Function and Adaptation of Acidophiles in Natural and Applied Communities

Home , Acidianus infernus, Acidiplasma, Acidithiobacillus caldus, Acidophile, Aciduliprofundum boonei, Ferrithrix

Stephan Christel Stephan

Linnaeus University Dissertations No 328/2018

Stephan Christel and Applied Communitiesand Applied Function and Adaptation of Acidophiles in Natural

Function and Adaptation of Acidophiles in Natural and Applied Communities

Lnu.se ISBN: 978-91-88761-94-1 978-91-88761-95-8 (pdf )

linnaeus university press Function and Adaptation of Acidophiles in Natural and Applied Communities Linnaeus University Dissertations No 328/2018

FUNCTION AND ADAPTATION OF ACIDOPHILES IN NATURAL AND APPLIED COMMUNITIES

STEPHAN CHRISTEL

LINNAEUS UNIVERSITY PRESS

Abstract Christel, Stephan (2018). Function and Adaptation of Acidophiles in Natural and Applied Communities, Linnaeus University Dissertations No 328/2018, ISBN: 978-91-88761-94-1 (print), 978-91-88761-95-8 (pdf). Written in English. Acidophiles are organisms that have evolved to grow optimally at high concentrations of protons. Members of this group are found in all three domains of life, although most of them belong to the Archaea and Bacteria. As their energy demand is often met chemolithotrophically by the oxidation of basic ions 2+ and molecules such as Fe , H2, and sulfur compounds, they are often found in environments marked by the natural or anthropogenic exposure of sulfide minerals. Nonetheless, organoheterotrophic growth is also common, especially at higher temperatures. Beside their remarkable resistance to proton attack, acidophiles are resistant to a multitude of other environmental factors, including toxic heavy metals, high temperatures, and oxidative stress. This allows them to thrive in environments with high metal concentrations and makes them ideal for application in so-called biomining technologies. The first study of this thesis investigated the iron-oxidizer Acidithiobacillus ferrivorans that is highly relevant for boreal biomining. