 1860s to 1962 when Ag, Au, Cu, Fe, Zn and pyrite were recovered. The brittle rhyolite bedrock of the site creates a fracture-flow type hydrologic conditions that has little buffering capacity. The massive deposit (95 % pyrite) located at, or above the water table was excavated by tunnels and shafts where airflow generated by heat production from pyrite oxidation produces a subsurface temperature of 50ºC (Nordstrom & Alpers, 1999; Nordstrom et al., 2000). High availability of oxygen and moisture as well as a mild to high temperature range benefits a wide variety of iron and S0 oxidisers (Bond et al., 2000; Edwards et al., 1999a; Edwards et al., 1999b; Schrenk et al.,

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1998). In 1983 Iron Mountain was ranked as the third largest polluter in the State of California on National Priorities List for Environmental Protection Agency Superfund (Iron Mountain Mine Study Case, http://www.epa.gov/aml/tech/imm.pdf, accessed January 13, 2008). Approximately 2,500 tons of pyrite were annually weathered from the Richmond mine before large scale remediation efforts were initiated in the late 1980s (Nordstrom & Alpers, 1999). The AMD contained very high levels of metals and approximately 300 tons of dissolved Cu, Cd and Zn were released into the Sacramento river. Sudden increases in AMD release into the Sacramento river caused massive fish deaths and > 47,000 trout died in a single week in 1967 (Nordstrom & Alpers, 1999). AMD is gravely hazardous for most living organisms due to its high acidity and presence of heavy metals producing symptoms such as gill lesions in fish (Henry et al., 1999; Henry et al., 2001). Also, sheep refuse to drink synthetic AMD water even without being given an alternative due to dissolved iron rather than the pH (Horvath, 1985). AMD waters are lethal to seaweeds and invertebrates in laboratory and field conditions (Marsden & DeWreede, 2000; O'Halloran et al., 2008). Damage caused by heavy metals is detected in rodent liver, kidney and testis caught near the metal processing plants (Damek-Poprawa & Sawicka-Kapusta, 2003). Cd, Cu and Zn were shown to be lethal in high doses and decrease the survival in low doses to various vertebrates and invertebrates (Baker et al., 1998; Gheorghiu et al., 2007; Handy, 2003; Jianhua et al., 2006). Heterotrophic bacteria commonly occurring in non-acidic streams could not survive in streams polluted with AMD at pH <3 (Tuttle et al., 1968; Walton & Johnson, 1992). The environmental pollution caused by AMD is long- lasting and some areas have suffered its impact for hundreds of years (Demchak et al., 2004; Younger, 1997). Species number and abundance in marine and fresh waters remain decreased for several years after remediation or even total neutralisation of the AMD source (Simon et al., 2006; Zis et al., 2004).

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AMD is extremely toxic for neutrophilic organisms though it creates suitable conditions for a variety of acidophilic bacteria, archaea and algae that often possess genetic determinants for metal resistance (Dopson et al., 2001). An interesting characteristic of such biogeosystems is a gradual shift in pH, metal concentration, dissolved oxygen and organic carbon depending on the distance from the AMD source which is reflected in the changing composition of microbial communities. Moreover, sediment layers from different depths of AMD streams are populated by defined groups of bacteria, algae and archaea (Johnson et al., 2001; Rowe et al., 2007). Microbial communities analysed at different sites of Iron Mountain AMD (pH 0.77 to 1.31) were dominated by “” spp., Leptospirillum group I, II and III, Sulfobacillus spp. and Acidimicrobium spp.. A. ferrooxidans was not found suggesting its lack of involvement in pyrite dissolution at Iron Mountain (Baker & Banfield, 2003; Bond et al., 2000; Schrenk et al., 1998). On the other hand, data was obtained showing A. ferrooxidans to be important in colonisation of pyrite at circumneutral pH. When attached to pyrite bacteria trigger leaching reactions and push the pH down preparing conditions suitable for other acidophiles (Mielke et al., 2003). A. ferrooxidans dominates in oxygen-depleted mine water, Huelva Spain (pH 2.67) which was supposedly conditioned by facultatively anaerobic metabolism of A. ferrooxidans (Rowe et al., 2007). Studies of microbial communities of Tinto river (pH 2.1 to 4.7) showed A. ferrooxidans to be one of the most available species both in aerobic and anaerobic conditions (Gonzalez-Toril et al., 2003). In oxic wetlands receiving acid coal mine drainage waters (influent pH 2 to 3.9) A. ferrooxidans together with A. thiooxidans accounted for 34 % of the bacterial population detected by fluorescent in situ hybridisation (FISH) (Nicomrat et al., 2006a; Nicomrat et al., 2006b). A. ferrooxidans is often found in numbers lower than L. ferrooxidans in AMD and bioleaching set-ups (Rawlings et al., 1999). However, Johnson et al. (2001) reports that an isolate with 98 % similarity to A. ferrooxidans dominated

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Leptospirillum – like bacteria in all water samples taken at King’s Mine, Norway (pH 2.75 to 3.71). Although L. ferrooxidans is more tolerant to Fe3+ than A. ferrooxidans the latter has the advantage of being more resistant to low temperatures (Hallmann et al., 1992; Leduc et al., 1993; Rawlings et al., 1999). Thus, the presence of a dominant A. ferrooxidans at the subarctic climate zone where King’s Mine is located with temperatures remaining below zero for more than half a year is consistent with other studies. Interestingly, low temperature (7°C) enrichment cultures from the black schist Talvivaara deposit, Finland demonstrated the presence of A. ferrooxidans, A. thiooxidans and A. albertensis while at higher temperatures L. ferrooxidans and A. caldus were prevailing (Puhakka et al., 2007). It appears that A. ferrooxidans plays multiple roles in ecosystems based on iron and sulphur compounds as the main source of energy and its colonisation on pyrite decreases the pH in the initial step of leaching. It oxidises Fe2+ to Fe3+ under aerobic conditions and reducing Fe3+ by oxidation of RISCs under anaerobic conditions supporting iron cycling in AMD waters and remediation wetlands. Finally, it is likely that A. ferrooxidans is the main microorganism catalysing pyrite oxidation in boreal climates. Chemolithotrophic bacteria and archaea are not the only microorganisms found in AMD environments and algae such as Chlamydomonas sp., Euglena cf. mutabilis, Chlorella sp. as well as several others species were isolated from the Tinto river (Aguilera et al., 2006; Amaral Zettler et al., 2003; Rowe et al., 2007). No photosynthetic algae were found in eukaryotic Iron Mountain subsurface biofilms which is attributed to the absence of a phototrophic niche rather than the extremely low pH or high temperature. Curiously, the 16S rRNA gene sequences of Dodhideomycetes and Eurotiomycetes found in this ecosystem were highly similar to neutrophilic lineages which implies that either colonisation of AMD by these fungi occurred relatively recently or that AMD conditions are quite acceptable for these species and do not exert significant evolutionary pressure (Baker et al., 2004). Twenty-seven yeast species from

33 different AMD sampling points have been isolated from the Iberian Pyrite Belt region, Spain and Portugal (Gadanho et al., 2006). Many of these species belonged to Candida and Cryptococcus genera whereas, 48 % probably constituted new taxa. The authors note that the composition of yeast consortia is similar in related environmental stress conditions (pH and salt concentration etc.) and did not depend on geographical location (Gadanho et al., 2006). Similarly, principal-component analysis of 16S rRNA and gyrB genes from AMD microbial communities of Dexing copper mine, Jiangxi, China suggests that sites with similar geochemical characteristics harbour communities of a close structure (Yin et al., 2008). Heterotrophic, acidophilic bacteria have been isolated and characterised from AMD sites. Despite their ability to oxidise Fe2+ their contribution to the total iron oxidation is considered to be negligible. Nevertheless, under certain conditions these bacteria could be responsible for the larger part of the biomass production (Johnson et al., 1992; Wichlacz & Unz, 1981). Many approaches have emerged to prevent and/or mitigate the problem of AMD that have been divided into “source control” and “migration control” measures or in other words prevention and remediation (Johnson & Hallberg, 2005). The “migration control” group comprises both active and passive methods while “source control” alternatives include:-

flooding or sealing of a mine land or water covering of mine tailings / rock capping mineral waste blending with acid consuming material bioshrouding / biological source treatment application of biocidic agents solidification of tailings chemical coating

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All these methods have different degrees of efficiency, certain drawbacks and should be applied carefully taking into concern the geochemical characteristics of the site and nature of AMD source (Anonymous, 1994; Gatzweiler et al., 2001). For example, efflorescent salts that accumulate at the site could produce even heavier AMD if dissolved, which makes a widely-used method of mine flooding undesirable (Nordstrom & Alpers, 1999). Tailings covered with water release heavy metals from the surface layer even under anoxic conditions and major spills from malfunctioning and corroded tailings dams cause serious damage to environment and agriculture (Bjelkevik, 2005; Olias et al., 2006; Vigneault et al., 2001). Biological source treatment and bioshrouding are recently proposed methods for source treatment (Jin et al., 2007; Johnson et al., 2008). Biological source treatment is a down-hole injection of sulphate reducing bacteria (SRB) and organic substrate in electromagnetically determined underground AMD “hot spots”. Microbial sulphate reduction occurs naturally in AMD sites or at locations receiving acid mine flows (Herlihy & Mills, 1985; Tuttle et al., 1969a) and it has been suggested to be applied for amendment of AMD (Tuttle et al., 1969b). SRB are used to mitigate AMD in bioreactors and in situ providing efficient metal removal (Dvorak et al., 1992; Koschorreck et al., 2007; Neculita et al., 2007). Bioshrouding utilises inoculation of freshly ground tailings with heterotrophic, Fe3+ reducing acidophiles from the and Acidocella genera. Bacteria form a biofilm on the tailings surface and reduce Fe3+, the primary oxidant of AMD (Johnson et al., 2008). Measures of AMD remediation are summarised in Fig. 9.

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Active systems: aeration and lime addition Abiotic Passive systems: e.g. anoxic limestone drains

Active Off-line sulphidogenic bioreactors Aerobic wetlands Biological REMEDIATION Compost reactors wetlands Passive Permeable reactive barriers

Packed bed iron-oxidation bioreactor

Fig. 9 Biological and abiotic strategies for remediation of acid mine drainage waters. Adapted from Johnson & Hallberg (2005).

1.5 Biomining

1.5.1 Biomining as an industrial process The ability of microorganisms to oxidise Fe2+ and S0 is widely used for metal recovery from sulphidic ores, a process known as biomining. The term ‘bioleaching’ refers to recovery of the metal of interest when it is chemically bound by sulphide (e.g. copper in chalcopyrite (CuFeS2)) and ‘biooxidation’ is the biomining of a metal of interest which is not chemically bound in the sulphide (e.g. gold particles embedded in arsenopyrite). Biomining is an increasingly developing industry (McCready, 1987; Songrong et al., 2002; Torma, 1985) and the following commercial-scale leaching techniques have been developed: in situ leaching, dump leaching, heap leaching, vat leaching and reactor leaching (Rossi, 1990). In situ leaching is a little utilised approach, limited mainly to bioleaching of copper and . The low grade deposit is blasted and bioleached at place making the capital and operational costs low but

36 significantly increasing the negative environmental impact of this technology (Rossi, 1990; Torma, 1985; Tuovinen, 1985). Dump and heap bioleaching are similar techniques exploiting metal recovery from the leachate collected in the base of a dump (heap) of crushed rock. However, a dump is an unorganised pile of mine waste or run-off-mine while a heap is an irrigated and aerated construction with drainage layer and watertight lining at the bottom (Fig. 10) (Groudev et al., 2001; Logan et al., 2007; Petersen & Dixon, 2007). The drawback of even highly engineered heaps is that bioleaching takes months and a large percentage of the target metals remains unleached (Petersen & Dixon, 2007). GEOCOAT® is a new technology involving heap construction of a specially prepared metal sulphide source that promises substantially increased oxidation time and higher metal output. The source for the heap construction is

Irrigation

Heap packing (ore)

Aeration

Drainage layer Solution collection

Fig. 10 A construction of an engineered heap (up arrows designate air flow and down arrows the liquid percolation). Adapted from Petersen & Dixon (2007).

37 particles of a bare rock 6 to 25mm in diameter with 0.5 mm thick coating of a fine milled ore (Harvey & Bath, 2007). In contrast, vat leaching is carried out in a series of concrete vats (tanks) filled with crushed ore where the leach solution is fed from the bottom of the vat and overflows to the next vat (Rossi, 1990). Additionally, bioleaching of high-value ores and concentrates is economically successful in stirred, aerated and temperature controlled reactors and constitute a highly developed biomining technology (Do Carmo et al., 2001; Rawlings, 2002; Van Aswegen et al., 2007). Stirred bioreactors with different kinds of impellers are mainly used in these processes but new designs like “aerated through bioreactor” or “low energy bioreactor” are being currently being developed (Rossi, 2001).

1.5.2 Acidophilic microorganisms in industrial bioleaching

For many decades A. ferrooxidans was considered to be the primary microorganism responsible for catalysis of metal sulphide oxidation due to their selection in culture-dependent analyses of bioleaching samples (Leveille et al., 2001; Mishra et al., 1983; Schippers et al., 1995). However, the development of culture independent microbial molecular techniques identified that other species were dominant in many bioleaching environments, especially bioreactors treating mineral concentrates (Hugenholtz et al., 1998; Johnson, 2001; Tan et al., 2007). Biodiversity in industrial bioleaching processes can be divided according to their growth temperature ranges into mesophiles at approximately 30°C, moderate thermophiles around 45°C and thermophiles at approximately 70°C (Norris, 2007; Rawlings, 2002). Typical mesophilic bioleaching microorganisms include the iron and S0 oxidisers A. ferrooxidans, “Sulfobacillus monseratensis” and intermedia; the iron oxidiser L. ferrooxidans; the sulphur oxidiser A. thiooxidans; and heterotrophic Acidiphilium spp. (Battaglia-Brunet et al., 2006; Norris et al., 2000; Rawlings & Johnson, 2007;

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Yahya & Johnson, 2002). Recently, L. ferrooxidans was reclassified and the new species Leptospirillum ferriphilum and Leptospirillum ferrodiazotrophum were formed. L. ferriphilum has a higher optimal growth temperature than L. ferrooxidans and L. ferrodiazotrophum was proposed as the main nitrogen fixer in an acidophilic AMD microbial community (Coram & Rawlings, 2002; Tyson et al., 2005). Consequently, L. ferriphilum is found in moderately thermophilic consortia together with A. caldus, Acidimicrobium ferrooxidans, and Sulfobacillus spp. (Dopson & Lindström, 1999; Karavaiko et al., 1987; Norris, 2007). Thermophilic stirred tank bioleaching consortia are dominated by archaea of Sulfolobus, Metallosphaera, Stygiolobus and Acidianus genera (Dopson et al., 2006; Mikkelsen et al., 2006). Mesophiles and moderate thermophiles are often isolated from heap leaching but thermophilic archaea of Thermoplasma and Sulfolobus genera are also present due to high temperatures measured in the deeper layers of a heap (Puhakka et al., 2007a; Rawlings & Johnson, 2007; Xie et al., 2007). The microbiology of biomining has several peculiar characteristics that are worth mentioning. The catalysis of mineral oxidation is carried out by mixed consortia in non sterile conditions (Rawlings, 2002; Rossi, 1990). It was noticed that adaptation of the culture to specific mineral, temperature and other factors is crucial for efficient bioleaching/biooxidation and though stirred tanks and heaps are supplied with the initial inoculum the microbial consortium composition, as a rule, changes during leaching due to the presence of indigenous microorganisms in the mineral and selective pressure of the system (Morin, 2007; Rawlings, 2007; Plumb et al., 2007). In stirred tanks where temperature, aeration and pH are maintained at the same level only a few species were found to be dominating (Mikkelsen et al., 2006). In heap bioleaching a temperature gradient across a heap creates multiple niches for survival of species with different growth temperature requirements (Plumb et al., 2007; Rawlings, 2007). Though bioleaching in stirred tanks and heaps are

39 dominated primarily by autotrophic microorganisms, heterotrophic and facultatively heterotrophic acidophiles and iron oxidisers also contribute to mineral sollubilisation. The relationships of autotrophs and heterotrophs in such systems are reciprocal; heterotrophs remove organic substances which are toxic to certain autotrophs (e.g. Leptospirillum spp.) and autotrophs provide necessary organic compounds to heterotrophic iron oxidisers (Okibe & Johnson, 2004).

