Genomics, Physiology, and Applications of Cold Tolerant

Maria Liljeqvist

Department of Molecular Biology Umeå University, Sweden Umeå 2012

Copyright © Maria Liljeqvist ISBN: 978-91-7459-472-0 Cover: Acidic stream in the Kristineberg mine. Photo: Jan-Eric Sundkvist Elektronisk version tillgänglig på http://umu.diva-portal.org/ Printed by: Department of Chemistry Printing Service, Umeå University Umeå, Sweden 2012

Till min älskade familj, ny som gammal ♥

Table of Contents

Table of Contents 1 Abstract 2 Sammanfattning på Svenska 3 Papers included in this thesis 4 Introduction 5 I. The importance of studying acidophiles 5 II. Fundamentals of 6 Industrial biomining 6 Metal oxidation - the two pathways 7 Acid rock drainage and 9 III. Biomining 10 spp. 10 Chemolithotrophic other than Acidithiobacillus spp. 13 Chemolithotrophic 14 Heterotrophic bacteria and archaea 15 Eukarya 15 Metagenomic analyses of acid mine drainage systems 15 IV. Cold tolerant acidophiles 16 Acidithiobacillus ferrivorans 16 Microorganisms in low temperature acidic environments 17 V. Cold tolerance and adaptation 18 Sensing the cold 19 The cold shock response 21 Temperature-dependent membrane modulation 21 Extracellular polymeric substances in cold adaptation 24 Dealing with ice formation in biological systems 25 Cold adaptation in Acidithiobacillus ferrooxidans 27 VI. oxidation at acidic pH 29 Ferrous oxidation in Acidithiobacillus ferrooxidans 29 Iron oxidation in other acidophilic bacteria 31 Iron oxidation in acidophilic archaea 31 VII. Inorganic compound oxidation in acidophiles 32 Oxidation of thiosulfate and polythionates 32 Oxidation of elemental sulfur and sulfite 34 Models of ISC oxidation in Acidithiobacillus spp. 35 Aims of this thesis 37 Results and Discussion 38 Genome encoded potential of a psychrotolerant (Papers I and III) 38 Substrate utilization by Acidithiobacillus ferrivorans SS3 (Papers II and III) 41 Metagenomic analysis of a low temperature acidic environment (Paper IV) 44 Low temperature ISC removal by psychrotolerant acidophiles (Paper V) 46 Concluding remarks 49 Acknowledgements 50 References 51

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Abstract

Psychrotolerant acidophiles have gained increasing interest because of their importance in biomining operations in environments where the temperature falls well below 10°C during large parts of the year. Acidithiobacillus ferrivorans is the only characterized acidophile with the ability to live a psychrotrophic lifestyle and is able to oxidize ferrous iron and inorganic sulfur compounds at low temperature. The A. ferrivorans SS3 genome sequence mirrors its low temperature chemolithotrophic lifestyle and indicates ecological flexibility. Two enzyme systems for the synthesis of the protective molecule trehalose as well as multiple cold shock proteins suggest its psychrotolerance. In addition to genes coding for ferrous iron and inorganic sulfur compound oxidation enzymes, candidate genes for molecular hydrogen utilization were identified. Also, A. ferrivorans SS3 was suggested to have the ability to switch between fixation and nitrogen uptake. Characterization of ferrous iron and inorganic sulfur compound oxidation during low temperature growth showed that both substrates were efficiently oxidized and revealed the potential of using ferric iron as an alternative electron acceptor. Gene transcript analyses also revealed constitutive expression of genes involved in ferrous iron oxidation and their rapid increase in expression when ferrous iron became available. Growth experiments further suggested ferrous iron was preferred over inorganic sulfur compounds during bioleaching. This phenomenon was especially evident during A. ferrivorans-mediated bioleaching of chalcopyrite as sulfur accumulated and eventually inhibited further leaching. A potential way to alleviate this problem is addition of low temperature, obligate inorganic sulfur compound oxidizing acidophiles. Although, these microorganisms have not been identified, analysis of a cold, acidic biofilm from Kristineberg mine suggested additional psychrotolerant inorganic sulfur compound oxidation oxidizers might be present. Psychrotolerant acidophiles from Kristineberg mine have also been demonstrated to remove inorganic sulfur compounds from mining process water at in situ temperatures. This use of indigenous microorganisms for removal of environmental pollutants is a big step towards greener mining.

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Sammanfattning på Svenska

Syra-älskande bakterier, s.k. acidofiler, är mikroorganismer som lever i ofta metallrika miljöer med väldigt lågt pH. Dom används industriellt i en process kallad biolakning där metaller utvinns från mineral genom den katalyserande förmågan hos dessa mikroorganismer. Acidithiobacillus ferrivorans är en nyligen karaktäriserad acidofil som oxiderar järn- och svavelföreningar vid låga temperaturer, den första och hittills enda av sitt slag. Den har visat sig väldigt användbar vid biolakning i kalla miljöer, såsom i Sveriges norra delar där medeltemperaturen sjunker under 10°C under stora delar av året. En inblick i den genetiska potentialen hos denna mikroorganism avspeglar dess livsstil samt avslöjar en bred ekologisk flexibilitet. Ett flertal gener kan sammankopplas med dess förmåga att överleva vid låga temperaturer, såsom gener för tillverkning av s.k. köldchockproteiner. Vid sidan om gener som kodar för järn- och svaveloxiderande enzymer återfinns potentialen att oxidera vätgas. A. ferrivorans verkar även kunna växla mellan att fixera kvävgas och ta upp kvävejoner ifrån sin omgivande miljö, beroende på den tillgängliga kvävekällan. Vidare karaktärisering av järn- och svaveloxidering vid låga temperaturer visade att båda typer av substrat kan oxideras effektivt men avslöjar även att svaveloxidering kan sammankopplas med reducering av järn istället för den traditionella syrgasen. Analys av genuttryck visade att gener för järnoxidering var kontinuerligt uttryckta och att en signifikant uppreglering sker så snart järn introduceras. Det visade sig även att järn verkar vara det föredragna substratet vid biolakning. Detta fenomen är speciellt uppenbart under biolakningsexperiment med A. ferrivorans då elementärt svavel succesivt ackumuleras och så småningom hindrar vidare frigörelse av metaller. En lösning på detta problem skulle kunna vara att tillsätta köldtoleranta mikroorganismer med enbart svaveloxiderande förmåga. Idag finns inte några sådana bakterier beskrivna, men analys utav en kontinuerligt kyld och sur biofilm isolerad ifrån Kristinebergsgruvan indikerar emellertid förekomsten av svaveloxiderande bakterier. Köldtoleranta acidofiler från Kristinebergsgruvan har även visat sig användbara vid industriellt avlägsnande av svavelföreningar från gruvindustriellt processvatten. Att använda naturligt förekommande mikroorganismer vid rening av miljöfarligt avfall är ett stort steg i riktning mot en grönare gruvindustri.

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Papers included in this thesis

I. Liljeqvist M, Valdes J, Holmes DS & Dopson M. Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS3. J Bacteriol (2011) 193:4304-4305.

II. Kupka D, Liljeqvist M, Nurmi P, Puhakka JA, Tuovinen OH & Dopson M (2009). Oxidation of elemental sulfur, tetrathionate, and ferrous iron by the psychrotolerant Acidithiobacillus strain SS3. Res Microbiol (2009) 160: 767-774.

III. Liljeqvist M, Rzhepishevska O. I. & Dopson M. Gene identification and substrate regulation provides insights into sulfur accumulation during bioleaching with the psychrotolerant Acidithiobacillus ferrivorans. Submitted Manuscript.

IV. Liljeqvist M, Ossandon F, Gonzalez C, Stell A, Valdes J, Holmes D. S. & Dopson M. Metagenomic analysis reveals adaptations to a psychrotrophic lifestyle in a low temperature acid mine drainage stream metagenome. Manuscript.

V. Liljeqvist M, Sundkvist J-E, Saleh A & Dopson M. Low temperature removal of inorganic sulfur compounds from mining process waters. Biotechnol Bioengin (2011) 108: 1251–1259.

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Introduction

I. The importance of studying acidophiles Iron- and sulfur oxidizing and/or reducing microorganisms are important in a variety of biogeochemical cycles due to their ability to catalyze metal solubilization and mineralization (Gadd, 2010). A consequence of sulfide mineral dissolution is the production of highly acidic and metal polluted solutions. The microorganisms that inhabit these environments often utilize oxidation/reduction systems to counteract the toxic effect of metals (Dopson, et al., 2003). The very same systems can also be applied in bioremediation processes of heavy metal contaminated solutions or soils (Takeuchi, et al., 2001, Magnuson, et al., 2010). The ability of acidophiles to catalyze metal solubilization from sulfide minerals is also exploited in the industrial process termed biomining . It is believed that biomining, rather than conventional mining, will be more extensively applied in the future as the availability of high grade ore deposits are becoming more limited (Brierley, 2008). A further consequence of the scarcety of mineral resources is that recycling of metals will become more important. For example, metals can be leached from spent lithium batteries and electronic waste with the help of microorganisms (Mishra, et al., 2008, Pradhan & Kumar, 2012). A big advantage of using microorganisms is that less or no energy is required. Acidophiles are a further source of acid-stable enzymes that have the potential to be used in a variety of industrial processes, such as biofuel production, degradation of polymers, as well as in the textile and food industries (Sharma, et al., 2012). The main benefit with using enzymes from extreme microorganisms, such as acidophiles and , is that they are functional under harsh conditions. One example is the use of α- amylases in the production of syrup from starch. The native pH of starch is around 3.2-3.6 and the current α-amylase used in the industry works optimally at pH 6.5 and requires Ca2+. This means that the pH has to be adjusted before treatment and Ca2+ has to be subsequently removed, resulting in additional costs (Crabb & Shetty, 1999). An acid-stable α- amylase, perhaps without the need for Ca2+, would reduce operational costs. Lithotrophic acidophiles might also provide a link to early earth when oxygen was unavailable and ecosystems were sustained by inorganic reactions of hydrogen-, sulfur-, iron-, and nitrogen compounds (Canfield, et al., 2006). It is believed that the early earth was hot and acidic and continuously impacted by meteorites (Nisbet & Sleep, 2001). In one study, phylogenetic analyses of modern thioredoxins (involved in disulfide reduction) and their reconstructed ancient counterparts reveals that ancient thioredoxins are much more stable than their modern counterparts, are highly resistant to temperature, and are active in acidic conditions (Perez- Jimenez, et al., 2011). Therefore, acid-stable enzymes might provide useful information about early earth enzymology. It has also been demonstrated that acidophilic microorganisms can grow on iron meteorites as the sole

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energy source (Gonzalez-Toril, et al., 2005). Environments inhabiting acidophilic microorganisms, such as Rio Tinto, Spain and Eagle plains, Canada, are also considered to be important terrestrial analogues of Mars (Fairen, et al., 2010).

II. Fundamentals of biomining Biomining is a process whereby microorganisms are utilized to catalyze the extraction of metals from sulfide minerals. It is an excellent complement to traditional smelting when the ore is complex or of low grade. Since traditional smelting is expensive, metal recoveries need to be large enough, or valuable enough to be profitable. Biomining is considerably cheaper and therefore, better suited for low grade and complex ores. Today, biomining operations are carried out worldwide and it is believed that they will be more extensively applied in the future (Brierley, 2008).

Industrial biomining Biomining can be carried out in a number of different ways that are utilized based upon considerations such as the metal(s) to be extracted, their concentration, and the costs. The various types of biomining technologies are:

• In situ leaching utilizes boreholes or hydraulic fracturing of the ore deposit without removing it from the ground, i.e. in situ. An acidic leaching solution is pumped into the ore and the metal-bearing leachate is then collected and processed. Despite the low operational costs, this method is rarely used. • Dump leaching is a simple and cheap method often using mine waste which is piled up and irrigated with an acidic solution. The leaching solution percolates through the mineral, solubilizing metals as it goes, and is collected at the base and further processed. • A more sophisticated version of dump leaching is heap leaching. Here, the mineral ore is crushed, often agglomerated to attach smaller particles to larger ore pieces, and carefully piled onto a waterproof base to form a heap. The heap is then aerated from underneath and irrigated from the top (as for dump bioleaching). Bioheap leaching is utilized in Talvivaara, Finland, for the recovery of nickel, copper, zinc, and cobalt (Fig. 1). Due to the climate conditions in northern Finland, where winter temperatures can reach as low as -30°C, extensive trials preceded the onset of this bioleaching operation. Today, approximately 50,000 tonnes of nickel and approximately 90,000 tonnes of zinc is produced annually (Talvivara annual report, 2011).

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Fig. 1. Photo from the top of the Talvivaara bioheap in Sotkamo, Finland. Photo: Olli Tuovinen.

• Reactor leaching is the most expensive biomining technology and is usually utilized for precious metals, such as gold. Pulverized mineral is mixed with an acidic, leaching solution in large tanks and microorgansisms added. The tanks are stirred and aerated, and have the potential to control variables such as pH, oxygen, and temperature. The optimized conditions results in increased leaching rates. This technique is exploited in the Barberton gold mines, South Africa, which produces approximately 2,800 kg of gold annually (Pan African annual report, 2011).

Metal sulfide oxidation - the two pathways The mechanism of how metals are solubilized from mineral ores has been extensively studied and has been the subject of many discussions. It was previously debated whether metal solubilization was a result of direct enzymatic oxidation of the mineral or if ferric iron contained in the leaching solution oxidizes the metal sulfide bond. The latter proved to be correct and in fact, sulfide minerals can be abiotically leached without the contribution of microorganisms. However, this process is extremely slow. The role of microorganisms is to re-generate the mineral attacking agents, protons and ferric iron (Sand, et al., 1995, Schippers, et al., 1996, Schippers & Sand, 1999). As microbial catalysis increases the rate-limiting step of ferrous iron

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oxidation by 500-fold, the microorganisms considerably enhance the leaching rate (Singer & Stumm, 1970). Metal sulfide minerals are principally leached via two independent mechanisms, depending on the acid solubility of the mineral, which in turn depends on the electron configuration (Schippers, et al., 1996, Schippers & Sand, 1999). If the valence electrons in the metal sulfide bond are derived only from the metal entity, they are acid-insoluble. In contrast, minerals with a contribution of valence electrons from both the metal and the sulfide entity are acid-soluble.

Fig. 2. Representation of the two biomining pathways. Adapted from Schippers and Sand (1999). Abbreviations: IOM = iron-oxidizing microbes; SOM = ISC-oxidizing microbes.

Acid-insoluble metal are leached via the so-called Thiosulfate pathway’ (Fig. 2A). The name derives from thiosulfate being the first inorganic sulfur compound (ISC) intermediate formed. ISC-oxidizing microorganisms then oxidize thiosulfate via tetrathionate, disulfane- monosulfonic acid, and trithionate into sulfuric acid. Further, and most importantly, iron-oxidizing microorganisms oxidize the ferrous iron into ferric iron, which can attack the chemical bond in the mineral and the catalytic cycle is closed. Hence, only iron-oxidizing microorganisms are responsible for catalytic dissolution of the mineral. Examples of metal sulfides that are leached via the thiosulfate pathway are , molybdenite, and tungstenite. In contrast, acid-soluble metal sulfides are leached via the Polysulfide mechanism’ (Fig. 2B). This mechanism is characterized by the formation of

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hydrogen sulfide, which is rapidly oxidized into polysulfides, which decompose into elemental sulfur in acidic solutions. Hence, leaching of acid- soluble metal sulfides is characterized by the accumulation of large amounts of elemental sulfur. The ferrous iron and the elemental sulfur are oxidized by iron- and ISC-oxidizing microbes into ferric iron and sulfuric acid, respectively. Since these types of minerals are acid-soluble the protons produced by ISC oxidation can also attack the mineral. Examples of minerals that are leached via the polysulfide mechanism are chalcopyrite, sphalerite, and galena. (Schippers, et al., 1996, Schippers & Sand, 1999).

Acid rock drainage and acid mine drainage As metal sulfide deposits are subjected to weathering, metals and ISCs are leached from the mineral. The presence of microorganisms further catalyzes this process, generating highly acidic, metal rich solutions, termed ‘acid rock drainage’ (ARD). However, within the mining industry the very same phenomenon has become an even greater problem. Through the action of mining, mineral deposits are exposed to air and water and the microorganisms weather the mineral, thereby generating so called ‘acid mine drainage’ (Thamdrup, et al. 1993). One of the most severe AMD sites in the world is Richmond mine located at Iron Mountain, California, USA where the pH of the AMD discharge has been measured at below 0 (Nordstrom & Alpers, 1999). This area was heavily mined for approximately 100 years during the 19th and 20th centuries mainly for gold, silver, copper, iron, and zinc. It is estimated that approximately 11.5 million tons of ore was removed from the original deposit that resulted in one of the largest AMD sites in the world. Unfortunately, the acidic effluent leaked into the Sacramento river and was responsible for significant fish deaths. The mine is no longer in use but subject to an extensive remediation process (Alpers, et al., 1992, Druschel, et al., 2004). Due to greater environmental awareness, mining companies are now subjected to tougher restrictions and are challenged to work towards ‘greener mining’. In October 2003, the Swedish environmental protection agency released a report stating that in year 2010, only 5% of Swedish lakes and 15% of rivers and streams should be affected by anthropogenic acidification (Swedish environmental protection agency, 2003). That goal was considered reached in 2008 and the main challenge today is to maintain this effort (Swedish environmental protection agency, 2011). As mentioned above, environmental acidification derived from mining activities is mainly due to the oxidation of ISCs derived from the mineral. During mineral processing these ISCs can be present in process waters and effluents and such liquids need to be ‘de-sulfurized’ before release in order to avoid downstream acidification of recipient waters. Present abiotic methods involve the use of hazardous chemicals with high operational costs (Ikumapayi, et al., 2009, Lagace, 2010). Alternative strategies must be considered and potential biotechnological solutions investigated.

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III. Biomining microorganisms All biomining microorganisms are acidophiles; a diverse group of microorganisms that thrive at low pH and in some cases are the direct cause of the environmental acidification. Acidophiles can be found in all domains of life; bacteria, archaea, and eukarya, and in a wide range of habitats including AMD sites. Some acidophiles demand a low pH to be able to survive, generally referred to as true acidophiles, while others can grow in a broader range of pH values (Johnson, 1998). The most studied acidophiles are iron- and/or ISC-oxidizing prokaryotes, mainly because of their importance in biomining. However, due to more sophisticated methods in molecular ecology, it has been discovered that non iron- and ISC-oxidizing prokaryotes are also quite abundant and these have gained more interest. Besides the biomining prokaryotes, which are still heavily studied, more focus has been put on acidophilic and their role in the establishment of AMD environments (Bacelar-Nicolau & Johnson, 1999). Further, it is becoming more evident that acidophilic are well established in AMD environments (Amaral Zettler, et al., 2002, Baker, et al., 2004, Baker, et al., 2009), even though they are considerably less studied.

Acidithiobacillus spp. Bacteria belonging to the genus Acidithiobacillus are by far the most studied acidophiles. They are Gram-negative , previously thought to be a member of the Gammaproteobacteria but have recently been suggested to have arisen after the divergence of Alphaproteobacteria but before the Beta/Gammaproteobacteria split (Williams, et al., 2010). Acidithiobacillus spp. are subdivided into 5 distinct species: Acidithiobacillus albertensis, Acidithiobacillus caldus, Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans. Each species consists of several strains with various characteristics. A. ferrooxidans is the most studied acidophile and is often referred to as the model of the acidophile community. The species consists of multiple strains with great variability and it has been questioned whether these strains actually comprise multiple species (Harrison, 1982, Harrison, 1984, Karavaiko, 2003, Ni, et al., 2007, Ni, et al., 2008, Ni, et al., 2008). For example, some of the species classified as A. ferrooxidans should probably be more accurately classified as A. ferrivorans (Schippers, 2007, Amouric, et al., 2011). A. ferrooxidans obtains carbon autotrophically and oxidizes various ISCs, ferrous iron, and molecular hydrogen. In addition it has been shown to be able to fix atmospheric nitrogen (Mackintosh, 1977, Norris, et al., 1995). Old data suggests that A. ferrooxidans is a facultative however; it is nowadays believed that these early reports are the result of contamination by heterotrophic acidophiles (Harrison, 1984). Two strains of A. ferrooxidans have been sequenced; A. ferrooxidans ATCC23270 (Valdes, et al., 2008) and A. ferrooxidans ATCC 53993 (NC_011206, direct submission). An additional strain, A. ferrooxidans DSM 16786, has been patented and sequenced by Biosigma SA, Chile, but the sequence is not publicly available (Levican, et al., 2008).

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Table 1. Examples of acidophilic bacteria and their main characteristics. Species S0 Fe2+ Carbon Temp. Proteobacteria Acidicaldus organivorans X - Hetero mT Acidiphilium acidophilum X - Hetero M Acidiphilium angustum - - Hetero ? Acidiphilium cryptum X - Hetero M Acidiphilium multivorum - - Hetero M Acidiphilium organivorum - - Hetero M, Tt Acidiphilium rubrum X - Hetero ? Acidisphaera rubrifaciens - - Hetero M Acidithiobacillus albertensis X - Auto M Acidithiobacillus caldus X - Mixo mT Acidithiobacillus ferrivorans X X Auto M, Pt Acidithiobacillus ferrooxidans X X Auto M Acidithiobacillus thiooxidans X - Auto M Acidocella aminolytica - - Hetero M Acidocella facilis - - Hetero M Acidocella mathanolica - - Hetero M ‘Thiobacillus plumbophilus’ X - Auto M ‘Thiobacillus prosperus’ X X Auto M Thiomonas cuprina X - Mixo M Nitrospira Leptospirillum ferrooxidans - X Auto M Leptospirillum ferriphilum - X Auto M, Tt ‘Leptospirillum ferrodiazotrophum’ ? X Auto M Leptospirillum thermoferrooxidans - X Auto mT Acidobacterium capsulatum - - Hetero M Acidimicrobium ferrooxidans - X Mixo mT ‘Ferrimicrobium acidiphilum’ - X Hetero M Firmicutes Alicyclobacillus acidiphilus ? - Hetero mT Alicyclobacillus acidocaldarius - - Hetero T Alicyclobacillus acidoterrestris - - Hetero mT Alicyclobacillus cycloheptanicus - - Hetero mT Alicyclobacillus disulfidooxidans X X Mixo M, Pt Alicyclobacillus herbarius ? - Hetero T Alicyclobacillus hesperidum ? - Hetero mT Alicyclobacillus promorum ? - Hetero mT Alicyclobacillus sendaiensis ? - Hetero mT Alicyclobacillus tolerans X X Mixo M Alicyclobacillus vulcanalis - - Hetero mT ‘Caldibacillus ferrivorus’ X X Mixo mT Sulfobacillus acidophilus X X Mixo mT ‘Sulfobacillus montserratensis’ X X Mixo M Sulfobacillus sibiricus X X Mixo mT Sulfobacillus thermosulfidooxidans X X Mixo mT Sulfobacillus thermotolerans X X Mixo M, Tt Abbreviations: Auto=autotroph; Hetero=; Mixo=mixotroph or facultative autotroph; M=; mT=moderate ; Pt=psychrotolerant; Tt=thermotolerant; T=thermophile; X=positive; -=negative; ?=no data available; S0=elemental sulfur; Fe2+=ferrous iron

A. ferrivorans is the newest member of the Acidithiobacillus genus and was previously considered to be a cold-tolerant strain of A. ferrooxidans (Hallberg, et al., 2010). It grows in the range of 5°-37°C and is therefore the only species in the genus shown to be able to grow below 10°C. It is similar to

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A. ferrooxidans and only differs in a small number of aspects, mainly the temperature range of growth (Kupka, et al., 2007, Hallberg, et al., 2010). A. ferrooxidans and A. ferrivorans are the only species in the genus able to oxidize ferrous iron.

Table 2. Examples of acidophilic archaea and main characteristics. Species S0 Fe2+ Carbon Temp. Euryarchaeota ‘ acidarmanus’ - X Mixo M, Tt Ferroplasma acidiphilum - X Mixo M ‘Ferroplasma cupricumulans’ X X Mixo mT ‘Ferroplasma acidarmanus’ - X Mixo M, Tt Picrophilus oshimae - - Hetero T Picrophilus torridus - - Hetero T Thermoplasma acidophilum - - Hetero mT Thermoplasma volcanicum - - Hetero mT Acidianus ambivalens X - Hetero T Acidianus brierleyi X X Mixo T Acidianus Infernus X X Auto T hakonensis X ? Mixo T Metallosphaera prunae X X Mixo T Metallosphaera sedula X X Mixo T acidocaldarius - - Hetero T Sulfolobus metallicus X X Auto T Sulfolobus shibatae X - Hetero T Sulfolobus solfataricus - - Hetero T Sulfolobus tokodaii X - Hetero T Sulfolobus yangmingensis X ? Mixo T Sulfurisphaera ohwakuensis X - Hetero T Sulfurococcus mirabilis X X Mixo T Sulfurococcus yellowstonensis X X Mixo T Abbreviations: Auto=autotroph; Hetero=heterotroph; Mixo=mixotroph or facultative autotroph; M=mesophile; mT=moderate thermophile; Pt=psychrotolerant; Tt=thermotolerant; T=thermophile; X=positive; -=negative; ?=no data available; S0=elemental sulfur; Fe2+=ferrous iron

A. caldus is the only moderately thermophilic species within the genus and, depending on the strain, grows in the range of about 32°-55°C with optimal growth at around 45°C (Norris, et al., 1986, Hallberg & Lindström, 1994, Hallberg, et al., 1996). It oxidizes various ISCs as well as molecular hydrogen and can fix atmospheric carbon. It has been shown to grow mixotrophically using yeast extract or glucose with higher growth yields than with CO2 alone (Norris, et al., 1986, Hallberg & Lindström, 1994). The genome sequence of the type strain, A. caldus ATCC 51756, was published in 2009 (Valdes, et al., 2009) and another strain, A. caldus SM-1, was published in 2011 (You, et al., 2011). A. albertensis consists of only 2 reported strains: A. albertensis ATCC 35403 (Bryant, et al., 1983) and BY-05 (Xia, et al., 2007), and is the least studied species in the genus. It oxidizes various ISCs and fixes carbon from the atmosphere. Organic substrates such as glucose, peptone, and yeast extract do not support growth of strain BY-05. It has an optimum growth temperature of about 30°C and is able to grow between 10°-35°C, although lower temperatures have not been tested (Xia, et al., 2007). Unfortunately, the original isolate has been lost (Kelly & Wood, 2000).

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A. thiooxidans is physiologically similar to A. albertensis but is regarded as a separate species mainly because of the absence of the dense glycocalyx present in A. albertensis (Bryant, et al., 1983). In 2011, the genome of A. thiooxidans ATCC 19377 was sequenced and published (Valdes, et al., 2011).

Chemolithotrophic bacteria other than Acidithiobacillus spp. Besides Acidithiobacillus spp., there is a wide range of acidophilic bacteria that can oxidize iron and/or ISCs and therefore are of importance for the bioleaching process. They are not as well studied as A. ferrooxidans, but more data is becoming available which is extremely important to fully understand the true potential of the biomining and acidophile communities. Thiobacillus prosperus’ is a Gram-negative motile rod (Huber & Stetter, 1989) that is most probably wrongly classified. The genus Thiobacillus belongs to the β-subclass of proteobacteria and T. prosperus groups together with the γ-subclass (Goebel, et al., 2000, Kelly & Wood, 2000). In either case, it is of great importance because of its high tolerance to salt. Some strains can grow in up to 6% NaCl (Huber & Stetter, 1989, Nicolle, et al., 2009) and some even require salt in order to grow (Nicolle, et al., 2009). Most other acidophilic species are highly sensitive to salt (Baker-Austin & Dopson, 2007, Zammit, et al., 2012). This poses a special problem for biomining operations in some regions of the world, e.g. Australia, where the fresh water supply is limited. Leptospirillum spp. are a group of Gram-negative spirilla-shaped bacteria belonging to the Nitrospirae phylum. They are autotrophic ferrous iron oxidizers and are completely unable to oxidize any ISCs. The genus comprises five species; Leptospirillum ferrooxidans, Leptospirillum ferriphilum, Leptospirillum rubarum, Leptospirillum ferrodiazotrophum, and Leptospirillum thermoferrooxidans. They are mesophilic to moderately thermophilic (Schippers, 2007). Leptospirillum spp. are considered important because they are able to withstand higher redox conditions than A. ferrooxidans and can continue to oxidize ferrous iron even at high redox potential, which is of importance for tank biomining operations (Rawlings, et al., 1999). L. ferriphilum is a thermotolerant mesophile that was shown to dominate tank bioleaching operations at 35-50°C. L. thermoferrooxidans is the only species in the genus described as moderately thermophilic. Unfortunately, the original isolate has been lost (Golovacheva, et al., 1992, Coram & Rawlings, 2002). Thiobacillus plumbophilus (Drobner, et al., 1992) and Thiomonas cuprina (formerly Thiobacillus cuprinus) (Huber & Stetter, 1990, Moreira & Amils, 1997) are somewhat special because they are unable to oxidize ferrous iron and most ISCs (Th. cuprina can oxidize elemental sulfur) except hydrogen sulfide (H2S). Still, they are able to catalyze the dissolution of galena (PbS) and other metal sulfides in the case of Th. cuprina. Th. cuprina is also able to oxidize various organic substrates. Unfortunately, very little research has been made on these two species. Other than the Gram-negative bacteria, there are a variety of Gram- positive bacteria able to oxidize ferrous iron and ISCs under acidic conditions. Acidimicrobium ferrooxidans (Clark & Norris, 1996) and

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Ferrimicrobium acidiphilum (Johnson, et al., 2009) are two closely related ferrous iron oxidizing Actinobacteria. Am. ferrooxidans is the only species in its genus and its genome was sequenced in 2009 (Clum, et al., 2009). Several species from the genus Sulfobacillus have been isolated from various mine sites and they are mostly moderately thermophilic. They usually oxidize ferrous iron and/or ISCs in the presence of yeast extract and some species have been shown to grow via anaerobic ferric iron reduction (Schippers, 2007). Sulfobacillus acidophilus is the only species in the genus that has been sequenced: strain TPY (Li, et al., 2011) and strain DSM 10332 (NC_016884, direct submission).

Chemolithotrophic archaea Metal solubilizing archaea are either ferrous iron oxidizing Euryarchaeota from the family Ferroplasmaceae or thermophilic iron and ISC oxidizing Crenarchaeota from the family . So far, the genus Ferroplasma consists of three characterized species; Ferroplasma acidiphilum (Golyshina, et al., 2000), 'Ferroplasma acidarmanus' (Dopson, et al., 2004) and Ferroplasma thermophilum (Zhou, et al., 2008). They are extremely acidophilic, with a pH optimum usually below 2.0, and are able to grow as low as pH 0 (Dopson, et al., 2004). Usually, they grow mixotrophically on ferrous iron and an organic substrate, such as yeast extract, even though F. acidiphilum has the ability to grow autotrophically (Golyshina, et al., 2000). Acidiplasma cupricumulans (formerly F. cupricumulans) was originally described in 2006 (Hawkes, et al., 2006) and reclassified in 2009 together with the announcement of a new member of the family; Acidiplasma aeolicum (Golyshina, et al., 2009). Ap. cupricumulans and Ap. aeolicum grow mixotrophically on ferrous iron and yeast extract, but only Ap. cupricumulans has been shown to solubilize metals from sulfide minerals (Hawkes, et al., 2006). Iron and ISC oxidizing archaea within the Sulfolobaceae are all thermophilic with growth optima between 65-90°C. These are especially advantageous in bioleaching operations at higher temperatures where metal solubilization is usually more efficient. Several species have been described and they belong to four different genera; Acidianus spp., Metallosphaera spp., Sulfolobus spp., and Sulfurococcus spp. Some species only catalyze metal dissolution weakly, whereas some isolates, i.e. Acidianus brierleyi, Sulfolobus metallicus, and Metallosphaera spp., are efficient biomining species. Sulfolobus acidocaldarius and Sulfolobus solfataricus have been reported to solubilize metals from metal sulfide ores in some studies (Kargi & Robinson, 1985, Tobita, et al., 1994, Vitaya, et al., 1994) but in another study demonstrated not to have that ability (Larsson, et al., 1990). Species within Sulfolobus and Acidianus have been shown to host viruses and this topic has gained increasing interest. Archaeal viruses seem to be exclusively DNA viruses, mainly dsDNA viruses with only one being a ssDNA virus (Mochizuki, et al., 2012). However, most of the archaeal viruses found so far belong to novel viral families and have shown to have exceptional morphologies that have previously not been observed. Sulfolobus spp. have shown to host fusiform, droplet-shaped, linear, and spherical viruses of

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various viral families while Acidianus spp. host fusiform, bottle-shaped, and linear viruses (Prangishvili, et al., 2006).

Heterotrophic bacteria and archaea Obligately heterotrophic microorganisms do not directly assist in the solubilization of metals from sulfide mineral ores. However, they are readily found in close association with the chemolithotrophs in acidic mining environments, and in some cases have been difficult to remove from cultures (Harrison, 1984). They are considered important for the bioleaching activity because they remove inhibitory organic compounds produced by the (Nancucheo & Johnson, 2010). Acidiphilium spp. are heterotrophic bacteria, even though some species have the ability to oxidize ISCs and reduce ferric iron (Hallberg & Johnson, 2001). Ferric iron reduction is considered to be another important contribution from heterotrophic bacteria, since it re-delivers ferrous iron to the autotrophs (Schippers, 2007). Acidiphilium cryptum JF-5 also reduces chromate, which might have implications for bioremediation of metal contaminated industrial liquids (Magnuson, et al., 2010). Ferric iron reduction has also been shown for the moderate thermophile Acidicaldus organivorans (Johnson, et al., 2006). Actually, ferric iron reduction seems to be a widespread phenomenon among heterotrophic acidophiles. Several species from the genera Acidiphilium, Acidocella, and Acidobacterium have been shown to catalyze the dissimilatory reduction of schwertmannite, a ferric iron mineral (Coupland & Johnson, 2008). There are also a number of bacteria and archaea that oxidize ferrous iron and ISCs but where metal solubilization activity has not been demonstrated.

Eukarya The presence of eukaryotic microorganisms in acidic mine polluted environments has been known for several years. However, little is known about their function and contribution in these environments and few have been properly characterized. Studies on the eukaryotic profiles in Rio Tinto, Spain (Amaral Zettler, et al., 2002), and at Iron Mountain, California (Baker, et al., 2004, Baker, et al., 2009), show quite a vast diversity of different eukaryotic microbes. In fact, the eukaryotic diversity in Rio Tinto far exceeds that of the prokaryotic (Amaral Zettler, et al., 2002). It has been shown that protists graze on acidophilic bacteria in culture (McGinness & Johnson, 1992, Johnson & Rang, 1993) and this might be a widespread phenomenon among acidophilic eukaryotes. It is also likely that they play a crucial role in carbon cycling in these nutrient poor environments (Baker, et al., 2009). Interestingly, neutrophilic bacteria have been found as endosymbionts in acidophilic protists (Baker, et al., 2003).

Metagenomic analyses of acid mine drainage systems Metagenomics is the isolation, sequencing, and characterization of community DNA in an environment and allows for elucidation of both the phylogeny and metabolic potential of mixed populations (Handelsman,

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2004). The sensitivity of the technique allows for identification of less abundant species that may otherwise go unnoticed. This technique has been applied to acidic environments including an arsenic rich ecosystem in Carnoulès, France (Bertin, et al.), a biofilm cave in the Frasassi cave system, Italy (Jones, et al.), and a low pH acid mine drainage system at Iron Mountain, California (Tyson, et al., Dick, et al., Goltsman, et al.). These studies evaluated the microbial community and were able to reconstruct partial or near full genomes of the most abundant species. To gain a deeper insight into the metabolic potential of the community, metagenomics can be combined with metaproteomic- and metatranscriptomic techniques. While genomics give information about the metabolic potential of a sample, proteomics (protein) and transcriptomics (mRNA) give information about active metabolic pathways. A combination of these techniques applied to an environmental sample is a very powerful tool as it reveals how the community responds to changing environmental conditions (Goltsman, et al., Bertin, et al.). This strategy was applied to Carnoulès Oxydizing Wetland, an arsenic-rich acid mine drainage system in France (Bertin, et al.). They found that this system was dominated by seven bacterial strains, whose genomes they reconstructed. Five of the strains represent yet uncultivated bacteria, whereby two represent a novel bacterial phylum, called ‘Candidatus Fodinabacter communificans’. Combined with metaproteomics they identified proteins involved in arsenic resistance, fixation, and iron- and ISC oxidation systems, among others. They were also able to assign each of these pathways to specific strains allowing for the construction of a metabolic model system of this particular ecosystem. Understanding of how an ecosystem functions can provide important information about how to design an efficient bioremediation system for that particular environment (Bertin, et al., 2011).

IV. Cold tolerant acidophiles Low ambient temperature might limit biotechnological operations in cold environments, such as biomining at high altitudes or at low or high latitudes. This would decrease the kinetic rates of iron- and ISC oxidation by mesophilic microorganisms, rendering the whole process uneconomical. Therefore, efforts have been made to find microorganisms able to efficiently catalyze these processes at low temperature. To date, there is no evidence of true psychrophilic acidophiles, however, there are a number of studies where cold tolerant acidophiles have been identified and this area is an expanding topic.

Acidithiobacillus ferrivorans A. ferrivoransT N0-37 (DSM22755) was isolated from an abandoned Norwegian copper mine in 2001 (Johnson, et al., 2001) but wasn’t properly described until 2009 (Hallberg, et al., 2010). Just like A. ferrooxidans, A. ferrivorans is an obligate autotroph with the potential to fix atmospheric nitrogen. It oxidizes iron and ISCs aerobically and is able to respire ferric iron with elemental sulfur anaerobically. It grows in the range of 4°-37°C with optimum growth at 27°-32°C and is therefore considered

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psychrotolerant (Hallberg, et al., 2010). The same study further classified the strains CF27 (Blake & Johnson, 2000), Peru6, and OP14 (Ghauri, et al., 2007) as A. ferrivorans. Phylogenetic studies, analyzing a number of different genes (rrs, atpD, nifH, recA, rusA/B, hip/iro and their concatenation) shows that psychrotolerant A. ferrivorans strains cluster together, distinct from mesophilic Acidithiobacillus strains (Amouric, et al., 2011). This further supports their re-classification as a separate species. A. ferrivorans SS3 was isolated from Norilsk, Siberia and was originally described as a psychrotolerant strain of A. ferrooxidans. It oxidizes ferrous iron and tetrathionate as well as catalyzing the leaching of pyrite/arsenopyrite and chalcopyrite at 4°C considerably faster than A. ferrooxidansT (Dopson, et al., 2007, Kupka, et al., 2007). Two additional strains were isolated in the same study; SS5 from Norilsk, Siberia and SK5 from Kristineberg, Sweden. Together with SS3, the three isolates are all phylogenetically similar to the strains that were classified as A. ferrivorans in 2009 (Kupka, et al., 2007, Hallberg, et al., 2010). A. ferrivorans has also been isolated from Cae Coch pyrite mine, Wales, UK (Kimura, et al., 2011) and from a Codelco-Andina old pond, Andes, Chile (Escobar, et al., 2010). Further, if the rrs gene sequence of A. ferrivoransT is blasted against all available Acidithiobacillus sequences, a number of hits with 98-100% identity, denoted as ‘A. ferrooxidans’ or ‘uncharacterized Acidithiobacillus clone’, appears. All this taken together suggests that A. ferrivorans is far more widespread than it is credited for.

Microorganisms in low temperature acidic environments Low temperature, acidic environments are extremely poorly characterized. One study investigated the microbial diversity in a low temperature abandoned underground pyrite mine (Kimura, et al., 2011). The sample site is located in the lower reaches of the Cae Coch mine, Conwy valley, Wales, that has had a stable temperature of about 8.5°C for the last 30 years. Biological samples were taken from seven different locations and they all proved to be extremely acidic (pH <2.5) and showed a limited microbial diversity. Active microbial communities inside water droplets from the mine roof contained A. ferrivorans and L. ferrooxidans in a 19:1 ratio. Microbial stalactites were dominated by Betaproteobacteria, in particular ‘Ferrovum myxofaciens’ but also a Gallionella-related clone. The stalactites also contain small numbers of actively growing Alpha- and Gammaproteobacteria. A more detailed analysis showed that the Alphaproteobacteria included Acidiphilium-like bacteria and a Sphingomonas sp.. ‘Fm. myxofaciens’ was also the most abundant, actively growing species in the acid streamers. However, from this sample they were also able to isolate an obligatory acidophilic bacterium with 99.3% identity to the neutrophilic Sphingomonas echnoidesT. Samples from the mine water pool mainly contained Gammaproteobacteria and Actinobacteria while the bottom of the pool also contained an uncultured euryarchaeote (Kimura, et al., 2011). The listed species were identified by FISH or in some cases by colony isolation suggesting they constitute the active flora and therefore are most likely to be psychrotrophic (although low temperature growth experiments were not

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carried out to confirm this). It is also notable that only iron oxidizers and no ISC oxidizers were identified (Kimura, et al., 2011). (Acidi-)thiobacillus-like species were observed in sulfidic ore samples obtained from the Citronen Fjord area, Greenland. Further experiments showed both autotrophic and heterotrophic activities, as well as iron solubilization, in enrichment cultures below 10°C, with optimum activity around 20°-25°C (Langdahl & Ingvorsen, 1997). Similarly, acidophilic iron- oxidizing bacteria were enriched at 4°-37°C from water samples taken from Keretti copper mine, Finland. The 4°C enrichment had a generation time of 72h whereas a 19°C enrichment had a generation time of only 56h when grown at 4°C (Ahonen & Tuovinen, 1989). The same samples also oxidized ISCs at <10°C (Ahonen & Tuovinen, 1990). Even though these studies show microbial activity at low temperatures, identification of the responsible species was not carried out. The ISC-oxidizer Alicyclobacillus disulfidooxidans (formerly Sulfobacillus disulfidooxidans) grows at 4°C with an optimum growth temperature of 35°C (Dufresne, et al., 1996, Karavaiko, et al., 2005). Unfortunately, the original culture was most likely impure and no further studies have been carried out (Schippers, 2007). It has also been shown that some of the “mesophilic” strains of A. ferrooxidans group together with the psychrotolerant strains of A. ferrivorans indicating their possible misclassification (Escobar, et al., 2010, Mykytczuk, et al., 2010, Mykytczuk, et al., 2011). Also, many older reports classified acidophilic psychrotolerant isolates as A. ferrooxidans based on appearance and biochemical studies, although no phylogenetic analysis was carried out (Ferroni, et al., 1986, Berthelot, et al., 1993, Leduc, et al., 1993). It is also worth noting that A. ferrooxidans strains have great variability (Harrison, 1982, Harrison, 1984, Karavaiko, 2003, Ni, et al., 2007, Ni, et al., 2008, Ni, et al., 2008) and it is suggested that some isolates should be re- classified into distinct species (Kelly & Wood, 2000).

V. Cold tolerance and adaptation Life at low temperature faces a variety of challenges due to a decrease in the energy state of matter at lower temperatures. This means that all physical and chemical movements are slowed down. For microorganisms, a lowering of the temperature has several consequences: i. a reduction of all biochemical reaction and dissolution rates; ii. an increase in the viscosity of aqueous environments; iii. decreased flexibility of macromolecular biocomplexes; and iv. increased concentrations of reactive oxygen species. These are basic challenges that need to be overcome in order to grow and survive in the cold. Microbial adaptation to low temperature has been fairly well studied and a number of different cold-adapted microorganisms have been discovered. These microorganisms are usually categorized into two classes: the true , growing optimally at temperatures below 15°C, and psychrotolerant microorganisms that preferentially grow at higher temperatures but are also able to grow at temperatures below 10°C. A number of mechanisms for adaptation to cold environments have been proposed and even though they vary from organism to organism, some general trends are observed.

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Sensing the cold In order to make adjustments in response to a decrease in temperature the cell needs to sense that change. There are a number of ways of how can sense a decrease in temperature:

• Through alterations in membrane fluidity. • Through alterations in DNA conformation. • Through alterations in RNA conformation. • Through alterations in translation. • Through alterations in protein conformation.

Sensing cold by changes in membrane fluidity. Upon temperature downshift, biological membranes exhibit a decrease in membrane fluidity, thereby causing a rigid and stiff membrane. Consequently, this might alter the conformation of membrane proteins making them dysfunctional. However, an alteration in membrane protein conformation is one of the ways in which microorganisms sense a temperature decrease. Instead of disabling the protein function, a cold sensor, inactive under normal conditions, gains function through this rigidification and becomes activated. By reducing membrane fluidity by artificial methods (instead of temperature) it has been shown that cold-inducible genes are activated, suggesting that the fluidity state of the membrane is the primary signal for cold sensing (Vigh, et al., 1993, Inaba, et al., 2003). In the cyanobacterium Synechocystis sp. PCC6803, cold sensing appears to be perceived by the membrane-bound two-component signal transduction system Hik33/Rer1, thereby activating downstream target genes. However, the picture is slightly more complicated as the Hik19 soluble histidine kinase and another response regulator, Rer26, were also found to be involved in cold perception. Further, Rer1 functions downstream of both Hik33 and Hik19. These two- component systems affect some cold-inducible genes but not all, suggesting crosstalk between several pathways, some still undiscovered (Suzuki, et al., 2000, Suzuki, et al., 2000, Browse & Xin, 2001, Suzuki, et al., 2001, Mikami, et al., 2002, Inaba, et al., 2003). Another membrane-associated cold sensing system is present in Bacillus subtilis. The membrane-associated two-component signal transduction system DesK/DesR senses the temperature downshift through the decreased fluidity state of the membrane, activating genes responsible for desaturation of the membrane. A desaturation of membrane lipids in turn increases the membrane fluidity, thereby constituting a sophisticated regulatory loop for sensing and responding to low temperature (Aguilar, et al., 2001, Mansilla, et al., 2003, Cybulski, et al., 2004, Mansilla & de Mendoza, 2005). Sensing cold by conformational changes in DNA. A decrease in temperature influences DNA packing and causes negative supercoiling and nucleoid compaction in B. subtilis (Grau, et al., 1994, Weber, et al., 2001). However, it is unknown whether temperature directly affects DNA conformation or indirectly by affecting the activity or synthesis of DNA binding proteins. Several DNA-binding proteins are up-regulated during

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Escherichia coli cold shock, such as DNA gyrase (Jones, et al., 1992), histone-like protein H-NS (La Teana, et al., 1991) and HU (Wada, et al., 1988, Giangrossi, et al., 2002), and topoisomerase I (Woldringh, et al., 1995). The topoisomerase type I and II (DNA gyrase) and H-NS are regulators of DNA supercoiling (Drlica, 1992, Tse-Dinh, et al., 1997, Williams & Rimsky, 1997, Dorman, et al., 1999) and it has been demonstrated that cold-inducible gene expression is inhibited when negative supercoiling is inhibited (Prakash, et al., 2009). Direct or indirect temperature-dependent DNA conformational changes have the potential to serve as an excellent thermo-sensor and the exact mechanism remains to be elucidated. Sensing cold through mRNA stability and protein synthesis. RNA molecules have a tendency to form complex secondary and tertiary structures (Andersen & Delihas, 1990) and therefore, have the potential to be influenced and modified by temperature. It is known that several pathogens utilize temperature-dependent differences in mRNA structure, so called thermosensors, to control translation. Expression of virulence genes in Yersinia pestis and Listeria monocytogenes are thermally controlled by the proteins LcrF and PrfA, respectively. Their mRNA have 5’-UTRs (un- translated regions) which form a secondary structure that masks the ribosomal binding site at low temperature but opens up for translation at 37°C (Hoe & Goguen, 1993, Johansson, et al., 2002). A similar mechanism has been demonstrated for the E. coli cold shock protein A (CspA). CspA is highly induced upon cold shock and reaches up to 2% of the total protein concentration (Brandi, et al., 1996). It has previously been shown that CspA induction is mainly regulated by post-transcriptional mechanisms. CspA mRNA is drastically stabilized upon cold shock and is selectively translated by the ribosome (translational bias) (Brandi, et al., 1996, Goldenberg, et al., 1996). However, it wasn’t until 2010 that the actual mechanism behind its efficient translation during cold shock was revealed. Giuliodori, et al. (2010) showed that CspA mRNA forms a secondary structure at 37°C that sequesters both the ribosomal start site and the AUG start codon. However, at 10°C CspA mRNA undergoes conformational changes such that the ribosomal binding site is revealed. It has also been shown that CspA mRNA binds multiple copies of the RNA-binding protein Hfq at its 3’-end in vitro. This might have implications on the accessibility of CspA mRNA to RNAses during cold shock (Hankins, et al., 2010). It has also been suggested that the ribosome itself senses temperature variations by changes in translational efficiency. When E. coli is subjected to antibiotics targeting the ribosome it induces a response similar to the heat or cold shock response, depending on the antibiotic. Further, the stress alarmone (p)ppGpp, was shown to decrease upon temperature downshift (VanBogelen & Neidhardt, 1990). Subsequent analysis showed that high levels of (p)ppGpp decrease cold shock induction whereas the absence of (p)ppGpp increases the cold shock induction upon temperature downshift. (p)ppGpp is synthesized by RelA which is found closely associated with the ribosome (Jones, et al., 1992). However, cold sensing via the ribosome is otherwise poorly understood.

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Cold sensing by alterations in protein conformation. Temperature-induced conformational changes in protein have been observed in Salmonella typhimurium. TlpA was shown to oligomerize and bind DNA at 28°C, thereby suppressing its own expression. But when the temperature is increased, TlpA oligomerization decreases and the target gene is activated. The function of TlpA is unknown but some relation to virulence is suggested due to its location on virulence plasmids. A motif present in the tlpA promotor has been observed in other virulence-related genes but so far there is no evidence of TlpA regulating these genes (Gulig, et al., 1993, Hurme, et al., 1996, Hurme, et al., 1997). The evidence of ‘protein thermometers’ has also been shown in Li. monocytogenes, where flagellar motility is observed at ambient temperatures (26°-28°C) but repressed at 37°C. Two genes control synthesis of flagellum genes: the transcriptional repressor MogR and the anti- repressor GmaR. GmaR and MogR form a stable complex under ambient temperatures, allowing transcription of flagellum genes. When the temperature increases, GmaR changes conformation which destabilizes the GmaR:MogR complex, releasing MogR to fulfill its function as a transcriptional repressor of the flagellum genes (Kamp & Higgins, 2011).

The cold shock response When living organisms encounter a temperature decrease, they usually enter a physiological state referred to as “cold shock”. This condition usually, but not in all cases, means that synthesis of most cellular proteins are inhibited and the cells temporarily enter a growth arrest. The cold shock is a direct result of impaired cellular functions caused by the decrease in temperature, such as: i. loss of membrane fluidity and function; ii. negative supercoiling and stabilization of DNA; iii. stabilization of misfolded RNA structures; and iv. rigidification, misfolding, and methylation of proteins. These changes induce a transient induction of proteins and enzymes necessary to counteract these challenges. The repertoire of cold-induced proteins is unique to each organism. Examples of proteins commonly induced upon cold shock in bacteria are RNA chaperones and helicases such as cold shock domain proteins and DExD-box helicases, ribosome-associated proteins and translation factors, DNA modifying enzymes, and protein chaperones. They all have one particular goal: to restore cellular functions and resume growth (Phadtare & Inouye, 2008).

Temperature-dependent membrane modulation Restoration of membrane fluidity is of crucial importance to maintain the membranes function as a selective barrier between the living cell and the outside world. Changes in temperature influences its fluidity and therefore, disabling most of its functions. A living cell needs to be able to modify its membrane and adjust it accordingly in order to survive. This can be achieved in several ways, some more common than others:

• Increasing the levels of unsaturated fatty acids. • Altering the size and charge of the polar head groups.

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• Changing the proportion of short and long chain fatty acids. • Increasing fatty acid chain branching. • Changing the proportion of cis and trans fatty acids. • Changing the composition of carotenoids.

Membrane proteins themselves influence membrane fluidity such that the lipids lose motional freedom upon interaction with membrane proteins (Takeuchi, et al., 1978, Takeuchi, et al., 1981). In Bacillus caldotenax, a shift from 65° to 45°C induced a change in the membrane protein content (Hasegawa, et al., 1980). It has also been suggested that certain heat shock proteins can modify the membrane fluidity upon interaction with membrane lipids and other proteins during heat shock (Torok, et al., 1997). Alterations in fatty acid desaturation and isomerization. Changes in fatty acid desaturation and isomerization are by far the most common membrane modifications in response to low temperature. Anaerobic fatty acid desaturation as a response to temperature decrease has been mostly studied in E. coli that increases its cis-unsaturated vaccenic acid and decreases its saturated palmitic acid (Marr & Ingraham, 1962). Anaerobic synthesis of E. coli unsaturated fatty acids is dependent on FabA, FabB (KAS I), and FabF (KAS II). FabA is the key enzyme and introduces a cis double bond into a 10-carbon intermediate. FabB and FabF are elongating enzymes responsible for synthesis of ≤C16:1 fatty acids and C18:1 fatty acids, respectively. Therefore, FabF is responsible for production of cis- vaccenic acid (Garwin, et al., 1980, Cronan & Rock, 1996). This type of unsaturation is dependent upon de novo synthesis of the acyl chain. It has also been shown that increased levels of cis-vaccenic acid at low temperature is independent of de novo synthesis of mRNA and protein, suggesting that FabF activity is the main point of regulation (Garwin & Cronan, 1980). Aerobic desaturation of fatty acids is the most common desaturation method and has been mainly studied in B. subtilis and cyanobacteria. This type of unsaturation is performed by membrane-bound desaturases working on a pre-existing lipid moiety and requires both oxygen and reducing equivalents. Aerobic desaturation introduces a double bond at a specific spot on a wide variety of fatty acids. In the case of B. subtilis, cis desaturation is introduced at the Δ5 position by the enzyme DesA (Altabe, et al., 2003). The expression of desA is tightly regulated by the membrane-associated two- component system DesK/DesR. It is transiently induced upon cold shock and is undetectable at 37°C (Shivaji & Prakash, 2010). In cyanobacteria, there are several enzymes responsible for fatty acid desaturation although not all are necessary for growth at low temperature. In Synechocystis sp. PCC 6803, three desaturases are induced upon cold shock, DesA, DesB, and DesD that cause double bonds at the Δ12, ω3, and Δ6 positions, respectively (Los, et al., 1993, Los, et al., 1997). However, desD mutants and desB/D double mutants show no differences in growth at low or high temperature. In contrast, a desA mutant and desA/D double mutant has impaired growth at low temperature. Also, inactivation of DesC, responsible for desaturation at the Δ9 position, appears to be vital at all temperatures (Wada & Murata, 1989, Murata & Wada, 1995, Tasaka, et al., 1996).

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There are two forms of fatty acid unsaturation, cis- and trans unsaturation (Fig. 3). Both types generate more membrane fluidity than saturated forms, but cis unsaturation has the most fluidizing effect because of the rigid 30°C kink in the acyl chain (Heipieper, et al., 2003). In some psychrophilic bacteria the amount of trans-fatty acids increases with increasing temperature. In Pseudomonas syringae Lz4W, it was shown to be due to another type of enzyme, a cis-trans isomerase, converting cis-fatty acids into trans-fatty acids upon temperature up-shifts (Kiran, et al., 2004, Kiran, et al., 2005). A cis-trans isomerase can work in both directions, but transforming trans into cis is energy-dependent (Heipieper, et al., 2003). Another type of isomerization that affects membrane fluidity is fatty acid branching. Branched and cyclic fatty acids increase the fluidity of the membrane by disturbing the packing order to a higher degree than their straight, un-branched counterparts (Grogan & Cronan, 1997). The most common types of branching occur at the second and third last carbon of the acyl chain and are referred to as iso-branching and anteiso-branching, respectively (Fig. 3). An increase in anteiso-branched fatty acids and a concomitant decrease in iso-branched lipids upon temperature downshifts occurs in Flavobacterium thermophilum (Oshima & Miyagawa, 1974), Bacillus T1 (Chan, et al., 1973), and B. subtilis (Suutari & Laakso, 1992). The exact mechanism for temperature-dependent alteration of the iso- and anteiso branching ratio is unclear but it is known that they need to be synthesized de novo.

Fig. 3. Different types of fatty acyl chain isomers commonly associated with membrane adaptation to temperature changes. R: Un-specified polar lipid head group.

Alterations in lipid polar head groups, carotenoids, and fatty acyl chain length. Changes in the polar head groups as a response to low temperature is less efficient and therefore fairly uncommon. However, their size and charge can modify the packing of glycerophospholipids in the membrane and thereby cause loose packing of the membrane. This strategy has been observed in B. caldotenax (Hasegawa, et al., 1980).

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Carotenoids usually confer rigidity to a membrane, with polar carotenoids providing more rigidity than non-polar carotenoids. The psychrophilic bacteria Sphingobacterium antarcticus and Micrococcus roseus increase their content of polar carotenoids in response to a temperature downshift, thereby increasing membrane stability. This might be explained by Sp. antarcticum increasing the biosynthesis of unsaturated and branched chain fatty acids at low temperature. In this case, the purpose of the carotenoids is to facilitate membrane stabilization to overcome the fluidizing effect of the unsaturated fatty acids. In fact, the fluidity state of the membrane is equal in cells growing at 5° and 25°C (Jagannadham, et al., 1991, Jagannadham, et al., 1996, Jagannadham, et al., 1996, Chattopadhyay, et al., 1997, Jagannadham, et al., 2000). A change in fatty acid change length has only been observed in actively growing cells during log phase (Denich, et al., 2003) and therefore, might not be the optimal way of modulating membrane fluidity in response to a temperature shift. Shorter fatty acids cannot form stable hydrophobic interactions with other lipids and membrane proteins and are unable to span the lipid bilayer. By increasing the amount of short fatty acids the fluid state of the membrane can be maintained at lower temperatures.

Extracellular polymeric substances in cold adaptation The role of exopolymers in cold adaptation is a fairly unexplored topic. However, psychrophilic bacteria isolated from Antarctic environments produce extensive amounts of exopolymers (Frias, et al., 2010). Extracellular polymeric substances (EPS) are complex organic materials secreted by microorganisms. They form gel-like matrices that are hydrated to varying degrees depending on the environmental conditions (Verdugo, et al., 2004). Of special interest in this context is that EPS can provide a buffer zone around the cells, protecting them from unfavorable environmental changes (Decho & Lopez, 1993, Schlekat, et al., 1998). It is known that synthetic polymers, such as ethylene glycol, function well as cryoprotective agents (Michelmore & Franks, 1982, Allegretto, et al., 1992). Polyhydroxyl compounds, such as glycerols and , are even more effective in ice formation inhibition. Therefore, the additive effect of high molecular weight polymers together with polyhydroxyl compounds would further decrease the freezing point (Sutton, 1991). As a result, it is believed that naturally produced EPS would be an excellent cryoprotector. Supporting data that EPS is important in cold adaptation is that the psychrophilic bacterium Colwellia psychroerythraea 34H genome sequence contains a preponderance of genes involved in the production and secretion of widely diverse polymeric substances (Methe, et al., 2005). Interestingly, it has been found that EPS from Antarctic bacteria contain large numbers of membrane vesicles and that membrane vesicle production is higher at low temperature. Proteomic analysis of vesicle protein content mostly revealed outer membrane and periplasmic proteins related to nutrient processing and transport. The vesicles are hypothesized to be a source of degradative enzymes that help to concentrate nutrients around the cell (Frias, et al., 2010).

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Dealing with ice formation in biological systems Macromolecular structures in the cell such as membranes, proteins, and nucleic acids are dependent upon interaction with water molecules for their functionality. Therefore, a decreased accessibility of water in biological systems poses a problem for the function and survival of the cell. Such a problem arises when the temperature decreases and aqueous water starts to transition into ice crystals (Beall, 1983). Microorganisms often deal with this problem by producing proteins and/or saccharides that interfere with ice crystal formation (Kawahara, 2002). Cryoprotective proteins. It has been shown that some proteins serve a protective role for other enzymes. For example, albumin prevents freeze inactivation of lactate dehydrogenase by surrounding the enzyme and protecting it from denaturation and aggregation (Tamiya, et al., 1985). Similarly, Pantoea agglomerans NBRC12686 produces a 29 kDa cryoprotective protein, CRP, that protects freeze-labile enzymes such as lactate dehydrogenase, alcohol dehydrogenase, and isocitrate dehydrogenase against freezing (Tamiya, et al., 1985, Koda, et al., 2001). One of the cold- acclimation proteins of Pantoea ananatis KUIN-3 is the Hsc25 chaperone that has a similar activity as GroEL but a higher affinity for cold-denatured proteins (Kawahara, et al., 2000). Ice nucleation and anti-nucleating molecules. Formation of an ice nucleus is the critical point of ice crystal formation. As soon as an ice nucleus has formed, water molecules can attach and the ice crystal starts to grow. One strategy that some microorganisms utilize as a response to low temperature is to inhibit ice nucleation and thereby, the formation of ice crystals. Acitenobacter calcoaceticus KINI-1 produces a 550 kDa anti- nucleating protein (Kawahara, et al., 1996) and Bacillus thuringiensis produces an anti-nucleating polysaccharide of 1300 kDa (Yamashita, et al., 2002). However, these particles are poorly characterized. Furthermore, some plant-associated microorganisms such as Pesudomonas fluorescens and P. syringae have an ice-nucleating activity that induces frost damages in crops and enhances survival of the bacterium on the surface of the leaf after freezing and thawing (Hirano, et al., 1982, Lindow, et al., 1983). However, it has been observed that coniferous trees contain hinokitiol on the surface of their leaves, which has a anti-nucleating activity against P. fluorescens (Kawahara, et al., 2000). Anti-freeze proteins. Anti-freeze (glyco-) proteins lower the freezing point of water without effecting the melting point, an activity referred to as thermal hysteresis. They can also inhibit ice recrystallization in the frozen state. Anti-freeze (glyco-) proteins have mostly been found within Eukarya (polar fish, insects, plants, and fungi) (Hew & Yang, 1992, Duman & Olsen, 1993). However, anti-freeze activity has been demonstrated in a variety of bacteria (Duman & Olsen, 1993, Gilbert, et al., 2004, Kawahara, et al., 2004) including the Antarctic bacterium Marinomonas primoryeansis that produces a hyperactive, Ca2+-dependent anti-freeze protein (Gilbert, et al., 2005). Compatible solutes. Compatible solutes are commonly accumulated in a range of bacteria as a response to different stresses, such as cold. They are

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usually sugars or amines and protect cells from damage by preventing denaturation and aggregation of proteins. Accumulation within cells occurs in two ways: either the bacteria produce and accumulate compatible solutes or they are taken up from the surroundings. B. subtilis accumulates a range of compatible solutes such as glycine betaine and l-carnitine and thereby, enhances growth at low temperatures. This accumulation is due to the expression of known Opu transporters (Hoffmann & Bremer, 2011). Several bacteria can produce compatible solutes themselves. In addition, the soil bacterium Virgibacillus pantothenicus increases expression of the ectoine biosynthetic genes, ectABC, upon a temperature decrease (Kuhlmann, et al., 2008). Trehalose (also, mycose or tremalose) is a disaccharide composed of two α-glucose molecules connected with a α,α-1,1-glucosidic bond. It acts in the cell as a stabilizer and protectant of membranes and proteins. E. coli produces trehalose in response to low temperature and its synthesis is essential for viability at low temperatures (Kandror, et al., 2002). However, trehalose also protects against dehydration, heat, and oxygen radicals (Elbein, et al., 2003). Oxygen radicals are a common source of stress induced by a temperature decrease due to the increased solubility of oxygen at low temperatures. Therefore, microorganisms that produce trehalose at decreased temperatures can “kill two birds with one stone”. Accumulating trehalose can have other benefits. In addition to several heterotrophic microorganisms utilizing trehalose as a carbon and energy source, it has also been shown to serve as carbon and energy reserves in insects (Becker, et al., 1996) and fungi (Thevelein, 1984) (Rosseau, et al., 1972). It has also been proposed to act as a sensing compound or a growth regulator. This proposition is based on the observation that tobacco and rice plants transformed with trehalose synthesizing genes showed severe growth defects. However, recent research has shown that it is trehalose-6-phosphate that is the coordinating factor between metabolism and development (Paul, et al., 2008). Trehalose is also the basic component of the cell wall in mycobacteria and corynebacteria (Lederer, 1976). Trehalose can be synthesized via five different pathways (Fig. 4) and is degraded via the activity of trehalase (Avonce, et al., 2006):

• The TPP/TPS pathway consists of two enzymes, sometimes fused into a single protein. First, trehalose-6-phosphate synthase (TPS) produces trehalose-6-phophate from UDP-glucose and glucose-6- phophate. Then, trehalose-6-phosphate phosphatase (TPP) dephosphorylates trehalose-6-phosphate to produce trehalose. This pathway is the most common in nature, existing in organisms from all domains of life. The TPS/TPP pathway is the only pathway of trehalose biosynthesis in multicellular organisms. • Trehalose synthase (TS) is an isomerase that converts maltose into trehalose. It was originally found in Pimelobacter sp. but occurs in several microorganisms, such as Streptomyces avermitilis. • The TreY/TreZ pathway was discovered in thermophilic archaea from the genus Sulfolobus. It converts maltodextrines to trehalose by two enzymatic steps. First, maltooligosyl trehalose synthase

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(TreY) transglycosylates the last glucose moiety of maltodextrines forming a trehalose moiety at the end of the polymer. Second, maltooligosyl trehalose trehalohydrolase (TreZ) catalyzes the hydrolytic release of trehalose from the polymer. • Trehalose phosphorylase (TreP) catalyzes the reversible hydrolysis of trehalose (plus inorganic phosphor) generating glucose and glucose- 1-phosphate. Whether this pathway works in trehalose synthesis or hydrolysis is still uncertain since its activity has only been demonstrated in vitro. This pathway, together with the TPS/TPP pathway is the only way of synthesizing trehalose in eukaryotes. • Trehalose glycosyltransferring synthase (TreT) catalyzes the reversible formation of trehalose from ADP(UDP/GDP)-glucose and glucose. It was first found in the hyperthermophilic archaeon Thermococcus litoralis and has the highest activity on ADP-glucose, but can also use UDP- or GDP-glucose. • Trehalose is degraded by trehalase into two D-glucose molecules.

Fig. 4. Illustration of the five pathways of trehalose biosynthesis: A) the TS pathway; B) the TPS/TPP pathway; C) the TreY/TreZ pathway; D) the TreT pathway; and E) the TreP pathway.

The TS, TreY/Z, and TreT pathways have only been demonstrated in bacteria and archaea.

Cold adaptation in Acidithiobacillus ferrooxidans Although acidophilic microorganisms inhabit cold mining impacted environments, little is known about how they cope with the decreased temperature. To date, two studies characterizing the response of mesophilic

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and psychrotolerant A. ferrooxidans strains to low temperature have been published (Mykytczuk, et al., 2010, Mykytczuk, et al., 2011). The authors of these studies note that the psychrotolerant strains of A. ferrooxidans might be more accurately designated A. ferrivorans. However, phylogenetic analysis suggests that they belong to A. ferrooxidans group II according to the classification by Amouric, et al. (2011). Analysis of membrane fluidity and fatty acid composition in response to low temperature was performed on a selection of mesophilic and psychrotolerant A. ferrooxidans strains. When the strains were grown at 25°C compared to 15°C, no general trend in membrane fluidity could be observed. However, the psychrotolerant strains had significantly decreased membrane fluidity when grown at 5°C compared to 15° or 25°C. Analysis of fatty acid composition revealed that all strains respond to lower temperature by increasing the amount of unsaturated fatty acids while decreasing their saturated and cyclic fatty acids. Specifically, psychrotolerant strains decreased their 12:0 (dodecanoic acid) fatty acids while increasing 16:1 w7c (cis-Δ9-hexadeconic acid) fatty acids at 5°C. The most prominent change among the mesophilic strains was a decrease in cyclopropane 19:0 fatty acid at 15°C (Mykytczuk, et al., 2010). Changes in the proteome in response to low temperature have also been investigated in one mesophilic (25° vs. 15°C) and one psychrotolerant (25° vs. 5°C) strain of A. ferrooxidans. Both strains show increased expression of proteins involved in energy- and carbon metabolism, transcription, protein fate, protection against reactive oxygen species, and proteins with unknown function at low temperature. Some of these responses are described as cold- inducible in other microorganisms (i.e. PNPase, NusA, TF, and ribosomal protein L7-L12) (Phadtare & Inouye, 2008) while others are seemingly unique to A. ferrooxidans. Interestingly, the psychrotolerant strain showed alternate expression of two paralogous AhpC-Tsa family antioxidants at low and high temperature. Further, the mesophilic strain increased the levels of a third AhpC-Tsa family antioxidant protein at low temperature. Finally, a thioredoxin protein was increased in both strains at low temperature. Protection from reactive oxygen species is considered important due to the increased solubility of gases (i.e. oxygen) at low temperature. Additionally, the high levels of iron in metal-contaminated environments also induces reactive oxygen species formation by the means of Fenton chemistry (Koppenol, 1993). In accordance, the key iron metabolism protein rusticyanin was also up-regulated at low temperature. This might be a strategy to compensate for a possible reduction in enzyme activity. However, a second hypothesis is that increased rusticyanin expression helps to efficiently oxidize and remove iron as a way of reducing its toxic effect. The increased solubility of gases is also reflected by up-regulation of proteins involved in carbon dioxide and nitrogen fixation. The psychrotolerant strain also has increased levels of proteins involved in EPS formation. All the information taken together indicates that A. ferrooxidans utilizes similar, but not identical, strategies in adaptation to a decrease in temperature as in many other organisms (Mykytczuk, et al., 2011).

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VI. Iron oxidation at acidic pH Microbial iron oxidation is widespread in the environment, from lithotrophic iron oxidation under acidic conditions to photosynthetic iron oxidation under pH neutral conditions. Acidophilic iron oxidizing microorganisms face two main challenges: i. at neutral pH (i.e. inside the cytoplasm), ferrous iron rapidly oxidizes into ferric iron, which in turn precipitates into ferric oxides; ii. since the midpoint potential of ferrous/ferric is higher (+0.77V) than the midpoint potential of NAD+/NADH (-0.32V) electrons gained via autotrophic ferrous oxidation must be pushed ‘uphill’ against an unfavorable redox potential, i.e. energy has to be invested to produce NADH. Acidophiles solve these problems by oxidizing ferrous outside of the cell and shuffling the generated electron via a series of proteins either ‘downhill’ to the terminal oxidase where O2 is reduced to H2O or ‘uphill’ to generate NADH. It is believed that acidophiles use the natural proton gradient across the cell membrane as an energy source for the ‘uphill’ generation of NADH (Ingledew, 1982, Ferguson & Ingledew, 2008). Despite the fact that many acidophiles oxidize ferrous iron, the molecular mechanism remains poorly characterized in most species.

Ferrous oxidation in Acidithiobacillus ferrooxidans The best understood pathway of ferrous oxidation is described for A. ferrooxidans (Fig. 5). This model is primarily based on bioinformatic and transcriptomic data from iron-grown A. ferrooxidansT (ATCC23270) together with reports from other A. ferrooxidans strains (Quatrini, et al., 2009). However, since a lot of data has been retrieved from different strains of A. ferrooxidans that is in some cases conflicting, the current model is not necessarily supported in all A. ferrooxidans strains. This is highlighted in a phylogenetic study of several A. ferrooxidans and A. ferrivorans strains based on genes including rusA and B, hip, and iro (Amouric, et al., 2011). In A. ferrooxidansT, ferrous iron oxidation is proposed to be mediated by a high molecular weight cytochrome c (Cyc2) located in the outer membrane. Retrieved electrons are transferred via rusticyanin (Rus) either downhill or uphill. Electrons migrating via the downhill pathway are transported by a membrane-bound cytochrome c4 (Cyc1) to a terminal aa3-type cytochrome oxidase (CoxABCD). In addition, a protein with unknown function (Cup) has been shown to associate with Cyc1 in an unnamed strain of A. ferroxidans (Castelle, et al., 2008). All these proteins are expressed from the rus operon and are up-regulated in iron-grown cells. The uphill pathway includes electron transfer from Rus via another membrane-bound cytochrome c4 (CycA1), a cytochrome bc1 complex (PetABC1), and the quinone pool to the NADH dehydrogenase complex working in reverse. The genes encoding CycA1, a short-chain dehydrogenase of unknown function (SdrA1), and PetABC1 are transcribed from a single genetic locus termed the petI operon, also up-regulated in iron-grown cells. Hence, this model suggests that the bifurcation of electrons via either the downhill or the uphill pathway occurs at the level of Rus. However, Cup exhibits some similarities to Rus and because of its association with Cyc1 (Castelle, et al., 2008) it might also be involved in the electron transfer between Cyc2 and Cyc1. Other hypothesized

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functions include the delivery of copper to one of the copper-containing proteins Rus or the aa3 cytochrome oxidase (Quatrini, et al., 2006, Quatrini, et al., 2009) or working as a chaperone important for stability and activity of the aa3-type cytochrome c oxidase under acidic conditions (Castelle, et al., 2010).

Fig. 5. Model of ferrous oxidation in Acidithiobacillus ferrooxidansT. The flow of electrons is indicated with dashed lines and the direction of proton transport across the membrane with solid lines. Iron is oxidized by Cyc2 and retrieved electrons are shuffled via Rus to either the aa3 complex (downhill pathway) or the NADH complex (uphill pathway). Abbreviations can be found in the text. Illustration adapted from (Quatrini, et al., 2009).

Other genes up-regulated on ferrous include a 6 gene cluster (ctaSURTBA) located immediately downstream of the rus operon and two genes (regBA) also downstream of the rus operon but transcribed in the opposite direction. CtaABT are predicted to be involved in cytochrome aa3 oxidase biogenesis and CtaRUS presumably regulates biogenesis in an iron- responsive manner. RegAB is a predicted two-component signal transduction system possibly involved in redox sensing. Its function remains elusive but it has been hypothesized to assist in balancing electrons between the uphill and downhill pathway. Alternatively, RegAB could be involved in switching between aerobic and anaerobic growth or between iron and ISC oxidation (Quatrini, et al., 2009). In a recent study, RegA was shown to bind to upstream genetic sequences of both iron- and ISC induced genes. Additionally, iron was preferentially oxidized to ISC suggesting RegA to be an activator of iron-induced genes and a repressor of ISC-induced genes. However, more studies are necessary to confirm this (Ponce, et al., 2012). Most parts of the above described ferrous oxidation model also applies to other strains of A. ferrooxidans. Based on available data, A. ferrooxidans ATCC 33020 utilizes a similar iron oxidation mechanism (Appia-Ayme, et al., 1998, Bengrine, et al., 1998, Appia-Ayme, et al., 1999, Levican, et al., 2002, Yarzabal, et al., 2002, Yarzabal, et al., 2002, Yarzabal, et al., 2003, Yarzabal, et al., 2004, Bruscella, et al., 2007) and data obtained from A. ferrooxidans ATCC 19859 supports at least parts of this model (the rus

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operon has not been characterized in this strain). Also, genetic evidence suggests a similar iron oxidation model in strain ATCC 53993 (NC_011206, direct submission). The iron oxidation system was also shown to form a supercomplex spanning the inner and the outer membrane in an unnamed A. ferrooxidans strain (Castelle, et al., 2008). However, there are several reports where this particular iron oxidation system cannot be supported. It was first reported in 2003 and further analyzed in 2011 that the rus gene is present in two different forms, rusA and rusB, and different strains of A. ferrooxidans harbor different rus genes (Sasaki, et al., 2003, Amouric, et al., 2011). Further, the high-potential iron-sulfur protein (HiPIP) is involved in ISC metabolism (referred to as hip) in some strains (Bruscella, et al., 2005, Quatrini, et al., 2006, Bruscella, et al., 2007, Valdes, et al., 2008, Quatrini, et al., 2009) but described as the main iron-oxidizing enzyme (referred to as iro) in another (Fukumori, et al., 1988, Kusano, et al., 1992, Cavazza, et al., 1995). In addition, one strain contains neither rusA nor coxC (aa3-type cytochrome oxidase subunit III) but is still able to oxidize ferrous (Chen, et al., 2009). When strains of A. ferrooxidans and A. ferrivorans were tested for the presence of rusA and/or rusB and hip or iro and further phylogenetically analyzed, it was discovered that they clearly grouped into distinct clusters. Group I and II, including A. ferrooxidans ATCC 19859, 53993, 23270 (I), and 33020 (II) clusters, contain rusA and hip, while group III (A. ferrivorans) and IV contain rusB and iro (Amouric, et al., 2011). Taken together, this suggests that different strains of A. ferrooxidans utilize different iron oxidation systems.

Iron oxidation in other acidophilic bacteria A gene cluster similar to the rus operon in A. ferrooxidansT is present in the salt tolerant ‘T. prosperus’. It contains cyc2, cup, coxBACD, and rus but lacks the gene encoding the key enzyme cyc1. On the other hand, it cannot be excluded that cyc1 is present elsewhere in the genome or that its function is replaced by another enzyme, such as Cup. All the genes were up-regulated when incubated in the presence of ferrous compared to sulfur except for cyc2 and rus. Also, rus is transcribed independently from the other genes as opposed to A. ferrooxidans where they are transcribed as a single unit. Despite the discrepancies, ‘T. prosperus’ was proposed to utilize a similar pathway as described for A. ferrooxidans (Nicolle, et al., 2009). In Leptospirillum spp. iron oxidation is carried out by the membrane- bound Cyt572 (Jeans, et al., 2008) and is suggested to deliver electrons to Cyt579, which is a periplasmic cytochrome highly abundant in a Leptospirillum-dominated biofilm (Ram, et al., 2005, Singer, et al., 2008, Singer, et al., 2010). Further, genes encoding cytochrome cbb3-type oxidases and cytochrome bd-type oxidases can be found in genomes of Leptospirillum spp. (Tyson, et al., 2004).

Iron oxidation in acidophilic archaea Based on metagenomic, proteomic, and biochemical data, a speculative model of ferrous oxidation in Ferroplasma spp. has been generated. The model proposes iron oxidation to be mediated by the blue copper

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sulfocyanin, a protein similar to rusticyanin, and that electrons are delivered to a cytochrome cbb3-type oxidase (Tyson, et al., 2004, Dopson, et al., 2005, Allen, et al., 2007). In S. metallicus a 15-kb region containing the fox genes is up-regulated in ferrous grown cells. foxA had the highest expression level of them all and the gene product can also be found in membrane fractions prepared from pyrite- grown cells. The genome of the iron-oxidizing Sulfolobus tokodaii contains a similar gene cluster (ST2587-2603), whereas the non iron-respiring S. acidocaldarius and S. solfataricus does not (Bathe & Norris, 2007). The corresponding fox genes in S. tokodaii are annotated as hypothetical proteins except foxA, which is annotated as a cytochrome c oxidase polypeptide I (Kawarabayasi, et al., 2001). A similar gene cluster to that in S. metallicus is also found in Metallosphaera sedula (Auernik, et al., 2008). Parts of this gene cluster are significantly up-regulated on ferrous, while other parts are down-regulated. It is also noted the two paralogous copies of foxA are present in the genome that are inversely expressed on ferrous relative to each other (Auernik & Kelly, 2008).

VII. Inorganic sulfur compound oxidation in acidophiles ISC oxidation is a common feature of diverse chemolithotrophic bacteria and archaea and occurs over the entire temperature and pH range. Many of the ISC oxidation enzymatic reactions occur in the periplasm and as this compartment is exposed to the outside environment (i.e. acidic, basic, high salinity etc.), a wide diversity of pathways and enzymes is documented. Also, sulfur occurs in many forms and intermediates (-2 to +6 oxidation states) and some reactions can occur spontaneously making ISC oxidation difficult to study. Despite this, ISC oxidation is fairly well understood in many microorganisms.

Oxidation of thiosulfate and polythionates

2- Thiosulfate (S2O3 ) is one the most abundant reduced sulfur species on earth and is used as substrate by nearly all ISC oxidizers. However, it is unstable at acidic pH (< 4.0) and often decomposes to elemental sulfur (S0) and sulfite 2- (SO3 ) (Johnston & McAmish, 1973). Currently, there are at least three pathways known for complete oxidation of thiosulfate by different microorganisms. The so-called “S4I pathway” for thiosulfate oxidation is the least resolved 2- pathway. It is characterized by the formation of a tetrathionate (S4O6 ) intermediate and is believed to be the preferred pathway for acidophiles due to the acid-instability of thiosulfate (Ghosh & Dam, 2009). As opposed to thiosulfate, tetrathionate is quite acid-stable even though it often produces 2- pentathionate (S5O6 ) in the presence of other reduced sulfur species (Johnston & McAmish, 1973). Plausible enzymatic steps have been proposed based on biochemical and physiological data. Acidianus ambivalens oxidizes thiosulfate to tetrathionate by a membrane-bound thiosulfate:quinone oxidoreductase (TQO) (Muller, et al., 2004). It consists of two subunits, identical to DoxA and DoxD previously described as parts of the terminal quinol oxidase DoxABCDEF (Purschke, et al., 1997). The formed

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tetrathionate is hydrolyzed by tetrathionate hydrolase (designated as Tth or TetH in various publications). TetH has been characterized in several microorganisms and a periplasmic or extracellular location has been demonstrated (Bugaytsova & Lindstrom, 2004, Beard, et al., 2011, Protze, et al., 2011). TetH is believed to produce sulfate, thiosulfate, and elemental sulfur from tetrathionate (Meulenberg, et al., 1992, Hallberg, et al., 1996) even though differences have been documented (Sugio, et al., 1996, Tano, et al., 1996, Bugaytsova & Lindstrom, 2004). In either case, a thiosulfate:tetrathionate cycle is established. However, thiosulfate can also abiotically decompose into elemental sulfur and sulfite in the acidic periplasm and it cannot be completely ruled out that acidophiles take advantage of this characteristic. The main products of TetH are sulfate and - (HS2SO3 ). Disulfane monosulfonic acid is highly reactive and therefore subsequently forms thiosulfate and elemental sulfur (Steudel, et al., 1987). This is supported by periplasmic accumulation of long-chain polythionates and elemental sulfur in A. ferrooxidans (Steudel, et al., 1987). Complete oxidation of tetrathionate requires an active membrane in Tetrathiobacter kashmirensis (Dam, et al., 2007) and there might be unknown components of membranes that transiently interact with the disulfane monosulfonic acid before it reacts further. The best characterized pathway is probably “the thiosulfate oxidizing multi-enzyme system” (TOMES), that completely oxidizes thiosulfate to sulfate without the formation of free intermediates (Kelly, 1982, Kelly, 1989). The complete TOMES consists of seven proteins (SoxAX, SoxYZ, SoxB, and SoxCD) all of which are catalytically inactive by themselves (Ghosh & Dam, 2009). The heterodimeric SoxAX initiates the oxidative cycle by coupling the sulfane sulfur (-S-) of thiosulfate to a SoxY-cysteine residue on the SoxYZ complex. This forms a cysteine S-thiosulfate residue (reaction 1), from which - SoxB subsequently catalyzes the release of the terminal sulfone (-SO3 ) group as a sulfate molecule (reaction 2).

- - - - + (1) SoxZY-SH + SSO3 → SoxZY-S-SSO3 + 2e + H catalyzed by SoxAX

- 2- + (2) SoxZY-S-SSO3 + H2O → SoxZY-S-SH + SO4 + H catalyzed by SoxB

The sulfane sulfur of the SoxZY-cysteine persulfide is then further oxidized to SoxZY-cysteine-S-sulfate by the heterotetrameric Sox(CD)2 complex (reaction 3).

- - + (3) SoxZY-S-SH + 3H2O → SoxZY-S-SO3 + 6e + 7H catalyzed by Sox(CD)2

Lastly, SoxB hydrolyzes the sulfonate moiety regenerating SoxYZ (reaction 4).

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- 2- + (4) SoxZY-S-SO3 + H2O → SoxZY-SH + SO4 + H catalyzed by SoxB

One complete TOMES cycle releases 8 electrons, 10 protons, and 2 sulfate molecules per thiosulfate oxidized (Friedrich, et al., 2001). All 8 electrons are transferred to small c-type cytochromes and then to terminal oxidases. It has also been noted that other sulfur species such as sulfide, elemental sulfur, sulfite, and tetrathionate can feed into the TOMES at various stages (Friedrich, et al., 2001, Sauve, et al., 2007). The complete TOMES is mainly present in the Alphaproteobacteria. However, parts of the Sox complex, lacking SoxCD but still functional, have been demonstrated in various bacteria, such as Chlorobi, Aquificae, and Beta- and Gammaproteobacteria (Friedrich, et al., 2001, Friedrich, et al., 2005) and is referred to as core TOMES. The core TOMES yields only 2 electrons per thiosulfate instead of eight and the products are believed to be sulfate and elemental sulfur or polysulfide (Friedrich, et al., 2005). The third pathway is also fairly well characterized and called “the branched thiosulfate oxidation pathway”, which is characterized by the interaction of two distinct enzyme systems: oxidation to tetrathionate and complete oxidation to sulfate with accumulation of intra- or extracellular sulfur globules as a result (Dahl, 2008, Grimm, et al., 2008). This pathway is extensively characterized in the purple sulfur bacterium Allochromatium vinosum (Grimm, et al., 2008). Oxidation to tetrathionate appears to only occur under acidic conditions and is carried out by a thiosulfate dehydrogenase (Hensen, et al., 2006). Complete conversion to sulfate starts with thiosulfate oxidation by the core TOMES. The lack of SoxCD appears to result in sulfur accumulation. Extracellular sulfur globules are reductively activated and transferred to the cytoplasm where oxidation to sulfite is achieved by the reversed activity of the dissimilatory sulfite reductase, DsrAB. Released electrons are fed into the electron transport chain via DsrC and DsrMKJOP to the terminal acceptor, DsrJ. Sulfite is believed to be oxidized by the sequential reverse action of AMP-dependent APS reductase (AprBA) and ATP sulfurylase (Grimm, et al., 2008, Ghosh & Dam, 2009).

Oxidation of elemental sulfur and sulfite Elemental sulfur occurs as a substrate taken up from the environment or produced as an intermediate in ISC oxidation. It is produced by the activity of the core TOMES and TetH as described above, by Sqr (Sulfide:quinone reductase), flavocytochrome c-sulfide dehydrogenase (FCSD), as well as by the decomposition of thiosulfate at acidic pH (Ghosh & Dam, 2009). Oxidation of elemental sulfur is one of the most energy-yielding reactions among lithotrophic organisms and can be oxidized completely to sulfate by the Dsr system or it can feed into the Sox pathway, as described above. Further, elemental sulfur can be oxidized by sulfur oxygenase reductase (Sor) (Chen, et al., 2007, Liu, 2008). It has also been proposed that elemental sulfur can be non-enzymatically activated by glutathione (GSH) to yield glutathione persulfide (GSSH). The sulfane sulfur (-S-) of GSSH is then oxidized by a periplasmic sulfur dioxygenase (Rohwerder & Sand, 2003).

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Even though a sulfur dioxygenase activity has been demonstrated in Acidithiobacillus spp. and Acidiphilium spp. (Rohwerder & Sand, 2003), neither the gene nor the enzyme have been conclusively identified. Instead, it is proposed that the heterodisulfide reductase system (Hdr) in acidophilic bacteria and archaea catalyzes the oxidation of disulfide intermediates by working in reverse (Quatrini, et al., 2009). Elemental sulfur oxidation produces sulfite in the case of Sor and Hdr activity and to date, two pathways involved in sulfite oxidation have been identified. The APS reductase and ATP sulfurylase system is described above, while the other is sulfite oxidoreductase catalyzing the direct conversion of sulfite to sulfate (Kappler & Dahl, 2001).

Models of ISC oxidation in Acidithiobacillus spp. A. ferrooxidans sulfur metabolism has been extensively studied. Based on the model (Fig. 6A) proposed by Quatrini, et al. (2009), thiosulfate is oxidized by TQO to tetrathionate, which is further hydrolyzed by TetH in the periplasm. Elemental sulfur is produced by Sqr-mediated sulfide oxidation and also possibly by TetH. The produced sulfur is proposed to be activated by glutathione and further oxidized by the reversed action of the Hdr complex. Elemental sulfur oxidation by Hdr and subsequent electron transfer to terminal oxidases is possibly assisted by TusA, rhodanese (Rhd), and DrsE (all encoded in the same gene cluster and similarly regulated). The formed sulfite is possibly oxidized to sulfate by the subsequent action of an APS reductase and ATP sulfurylase (Sat). However, only a candidate gene for ATP sulfurylase (Sat) and not APS reductase has been found in A. ferrooxidans even though a sulfite oxidase activity has been demonstrated in several strains (Vestal & Lundgren, 1971, Sugio, et al., 1988, Sugio, et al., 1992, Suzuki, 1994). However, sulfite is metastable in mine-impacted environments and often non-enzymatically oxidizes to sulfate, thiosulfate, and glutathione S-sulfonate (Sugio, et al., 1987, Suzuki, et al., 1992, Harahuc & Suzuki, 2001). On the other hand, sulfite is proposed to be formed in the cytoplasm by the Hdr complex and enzymatic transformation of sulfite is therefore likely to occur (Quatrini, et al., 2009). The electrons gained via the different enzymatic steps are transferred via the quinol pool to the terminal oxidases. Several different types of terminal oxidases are suggested to mediate the reduction of oxygen, including bd-, bo3-, and aa3-type cytochrome oxidases. Furthermore, a high-potential iron-sulfur (Hip) protein and a small cytochrome c (CycA2) are suggested to mediate electron transfer between the bc1-complex and the aa3-complex. A model for ISC oxidation has also been proposed for A. caldus (Mangold, et al., 2011) (Fig. 6B). Similarly to A. ferrooxidans, the model consists of Sqr-mediated sulfide oxidation, TetH-mediated tetrathionate hydrolysis, and Hdr-mediated sulfur oxidation as well as similar terminal oxidases. However, A. caldus also contains the core TOMES (Valdes, et al., 2009, You, et al., 2011) responsible for thiosulfate oxidation and Sor involved in sulfur oxidation. A recent study carried out on A. caldus MTH-04 also proposes a similar model (Chen, et al., 2012). However, this model indicates the presence of a sulfur dioxygenase (SDO) involved in the core TOMES cycle and proposes thiosulfate oxidation

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in the cytoplasm by a rhodanese (von Wettstein-Knowles, et al.). Unfortunately, the study does not further characterize the SDO but rather refers to unpublished data. The recently sequenced genome of A. thiooxidans ATCC 19377 suggests that it utilizes a similar pathway as A. caldusT, with the exception of Sor (Valdes, et al., 2011).

Fig. 6. ISC oxidation models in (A) Acidithiobacillus ferrooxidansT and (B) Acidithiobacillus caldusT. The flow of electrons is indicated with dashed lines and the direction of proton across the membrane with solid lines. Abbreviations can be found in the text. Illustrations adapted from Quatrini et al, 2009 (B) and Mangold et al, 2011 (B).

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Aims of this thesis

The aims of this thesis were to elucidate the distribution and characteristics of psychrotolerant acidophiles and evaluate their potential use in biotechnological applications. The specific aims were to:

¾ Decipher the genetic potential of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS3 (Papers I and III). ¾ Characterize substrate utilization by A. ferrivorans SS3 and evaluate its role in bioleaching operations at low temperature (Papers II and III). ¾ Analyze the microbial community of an acidic, low temperature AMD stream to resolve the distribution and genomic characteristics of psychrotolerant acidophiles (Paper IV). ¾ Evaluate the use of psychrotolerant acidophiles in the development of a bioreactor system to remove sulfur species from mining process water (Paper V).