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2. AIMS OF THE STUDY

Heap leaching is a developing industry and there is a need for experimental studies of ore arrangement and management in the heap as well as analysis of microbial activity during heap bioleaching (Petersen & Dixon, 2007). The first aim of this study was to test whether the novel vacuum-positive pressure driven bioreactor was suitable for modelling of heap bioleaching.

Heap leaching in arctic and sub arctic environments has a big potential (Puhakka et al., 2007) though new technologies must be developed for bioleaching in cold temperatures. The second aim of this study was to characterise a new psychrotolerant strain of A. ferrooxidans which dominates heap leaching of black schist at low temperatures (Finland).

Occurrence of polythionates (tetrathionate and thiosulphate etc.) in cyanidation slurry increases cyanide consumption in industrial bioleaching (Van Aswegen et al., 2007). The third aim of this study was to investigate regulation of expression of tetrathionate hydrolase from A. caldus, a candidate microorganism to use in pre-cyanidation treatment.

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42

3. RESULTS AND DISCUSSION

PAPER I

A novel reactor design was presented that was used for bioleaching with an acidophilic mixed culture and its performance compared to bioleaching in a stirred tank reactor inoculated with the same culture. The new reactor utilised cycles of positive and negative pressure applied to a system of two connected vessels (Fig. 11).

Sensor controlling upper level of liquid

Compressed air pump Ground

Spray nozzle

Tube a

Vessel A One-way valve Vessel B One-way valve for aeration Tube b

Filter cloth (felt liner)

Fig. 11 Design of the novel reactor.

The design allowed the leachate to circulate between the vessels and drain through a layer of finely milled mineral (tailings) placed on the filter in vessel B and thus, simulate heap bioleaching. Resting periods between phases when positive or negative pressure was applied helped to lower energy consumption.

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The average leaching rate in the new bioreactor was 0.17 g Fe/l·day, while in the conventional stirred reactor it was 0.50 g Fe/l·day. The dissolved oxygen concentration in vessel A fluctuated from 8.4 to 9.4 mg/l depending on the phase of the cycle whereas; typical dissolved oxygen concentrations in commercial leaching bioreactors are between 1 to 2 mg/l (Van Aswegen et al., 2007 and Anthony Pinches, personal communication). The bioleaching proceeded for 83 days and the changes in microbial composition of the mixed culture was analysed by denaturing gradient gel electrophoresis (DGGE; Paper I, Fig. 4). By cloning and sequencing the 16S rRNA gene, clones related to A. thiooxidans, Leptospirillum OS7, L. ferriphilum and Sulfobacillus Y0017 were identified from both reactor types. Additionally, an archaeon similar to F. acidiphilum was identified from the novel bioreactor. Although many microorganisms were observed in the leachate (liquid media) no biofilm was found on the tailings (vessel B). During the resting phase of the cycle the leachate percolated through the vessel and thereby, wetted and aerated the tailings. It is possible that the leachate drained through the tailings too quickly and over drying of the mineral particles occurred during the resting and negative pressure phases (95 s) that prevented biofilm formation. Recent data on the importance of biofilms in bioleaching (contact indirect bioleaching) suggests that the results obtained from the novel bioreactor may be improved if biofilm growth could be promoted (Sand & Gehrke, 2006). To obtain a biofilm on the tailings, the resting time could be shortened or vessel B could be packed with a thicker layer of tailings to lower the percolation rate and facilitate bacterial attachment. By FISH, most of the active Leptospirillum spp. and Sulfobacillus spp. were found at the bottom of vessel B (Fig. 12). Fe3+ and protons generated by these bacteria oxidised the tailings during leachate circulation and thus, leaching was due to non-contact in-direct bioleaching. The bioreactor is suitable for modelling of heap bioleaching and microbial community studies in AMD environments.

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Fig. 12 FISH of the samples from the bottom of the vessel B. Leptospirillum spp. (spiral shaped; top right) and Sulfobacillus spp. (straight rods; bottom right) visualised with 16S rRNA probes (LEP 154 and SUL 228) labeled with Cy- 5. The left hand panels show corresponding fields visualised with DAPI.