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Results and Discussion

Genome encoded potential of a psychrotolerant acidophile (Papers I and III) Psychrotolerant A. ferrivorans spp. were previously thought to be cold tolerant strains of A. ferrooxidans but further differences were noted and the strains were subsequently reclassified as a separate species (Hallberg, et al., 2010). A. ferrivorans SS3 was isolated from the Norilsk area, Siberia, in September 2002. This area is characterized by permafrost and between 1934-1994 had a mean annual temperature of -9.6°C and a minimum of -53.1°C (Kupka, et al., 2007). A. ferrivorans SS3 grows and oxidizes ferrous iron and ISCs autotrophically at 5°C, making it psychrotolerant. Attempts to grow it on organic carbon sources were unsuccessful (Kupka, et al., 2007). To further highlight the uniqueness of A. ferrivorans and to get a deeper understanding of this , the A. ferrivorans SS3 genome was sequenced (Paper I). General features. A. ferrivorans SS3 genome consists of 3,21 Mbp (3,152,659 bp) with a GC content of 56.6%. The annotation revealed a total of 3,335 genes dispersed on a single chromosome. 3,093 genes encode proteins of which 986 were annotated as hypothetical proteins (NC_015942.1, updated from paper I). Chemolithoautotrophy. Its chemolithoautotrophic lifestyle is reflected in the genome by 2 copies of type I RuBisCO and 1 copy of type II RuBisCO. Associated with genes encoding one of the type I RuBisCO are genes encoding carboxysomal proteins and carbonic anhydrase. This suggests carbon dioxide (CO2) fixation by the means of the Calvin-Benson-Bassham cycle as has been described for other Acidithiobacillus spp.. Type I and II RuBisCOs have different catalytic properties and the different copies might be differentially expressed depending on the environmental conditions, as has been shown for A. ferrooxidans (Quatrini, et al., 2006). The genomic content suggests inorganic nitrogen can be assimilated either by fixing atmospheric nitrogen (N2) by the means of nitrogenase or by uptake of nitrate, nitrite, and ammonia from the environment. Several transport systems for inorganic nitrogen uptake were found as well as nitrate and nitrite reductases. No evidence of nitric oxide reductase was found indicating that A. ferrivorans SS3 cannot oxidize thiosulfate under denitrifying conditions as was first believed (Paper I). The A. ferrivorans SS3 genome contains genes encoding a group 3 bidirectional cytoplasmic [NiFe]- hydrogenase with accessory proteins and a group 4 membrane-associated respiratory [NiFe]-hydrogenase (Vignais, et al., 2001). This indicated that they have the ability to utilize molecular hydrogen, although it remains to be experimentally verified (Table 1). A more in-depth investigation of genes potentially involved in iron and ISC oxidation was carried out mainly using the published A. ferrooxidans (Quatrini, et al., 2009) and A. caldus (Mangold, et al., 2011) model systems (Paper III). The genetic configuration of genes encoding putative ferrous iron oxidizing proteins was highly similar to the closely related A. ferrooxidansT (Fig. 1a; Paper III). It contains the rus and petI gene clusters

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Table 3. Identified A. ferrivorans SS3 genes putatively involved in a psychrotolerant and chemolithoautotrophic lifestyle. Gene product Locus tag

CO2 fixation microcompartments protein acife_3138 microcompartments protein acife_3139 carboxysome peptide B acife_3140 carboxysome peptide A acife_3141 carboxysome shell carbonic anhydrase acife_3142 carboxysome structural protein CsoS2 acife_3143 ribulose bisphosphate carboxylase small chain acife_3144 ribulose bisphosphate carboxylase large chain acife_3145 ribulose bisphosphate carboxylase small chain acife_2231 ribulose bisphosphate carboxylase large chain acife_2232 ribulose bisphosphate carboxylase acife_1242 Nitrogen assimilation nitrogenase molybdenum-iron protein beta chain acife_1648 nitrogenase molybdenum-iron protein alpha chain acife_1649 nitrogenase iron protein acife_1650 respiratory nitrate reductase subunit gamma acife_1457 nitrate reductase molybdenum cofactor assembly chaperone acife_1458 nitrate reductase subunit beta acife_1459 nitrate reductase subunit alpha acife_1460 nitrite reductase (NAD(P)H) large subunit acife_2076 nitrite reductase (NAD(P)H) small subunit acife_2077 [NiFe]-hydrogenases (NiFe) hydrogenase subunit beta acife_1818 oxidoreductase FAD/NAD(P)-binding domain-containing protein acife_1819 NADH ubiquinone oxidoreductase 20 kDa subunit acife_1820 nickel-dependent hydrogenase large subunit acife_1821 hydrogenase maturation protease acife_1822 hydrogenase expression/formation protein HypE acife_1823 hydrogenase expression/formation protein HypD acife_1824 hydrogenase assembly chaperone hypC/hupF acife_1825 acylphosphatase acife_1826 hydrogenase accessory protein HypB acife_1827 hydrogenase nickel incorporation protein hypA acife_1828 NADH ubiquinone oxidoreductase 20 kDa subunit acife_1243 NADH-ubiquinone oxidoreductase chain 49kDa acife_1244 NADH/Ubiquinone/plastoquinone (complex I) acife_1245 hydrogenase-4 subunit E acife_1246 Trehalose biosynthesis trehalose synthase acife_0606 glycogen debranching protein GlgX acife_0607 malto-oligosyltrehalose trehalohydrolase acife_0608 malto-oligosyltrehalose synthase acife_0609 Cold shock proteins cold-shock DNA-binding domain-containing protein acife_0085 cold-shock DNA-binding domain-containing protein acife_0976 cold-shock DNA-binding domain-containing protein acife_2932 Membrane fluidity fatty acid desaturase acife_1712 cyclopropane-fatty-acyl-phospholipid synthase acife_0120 cyclopropane-fatty-acyl-phospholipid synthase acife_0809 hypothetical protein Acife_3160

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as well as the cta gene cluster and putative regulatory genes. However, some features deviated from the A. ferrooxidansT genomic configuration. The cta gene cluster did not contain the iron responsive regulator ctaR, although candidate genes can be found elsewhere in the genome. Also, the rus gene cluster contained a hypothetical protein of unknown function only present in A. ferrooxidans ATCC 53993 and not in the type strain. The A. ferrivorans SS3 genome further contained two additional copies of rus and a copy of the iron oxidase iro (Fig. 1a and Supplementary Table 3; Paper III). Iro has been described as the main iron-oxidizing enzyme in A. ferrooxidans JCM 7811 (Fukumori, et al., 1988, Kusano, et al., 1992, Cavazza, et al., 1995). A deeper analysis of the Rus protein sequences revealed that the Rus present in the rus gene cluster was a RusA- type, similar to A. ferrooxidansT, whereas the other two were RusB-types (Supplementary Fig. 1a; Paper III). The two different types of Rus are predicted to have different kinetic properties (Ida, et al., 2003) and only RusA has been directly connected to ferrous iron oxidation. Therefore, the involvement of RusB type enzymes in ferrous iron oxidation remains to be experimentally verified. Whether the differences observed in the genome is reflected in the actual iron oxidative pathway remains to be elucidated. A. ferrivorans genes involved in ISC oxidation revealed a genomic configuration distinct from that in A. ferrooxidans and A. caldus with apparent functional redundancy (Fig. 1b, Supplementary Tables 4 and 5; Paper III). Thiosulfate is oxidized via the DoxDA (TQO) system in A. ferrooxidans and by the core TOMES in A. caldus. Both systems were present in A. ferrivorans, although the core TOMES was incomplete and appeared to be dysfunctional. Therefore, thiosulfate was most likely oxidized by DoxDA forming tetrathionate. Tetrathionate is then predicted to be metabolized by TetH. Two homologues of TetH were identified, tetH1 and tetH2. While the TetH1 protein sequence clustered with described tetrathionate hydrolyzing enzymes, TetH2 clustered with a protein from Ac. ambivalens, previously shown not to have tetrathionate hydrolyzing activity (Protze, et al., 2011). In a similar manner, two copies of sulfide-quinone reductase were present, a type I (sqr1) and a type V (sqr2). Type I Sqr has been functionally and structurally characterized in several species, including A. ferrooxidans (Wakai, et al., 2004, Wakai, et al., 2007, Zhang, et al., 2009, Cherney, et al., 2010) while no bacterial type V Sqr has been shown to oxidize sulfide. This suggested that tetrathionate and sulfide were most likely metabolized by tetH1 and sqr1, respectively. The A. ferrivorans genome further contained 2 complete systems predicted to oxidize S0, the A. ferrooxidans Hdr system and the A. caldus Sor. It was also noted that a putative thiol:disulfide interchange protein and several rhodanese-like sulfur transferases were present in the genome. Sulfite, which is formed by both Hdr and Sor, is hypothesized to be oxidized to adenosine-5’-phosphosulfate (APS) by AprAB. APS is then predicted to be oxidized by Sat to sulfate and ATP. As in A. ferrooxidans, the sulfite oxidation system in A. ferrivorans appeared to be incomplete, as only a putative sat is found. Several electron transport systems putatively involved in energy conservation during ISC oxidation were present, including three cyo clusters encoding a cytochrome bo3 oxidase (although one appears to be incomplete), a petII cluster that

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encodes a further bc1 complex, a NADH complex cluster, and an ATP synthase complex cluster. Both A. ferrooxidans and A. caldus contain bd- type cytochromes suggested to be involved in ISC metabolism although no candidate genes were identified in A. ferrivorans. Cold tolerance. A cluster of genes involved in the synthesis of the protective molecule trehalose was identified (Table 1). The cluster constitute genes for trehalose synthase (TS), glycogen debranching protein GlgX, malto-oligosyltrehalose trehalohydrolase (TreZ), and malto-oligosyltrehalose synthase (TreY). The debranching enzyme has been shown to hydrolyze the side chain of glycogen into maltodextrin, which is then further processed by TreY and TreZ (Maruta, et al., 1996). The enzyme is denoted TreX in several organisms with a similar genomic configuration. Trehalose synthesis has been proven critical for low temperature growth in E. coli (Kandror, et al., 2002). The occurrence of two different enzyme systems for trehalose synthesis in A. ferrivorans SS3 indicates its importance although, whether it is involved in low temperature growth remains to be resolved. Except for the TreY/TreZ enzyme system, which is also present in A. ferrooxidans ATCC 53993, trehalose synthesizing enzymes are absent in all other sequenced Acidithiobacillus spp. Another interesting feature is that the A. ferrivorans SS3 genome encodes three DNA-binding cold shock proteins (Table 1), whereas other sequenced Acidithiobacillus spp. only contain one each. The A. ferrivorans SS3 genome also contains genes encoding a putative δ-12 desaturase, two cyclopropane-fatty-acyl-phospholipid synthases, and a hypothetical protein of the membrane-FADS-like superfamily (fatty acid desaturase-like superfamily). The latter having 35 % identity to a hypothetical protein in the cyanobacterium Nostoc sp. PCC7120 as the best identified homologue (Table. 1). Membrane desaturation systems and cold shock proteins are critical for low temperature growth in several microorganisms (Phadtare & Inouye, 2008, Shivaji & Prakash, 2010). However, encoding several cold shock proteins does not necessarily indicate psychrotolerance, as E. coli has seven cold shock proteins and cannot grow below 10°C (Phadtare & Inouye, 2008).

Substrate utilization by Acidithiobacillus ferrivorans SS3 (Papers II and III) A. ferrivorans SS3 has previously been demonstrated to oxidize ferrous iron and ISCs at low temperature (Kupka, et al., 2007). The initial characterization of A. ferrivorans SS3 demonstrated increased generation times with decreasing pH and lower ferrous iron oxidation rates below the optimum at around 25°C (Kupka, et al., 2007). However, ISC oxidation is far less characterized. Kupka, et al. (2007) show that tetrathionate can be used as energy source and sustain growth at 5° and 30°C, but further knowledge is lacking. Therefore, a set of experiments was designed to further characterize ferrous iron and ISC oxidation in A. ferrivorans SS3 (Papers II and III). Ferrous iron and ISC oxidation rates. Elemental sulfur and tetrathionate oxidation was measured over a temperature span of 5°-30°C. As in the case of ferrous iron oxidation, tetrathionate oxidation rates decreased with lower temperatures and showed maximum oxidation rates at

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25°-26°C (Fig. 1b, c; Paper II). Activation energies were calculated from Arrhenius plots and yielded 61 kJ mol-1 (Fig. 2a; paper II) when tetrathionate consumption was measured and 89 kJ mol-1 (Fig. 2b; paper II) when pH was measured. It was also noted that at 30°C a lag phase of < 12 h was observed, whereas at 5°C a 360 h (15 days) lag phase preceded tetrathionate oxidation (Fig. 1a; Paper II). Similar lag phases were observed when incubated with elemental sulfur. However, a lower temperature optimum of about 20°C was observed even though a distinct optimum peak was absent (Fig. 1d; paper II). The broad temperature optimum suggests that the oxidation rates were not solely dependent upon temperature. The available surface area that microorganisms can attach to in order for oxidation to occur might also have influenced the oxidation rates. The activation energy calculated from sulfate production was 110 kJ mol-1 (Fig. 2b; paper II). Activation energies for ISC oxidation were all higher than the 38 kJ mol-1 calculated for ferrous iron oxidation (Kupka, et al., 2007). Oxidation of elemental sulfur at 4°C showed a 30-day long lag phase (approximately 720 h) when pre-grown on ferrous iron. Once elemental sulfur oxidation started it proceeded with a rate constant of roughly 0.044 d-1 until the pH reached the prohibitively low value of 1.1 (Fig. 3a, b; paper II). This indicated that substantial elemental sulfur oxidation was delayed until the available ferrous iron was completely oxidized. When cells were grown with both ferrous iron and elemental sulfur, ferrous iron was oxidized immediately with a rate constant of 0.12 h-1 (Fig. 4a; paper II), which is in agreement with previous data (Kupka, et al., 2007). Whether the elemental sulfur was oxidized immediately or not remains obscure (Fig. 4b, c; paper II). In an attempt to elucidate whether elemental sulfur oxidation could be coupled to ferric iron reduction, cells were grown at 4°C with elemental sulfur and ferric iron. Sulfate and proton production could be measured after approximately 60 days indicating elemental sulfur oxidation occurred (Fig. 5a, b; paper II). When pH levels decreased so that simultaneous ferrous iron oxidation was inhibited, ferrous iron accumulated in the cultures (Fig. 5c; paper II). This indicated that elemental sulfur oxidation was coupled to ferric iron reduction. Hence, the decrease in pH due to elemental sulfur oxidation slowed down ferrous iron re-oxidation, while elemental sulfur-dependent ferric reduction continued. Several strains of A. ferrooxidans use ferric iron as an electron acceptor during oxidation of elemental sulfur and hydrogen and several different enzymes have been suggested to carry out ferric reduction. A. ferrivorans SS3 mixed substrate oxidation. Previous experiments suggested that ferrous iron might be preferentially oxidized to ISCs (Paper II). This would have substantial implications on bioleaching rates since these sulfur species may not be removed until the available ferrous iron has been oxidized. In the case of elemental sulfur, which is often formed as an intermediate during bioleaching (Fig. 1), it can form a solid layer on top of the mineral, inhibiting further leaching. To further elucidate a possible substrate preference, substrate competition experiments were designed. A. ferrivorans SS3 were grown in a chemostat with changing concentrations of the substrates ferrous iron and tetrathionate. During

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cultivation, real time quantitaive-PCR expression analyses were carried out at intervals to evaluate gene expression alterations as the available substrates changed. Samples were analyzed for expression of petA1 and petB1 for genes associated with ferrous iron oxidation and cyoB for RISC oxidation. During cultivation on tetrathionate alone a basal level of petA1 and petB1 gene expression was observed but the expression of cyoB was slightly greater than petA1 and petB1 (sample points 1 and 2 in Fig. 2; Paper III). When ferrous iron was introduced alongside tetrathionate a decrease in cyoB and increase in petA1 and petB1 gene expression was observed (sample point 3 in Fig. 2; Paper III). At the last sample point when only ferrous iron was present, cyoB expression decreased 6.6 fold compared to expression on tetrathionate alone whilst petA1 and petB1 expression increased 10.6 and 10.4 fold, respectively (sample point 4 in Fig. 2; Paper III). The results suggested substrate dependent expression of petA, petB, and cyoB in the presence of ferrous iron and tetrathionate although none of the genes were totally inhibited in presence of both substrates. Considering that the concentration of tetrathionate was maintained at the same level in the first three samples, the expression of petA and petB was likely to be induced by the presence of ferrous iron. This is consistent with previous studies showing petAB up- regulation on ferrous iron as a sole energy source (Quatrini, et al., 2006, Bruscella, et al., 2007). CyoB expression levels decreased when ferrous iron was introduced to the medium indicating down regulation of ISC genes. The fairly high basal expression of petAB during tetrathionate cultivation and its strong induction along with decreased cyoB levels upon introduction of ferrous iron again indicated that ferrous iron might be preferentially oxidized. During A. ferrivorans SS3 cultivation in batch culture inoculated with cells from chalcopyrite containing media (where both substrates are available), they preferentially oxidized the ferrous iron over tetrathionate (Fig. 3; Paper III). This supports the view that ferrous iron is the preferred substrate among ferrous iron oxidizing Acidithiobacillus spp. (Ponce, et al., 2012). Influence of preference for ferrous iron on sulfide mineral leaching rates. A. ferrivorans SS3 mediated oxidation of ferrous iron and ISC at low temperature is important for heap bioleaching in boreal environments. Despite the temporally low temperatures during winter oxidation can proceed, thereby aiding heat generation within the heap to sustain the activity of meso- and thermophilic microorganisms (Dixon, 2000). Since A. ferrivorans SS3 preferentially oxidizes ferrous iron to tetrathionate when pre-cultured on the sulfide mineral chalcopyrite we aimed to investigate how this phenomenon influences sulfide mineral leaching. Leaching experiments with the sulfide minerals pyrite (leached by the thiosulfate mechanism) and chalcopyrite (leached by the polysulfide mechanism) were carried out at 4°C using A. ferrivorans SS3 and a T7 mixed culture dominated by A. ferrivorans-like species. During chalcopyrite bioleaching the S0 increased to higher levels than for pyrite bioleaching (Supplementary Table 6, Fig. 4, Supplementary Fig. 3; Paper III). In the case of T7 leaching, it was also noted that when a substantial amount of S0 accumulated (after approximately 62 days), metal release ceased, resulting in 38% and 16% of leached iron and copper, respectively. The available S0

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was only consumed once the iron and copper concentrations no longer increased (after 62 days). Finally, once the S0 concentration decreased, bioleaching continued (Supplementary Fig. 3; Paper III). This suggested that S0 passivation of the chalcopyrite occurred and that S0 was only consumed once no ferrous iron was available. A similar trend was observed during chalcopyrite bioleaching by A. ferrivorans SS3 at room temperature, with almost six times as much sulfur being accumulated with chalcopyrite compared to pyrite (Supplementary Fig. 4; Paper III). S0 accumulation during chalcopyrite bioleaching supports that ferrous iron is preferentially oxidized by A. ferrivorans SS3.

Metagenomic analysis of a low temperature acidic environment (Paper IV) Understanding of the psychrotolerant acidophilic diversity is extremely limited with only one study to date characterizing the microbial diversity in a low temperature acidic environment (Kimura, et al., 2011). In addition, previous work indicates the need to identify psychrotolerant obligate ISC oxidizers (Paper III). We used a metagenomic approach to decipher the microbial diversity and physiological potential of a low temperature acidic mine stream (Paper IV). The stream is located in an abandoned mine shaft 250 m below surface in Kristineberg mine, Västerbotten county, Sweden. At the time of sampling (April 2010) the stream water had a temperature of 6°C, a pH of 2.5, and contained 16.8 mM ferrous iron. In November 2011 the temperature had increased slightly to 11°C suggesting that temperature fluctuates according to season but still remains low all year around. The sample was divided into biofilm and planktonic fractions and sequenced. In total, 19,0511 and 8,115 contigs with 30 and 18 fold coverage were assigned to the planktonic and biofilm fractions, respectively (Supplementary Table 1; Paper IV). Phylogeny of the metagenome sample. Kristineberg mine has been previously analyzed for its microbial content using denaturing gradient gel electrophoresis (DGGE) (Figs. 1 and 4; Paper V). Further, samples taken in November 2011 were plated in an attempt to isolate new psychrotolerant species. The identified species together with the localized 16S rRNA gene sequences from the metagenomic data were analyzed for their phylogenetic relationship (Fig. 1; Paper IV). A. ferrivorans was identified using all three techniques and was present in both the planktonic and the biofilm fractions. This indicated that the sample site was dominated by A. ferrivorans. In addition, 16S rRNA gene sequences identified from the metagenomes included one clone aligning with the low temperature ferrous iron oxidizing acidophile 'Fm. myxofaciens' (Kimura, et al., 2011), three clones that aligned with the Gram positive, ferrous iron oxidizing acidophilic heterotrophs from the genera Acidimicrobium and Ferrimicrobium (Clark & Norris, 1996, Johnson, et al., 2009); two clones aligning with an uncultured Alicyclobacillus sp. acidophile (Wisotzkey, et al., 1992); and finally clones aligning with the ferrous iron oxidizing neutrophile Gallionella capsiferriformans (NC_014394, direct submission) and the low temperature species Terriglobus saanensis (Männistö, et al., 2011).

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Binning of the metagenomic sequences revealed nearly 58% and 39% of the biofilm and planktonic sequences, respectively were assigned to Acidithiobacillus spp., with the majority binned as A. ferrivorans. The next most abundant strains were binned as the ferrous iron and ISC oxidizing A. ferrooxidans followed by the ISC oxidizing A. caldus (Supplementary Fig. 5; Paper IV). The presence of contigs attributed to A. ferrooxidans and A. caldus was surprising as they are mesophilic and moderately thermophilic, respectively. A low species diversity dominated by just 2 (Tyson, et al., 2004), 7 (Bertin, et al., 2011), and 1 (Jones, et al., 2012) strain(s) appears to be a typical trait of acidic environments. Although the environment was dominated by Acidithiobacillus spp. and in particular, A. ferrivorans there was also a significant "tail" of low abundance species. These included contigs assigned to the Gram negative, organotrophic acidophile Acidobacterium spp. (Acidobacterium capsulatum) (Kishimoto, et al., 1991). In addition, contigs were binned as the neutrophilic ferrous oxidizer Gallionella spp. (Gallionella capsiferriformans) capable of growth at 6°C but not 30°C (Emerson & Moyer, 1997). Finally, the metagenome also contains contigs assigned to Terriglobus saanensis which is an organotrophic acidobacterium isolated from northern Finland that was capable of growth at pH 4.5-7.5 and 4-30°C (Männistö, et al., 2011). This suggests that the sample site is mainly sustained by ferrous iron oxidation. However, sequences binned as A. caldus indicates the presence of obligate ISC oxidizers. Further, when the Kristineberg sample was enriched on tetrathionate at 6°C, the dominant species had a 98-99% 16S sequence identity to the obligate ISC oxidizer A. thiooxidans (Fig. 7).

Fig. 7. DGGE image of enrichment cultures partial 16S clones. T3= tetrathionate pH 3; T4=tetrathionate pH 4. Band No 1 identified as similar to Acidithiobacillus thiooxidans.

Biofilm formation and cold tolerance. Several genes possibly involved in biofilm formation were identified in the metagenome, such as cellulose synthase, various glycosyl transferases, and different UDP--

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transforming enzymes. Most of the biofilm-associated genes were found in Acidithiobacillus-designated contigs, suggesting they are the major biofilm forming species in the community. Biofilm formation and production of extrapolymeric substances can serve a cryoprotective role (Krembs & Deming, 2008) and may help the microbial community at Kristineberg mine to grow at low temperature. Other genes associated with a psychrotrophic lifestyle were found in the metagenome, including genes coding for enzymes involved in membrane desaturation, protection against reactive oxygen species, and cold shock proteins. In addition, all microbial trehalose biosynthetic pathways were present and binned to A. ferrivorans (TS, TreYZ, and TreT), A. ferrooxidans (TreP), Acidobacterium capsulatum (TS and TreP), and Terriglobus saanensis (TreT), possibly involved in cryoprotection. Chemolithoautotrophy. The metagenomic data also suggested that the biofilm fraction is mainly sustained by chemolithoautotrophy. Genes associated with ferrous iron-, ISC- and hydrogen oxidation, carbon dioxide fixation, and different nitrogen assimilation systems were found. In contrast, evidence of a heterotrophic lifestyle was more pronounced in the planktonic fraction. Organic carbon is typically low in acid mine drainage environments and the primary producers of organic carbon are most likely autotrophic organisms, such as Acidithiobacillus spp. Therefore, the relatively small number of heterotrophic bacteria in the biofilm fraction was expected. The role of the heterotrophs was likely to metabolize organic carbon that can be toxic to autotrophic acidophiles (Nancucheo & Johnson, 2010).

Low temperature ISC removal by psychrotolerant acidophiles (Paper V) Process water and effluents from mining operations treating sulfide rich ores often contain considerable concentrations of ISCs. This may cause environmental problems if released to downstream recipients due to oxidation to sulfuric acid by acidophilic microorganisms (Silver & Dinardo, 1981). Previous studies have identified mesophilic and moderately thermophilic (Rzhepishevska, et al., 2005) as well as psychrotolerant (Kupka, et al., 2007) ferrous and ISC oxidizing acidophiles from the Gillervattnet tailings water in Boliden, Sweden. This suggests year round ISC oxidation may occur and to further test this, phylogenetic analysis of the tailings area and an underground acid mine drainage site was carried out using DGGE. Two streams leading from the Gillervattnet tailings pond were analyzed: Brubäcken stream which was pH adjusted and had a pH of 6.9 and the Gillervattnet stream which was not pH adjusted and had a pH of 5. Identified microorganisms in the Gillervattnet stream included species similar to the psychrotolerant ferrous iron and ISC oxidizing acidophile A. ferrivorans OP14 (Hallberg, et al., 2010). Other species identified included microorganisms related to an acidophilic heterotrophic ferrous iron oxidizer Ferrithrix thermotolerans (Johnson, et al., 2009); a neutrophilic ferrous iron oxidizer Siderooxydans paludicola (Weiss, et al., 2007); and the acid- tolerant ferrous iron oxidizers Sideroxydans lithotrophicus, Sideroxydans lithoautotrophicus, and Ferritrophicum radicola (Weiss, et al., 2007,

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Duckworth, et al., 2009, Ludecke, et al., 2010) (Fig. 2, Supplementary Fig. 1, and Supplementary Table I; Paper V). Microorganisms identified from the Brubäcken stream included an acid tolerant thiosulfate oxidizing bacterium similar to Sulfurimonas autotrophica (Inagaki, et al., 2003) and an uncultured archaeon found from a cold sulfidic spring (Rudolph, et al., 2004) (Fig. 2, Supplementary Fig. 1, and Supplementary Table I; Paper V). The data suggested that low temperature ISC oxidation occurs in both treated and untreated effluent waters causing downstream acidification in recipient waters. Also, an acidic stream at 250 m depth in an abandoned mine shaft in Kristineberg mine was analyzed. DGGE analysis identified A. ferrivorans-like species and a species distantly related to a moderate thermophilic Sulfobacillus sp. Development of the bioreactor system. A bioreactor system was developed to investigate if the indigenous microbial flora could be used for removal of ISCs at in situ temperatures in northern Sweden. This has the potential to reduce operating costs by removing the need to heat the process water and reduce consumption of chemicals to treat the process waters. The bioreactor temperature was successively lowered from 37°C to 5°C under various reactor conditions (Table 1; Paper V). In order to promote biofilm formation and avoid wash-out of active microorganisms two types of biofilm carriers was tested in the main reactor: activated carbon and the MBBRTM carrier (AnoxKaldnes AB, Lund, Sweden). As the temperature was reduced below 19°C the main reactor containing activated carbon was inoculated with an enrichment culture from the Kristineberg mine. Efficient ISC removal was sustained as the main reactor temperature decreased all the way to 5°C. However, when the biogenerator reactor (used to replace the microbial population in the main bioreactor) temperature was decreased from 19°C to 5°C, increasing amounts of tetrathionate could be measured in the discharge (Tables 1 and 2; Paper V). This indicated that while ISC removal was successful at 5°C, the biogenerator needed to be maintained at higher temperatures. A second experiment using the MBBRTM carrier largely confirmed the previous results (Tables 1 and 2; Paper V). Phylogeny of ISC removing species. Along with the bioreactor development, the microbial populations responsible for ISC removal was phylogenetically analyzed using DGGE. As mentioned previously, the mine stream in the Kristineberg mine, which was enriched and used as inoculum for low temperature removal, contained mainly A. ferrivorans-like species but also species with low similarity to Sulfobacillus sp., suggesting a new species might have been present. In addition, based on similar band migrations to those found in the biogenerator the inoculum also contained T. plumbophilus-like and Frateuria-like species as well as an un-identified bacterial clone. Both T. plumbophilus and Frateuria sp. have been documented in mining-impacted environments (Drobner, et al., 1992, Coupland & Johnson, 2008). The Kristineberg inoculum was added to the reactors on day 29 and the population in R1 and R2 appeared similar as found in the inoculum. No additional species were observed after 49 days but some selection for A. ferrivorans and Frateuria sp. seemed to have occurred. Finally, the two A. ferrivorans-like clones were more active in the

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main reactor after 141 days suggesting they were responsible for the majority of the thiosalt oxidation (Figs. 2 and 4, Supplementary Table 3; Paper V). The data suggested that a significant part of the acid generating potential from ISCs can be removed from mining process waters and effluents by means of psychrotolerant microbial oxidation.

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Concluding remarks

¾ The A. ferrivorans SS3 genome contains a full repertoire of iron- and ISC oxidizing genes as well as genes for hydrogen oxidation, nitrogen and carbon dioxide fixation, indicating ecological flexibility. The genome also contained genes potentially involved in cold tolerance, such as trehalose synthesis and membrane desaturation.

¾ A. ferrivorans SS3 can oxidize iron and ISCs efficiently at low temperature and displayed the potential of utilizing ferric iron as an alternative electron acceptor.

¾ A. ferrivorans SS3 preferentially oxidized ferrous iron to ISCs that resulted in the accumulation of elemental sulfur during chalcopyrite bioleaching.

¾ Analysis of a cold, acidic biofilm revealed the presence of additional potentially psychrotolerant species, including ISC-oxidizers that may improve efficiency of bioleaching at low temperature.

¾ Psychrotolerant acidophiles can be used in low temperature removal of ISCs from mining process water.

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Acknowledgements

While I was studying science prep school (basåret in Swedish) I saw this documentary on TV about sulfur caves in Mexico. Huge colorful biofilms and stone eating bacteria. I was hooked. So, thank you Mark for fulfilling that dream and introducing me to the coolest bug of them all – Acidithiobacillus ferrivorans SS3! You dragged me through this adventure: pushed me when I was standing still, held me back when I got carried away and released me when I’ve had enough. I wish you all the best in the future and good luck with Anders! ‘blink blink’

My fellow bioleachers: Stefanie − ‘Vino tinto at Rio Tinto’. I’m going to miss you. You’ve been a great friend, a fun fellow co-worker and an excellent vice supervisor! I wish you all the best! Siv – gruppens stöttepelare. Äntligen har du ny dator och eget kontor! Joanna – you were truly the boss. Your great mind doesn’t compare. Shabir – ‘Puss puss’. ‘Acha, acha, acha’… Olena, du kommer alltid tillhöra oss, även om du jobbar med nåt annat skräp nu. All the former project students and guest workers - you made the lab less empty and the office less quite.

Department of MolBiol is the best department of all! Great service + Great people = Great science!

Jaakko and Olli for kindly having me in Tampere, working up my first publication, and Pauliina for helping and taking care of me during my stay. Jan-Eric – THANK YOU for letting me visit the absolutely, undoubtedly coolest place on earth: Kristineberg mine!

All my friends – I’m so greatful for having you guys in my life! From now on I shall spend more time with you.

Mamma. Pappa. Lillskrutt. Jag älskar er! Tack för att ni alltid finns där för mig! Tack för att ni alltid är så stolta över mig! Nu får ni här en riktig bok att visa upp istället för den sketna magisteruppsatsen! Olof – du är den bästa svågern jag någonsin kunde fått! Tack för att du tar hand om och älskar min allra finaste syster. Vi älskar dig tillbaka. Min älskade Jonas – vad kan jag säga? Utan dig är jag ingenting! Eller kanske snarare, med dig är jag allt! Jag är så evigt tacksam för att jag får känna dig. För att jag får leva med dig. För att jag får älska dig. För att du älskar mig. Från och med nu kommer jag alltid att finnas vid din sida – inget mer Umeå. Inget mer Tammerfors. Bara här. Med dig. Jag älskar dig.

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