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PAPER II

Knowledge of RISC metabolism and its regulation in acidophilic microorganisms might be useful for industrial bioleaching processes. Cyanidation is a common hydrometallurgical gold recovery process based on the reaction of gold with cyanide whereby, gold is converted into the water soluble aurocyanide complex that is collected by adsorption on carbon particles (International Cyanide Management Institute http://www.cyanidecode.org/cyanide_use.php; accessed 25.01.2008). Pre- treatment is necessary to extract gold from sulphide ores (typically arsenopyrite) in order to dissolve the mineral and liberate the gold. Polysulphides and polythionates (e.g. tetrathionate and thiosulphate) produced during mineral sulphide oxidation compete for cyanide in the cyanidation step according to the following reactions:

2- - 2- - S4O6 + 3CN + H2O → S2O3 +2HCN +SCN (Kelly et al., 1969) 2- - 2- - SxO3 + CN → SO3 +SCN (Van Aswegen et al., 2007)

Therefore, RISCs present in the slurry consume cyanide required for successful gold recovery and one solution to decrease its consumption is oxidation of the residual RISCs by acidophilic sulphur oxidisers. Reductions in cyanide consumption will not only reduce costs but also lower nitrate (the final SCN- breakdown product) release into the environment that can result in eutrophication of water bodies. Higher temperatures during pre-cyanidation treatment ensures more efficient RISC oxidation (Van Aswegen et al., 2007) and therefore, use of the moderately thermophilic sulphur oxidiser A. caldus in this step might be of interest. Tetrathionate hydrolase (TetH) has been purified and described as the enzyme responsible for tetrathionate decomposition

46

1000 bp

ISac1 rsrR rsrS tetH doxD

P0 P1

Fig. 13 Schematic representation of tetH gene cluster.

(Bugaytsova & Lindström, 2004). In this study, we investigated A. caldus TetH regulation and characterised the first gene cluster containing tetH (Fig. 13). Expression of the enzyme was compared during A. caldus cultivation on tetrathionate and S0 by Western-blot analysis (Paper II, Fig. 3) and Q-PCR. TetH expression during bioleaching was demonstrated in the Western blot confirming that investigations of TetH are relevant to bioleaching environments. It was demonstrated that TetH expression was transcriptionally regulated and was 233.5 ± 134 fold higher in tetrathionate grown cells compared those grown on S0. Genes coding for a putative transposase, a two component regulatory system (RsrRS) and DoxD were co-transcribed with tetH. P0 and P1 putative promoters (Fig. 13) were identified in the cluster and shown to increase transcription of β- galactosidase when cloned up-stream of the lac operon of pRW2 plasmid in E. coli. The transcription start site of the P1 promoter was mapped by primer extension (Paper II, Fig.4). TetH was similar to pyrrolo-quinoline quinine (PQQ) binding dehydrogenases ((Paper II, Fig. 2) and the presence of a quinone moiety was suggested for this protein based on the positive staining with NBT-glycinate. Previously, it was shown that tetrathionate oxidation is inhibited by electron transport uncouplers (Hallberg et al., 1996). This data taken together, led us to propose that TetH was feeding electrons from tetrathionate to the electron transport chain. Protein crystallisation experiments are on-going to elucidate the

47

A B

Fig. 14 Crystals of TetH in 3M MgSO4 (A) and 1.5M MgSO4, 2.5M NaCl and zwittergent 3-12 (B).

mechanism of tetrathionate oxidation reaction through analysis of its crystal structure. Initial crystals have been obtained (Fig. 14) but further optimisation of the crystallisation conditions is needed.

PAPERS III AND IV

Papers III and IV are tightly connected. In paper III we characterise A. ferrooxidans strain SS3 as the first psychrotolerant acidophilic chemolithotrophic iron oxidiser. In paper IV we investigate RISC oxidation during cold temperature leaching by A. ferrooxidans strain SS3 and T7 mix psychrotolerant cultures. Both studies were aimed at understanding and improving bioleaching processes in boreal climates. We show that strain SS3 was able to grow at 5°C by oxidising Fe2+ (Fig.15) with the rate constant ranging from 0.0162 to 0.0104 h-1 (generation time of 43-58 h) depending on the initial pH. A mesophilic A. ferrooxidans reference strain was substantially less efficient under the same conditions (Paper III, Fig. 2).

48

(mM) Oxidized iron

0 200 400 Hours

Fig. 15 Iron oxidation at 5°C. A. ferrooxidans SS3 at the initial pH 2.8 (■) and 2.0 (∆); mesophilic A. ferrooxidans reference strain at initial pH2.0 (◊); abiotic control (Χ). From paper III.

Tetrathionate was shown to support the growth of strain SS3 at both 5° and 30°C (Paper III, Fig. 5). 16S rRNA gene phylogeny showed strains SS3, SK3 and SK5 to be similar to several A. ferrooxidans isolates and clones acquired from arctic and sub-arctic environments (Paper III, Fig. 1). Also, several of these isolates are psychrotolerant suggesting that they may constitute a new species and experiments to test this hypothesis are ongoing. Bioleaching of different sulphidic ores at 5°C with strain SS3 showed that it was 5 to 6 times more efficient than the mesophilic A. ferrooxidansT (Dopson et al., 2007). Chemical analysis of leachate and solids of chalcopyrite and complex Talvivaara ore showed a transient formation of S0 in the reactors (Paper IV, Fig. 1). Considering that accumulation of S0 was accompanied by a halt in metal solubilisation we suspected that the S0 was responsible for mineral passivation.

49

100

90 0.40 Gene expression relative to 80 0.35 70 0.30 60 0.25 50 0.20 40 Substrate (mM) 0.15 30

0.10 rrs 20

10 0.05

0 0.00 0 2 4 6 8 10 12 14 16 Culture volumes

Fig. 16 Expression of A. ferrooxidans SS3 cyoB (black), petA (light grey) and petB (dark grey) genes in tetrathionate and Fe2+ limited continuous culture (all values are expressed as n-fold relative to rrs gene (5 x 10-4)). Lines represent theoretical changes in tetrathionate (dashed) and Fe2+ (solid) concentrations.

Almost no S0 was detected during pyrite leaching as its oxidation occurs via the thiosulphate mechanism (Schippers & Sand, 1999). Expression of the A. ferrooxidans SS3 genes involved in S0 and Fe2+ oxidation (Fig. 16) had the same pattern as for the mesophilic A. ferrooxidansT such that cyoB was up regulated on the RISC compound tetrathionate and petA and petB were up regulated on Fe2+ (Quatrini et al., 2006). When bacteria were cultivated on both substrates, intermediate levels of the expression for all three genes were measured (Paper IV, Fig. 2). The results suggest that transient oxidation of RISCs in strain SS3 can occur alongside with Fe2+ oxidation.

50

4. CONCLUSIONS

A novel bioreactor performed efficiently during the conditions tested for simulating heap bioleaching though it was not as efficient as a stirred reactor.

Oxidation of iron and catalysis of mineral dissolution by a novel psychrotolerant A. ferrooxidans strain SS3 was more efficient compared to A. ferrooxidansT at low temperatures.

S0 was transiently accumulated in low temperature bioleaching of chalcopyrite and chalcopyrite containing ore with psychrotolerant pure and mixed cultures potentially causing passivation of the mineral surface.

Expression of the genes proposed to be responsible for iron and S0 oxidation in A. ferrooxidansT had a similar pattern in strain SS3 suggesting that iron and RISC oxidation in both strains occurs through similar pathways.

During cultivation on mixed substrate (Fe2+ and tetrathionate) a transient expression of corresponding genes proposed to be involved in Fe2+ and tetrathionate oxidation was observed.

Expression of TetH was transcriptionally regulated in a substrate dependent manner. It was proposed that this enzyme belongs to a group of acidophilic dehydrogenases.

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52

5. ACKNOWLEDGMENTS

Six years at the Umea University were happy, interesting and intensive time for me and here comes the list of people who contributed to this feeling. I’m grateful to my supervisors, Börje Lindström and Mark Dopson; to Börje for taking me in this project and to Mark for helping me to get out of it. Both of the guys gave me a lot of freedom so I could go to different conferences and courses, sleep in the morning as much as I like and do whatever experiments. Mark only insisted that whatever experiments should eventually take a form of published articles, which is in fact a sober thinking in the modern science. I’d like to thank all the former and present members of my group: Siv Sääf who for me is an expression of stability, energy, good mood and perfect technical skills. The group can have a different supervisor but Siv is always Siv; Zhanna Bugaytsova, a woman of a strong character and an author of an inspiring article on the tetrathionate hydrolase; Rana Atahr, for her careful teaching and extremely polite instructions; Stefanita Mangold and Dr. Shabir, who arrived recently but it feels like we’ve been friends since long ago. I’d like to acknowledge my great co-workers and teachers Violaine Bonnefoy (for hosting me in her lab and opening my eyes on French food) and David Holmes (for taking my invitation and coming to Umeå from the other side of the world with a talk, lots of ideas and a stone egg), remote co-supervisor Olli Tuovinen for involving me in the psychrotolerant affairs, Jaakko Puhakka for a couple of creative discussions and Anna Kakksonen for FISHy guidelines which were actually quite precise, my teachers from Odessa Mechnikov University Ivanitsa V., Elinskaya N. and Vasilyeva T.. Thanks to Sven Bergström who sorted out my supervisors and sorted out me when it was needed; to my favourite dealer Johnny with his hit “ethanol for dates, methanol for blind dates”; “catch me if you can” guy Marek Wilczynski –for all reinstalling and total upgrading; our administrators Marita and Berit who despite their fragile appearance are real batmen in the tuff world of papers, numbers and signatures; Gunilla Jäger for eternal fight with toxic chemicals. Marionella, gracias, por la limpieza y por todo! Thanks to media department and especially to Agneta for all the important work they do. My best wishes to the office inhabitants of different periods Arunas, Dafne and Karina. Karrrinita, your vitality and sense of humor kept me going when I was a big ball with short legs (kept me rolling I meant). Thanks, to the helpful and jolly neighbours Eleonore, Lisandro, Linda, Odil, Joanna and Teresa, transcriptionally regulated by Vicky Shingler. Big hug to my teaching partners from whom I learned a great deal of molecular genetics, e.g. single malt is a

53 good stuff (Jocke), boiled cucumbers taste very good - no kidding (Bo) and that if you are tiered of sport it is better to do some other kind of sport (Anders). Hello to Chenito, Changchung and Tiago. It is important to discuss here my most pleasant membership in THE JOURNAL CLUB. Thanks to BEU and SNW for the leadership, to Annika and Jorgen for funny presentations, to Carlos for being dangerously good looking, to Klaudia and Anna for “primer extension intensive course”, to Jurate for being a friend anyway, to Stina for blodomloppet, to Ingve and Berit for smart advice, to Edmund for having his critical opinion on everything, to Kaloris for quiet enthusiasm, to Barbro for loud enthusiasm, to Connie for tricky hair styling, to Soni for a beautiful smile, to Hyun-Sook, Tianyan and others for questions, suggestions, interesting cakes and tasty papers. Thanks to the guys who stir the social life and organise non scientific activities in the department: Jenny, Christer, Lelle, Ignas, Sara, Maria, Pär, Mike and Betty; to Gosia for intelligent jokes and Monika Vestling for the vivid descriptions of labour and breast feeding; to Kristoffer Brännström for introduction to HPLC and to Per for corridor attacks. Many thanks to Marija Pinne for sharing so many things and skiing (snowboarding) trips. Hej to the people from up above, Mazan, Margaret, Olga, Gautam, Uwe and Tobias Hainzl. Tobias, may live our crystal venture! Regards to fellows from the other side, Gerhard for his wine and power drive in everything, his students “lazy bone” Marcus and sunny Marco Sani, Maribel for Spanish training and crazy squash, Ana Virel for the cute three language cocktaile and MEGA help, my first lab-mates in Umeå Mantas and Vovka and to Alvaro for unforgettable trip to Castellon. Thanks to the Russian gang, Elena, Asia, Zhenia and Misha for the beautiful sounds of the mother toungue; to Darius for birdwatching; to my ex room-mates Lijana and Aurelija ( we don’t live together anymore but you always have one room each in my heart); to my just exs, Burkhard and Denis - it was nice to meet you guys; to Frederick, the boy- friend of Lijana (who used to be my room-mate) and a perfect cook; I thank my sister Anna and my friends Valentina and Jana who lent me some money for a flight to Umeå otherwise I’d had to walk here. My parents should be acknowledged (Mila and Ivan) cause they are my parents☺ and probably it was them who planted the affection for biology in my head (it was a sheer brain washing!). Finally thanks to Patrik for his love, for letting me love him and for the best present I ever got in my life – our son Sasha. And to Patrik’s parents of course for all this terrific food and babysitting! To all people who read this I wish good health, true love and a lot of scientific achivements!

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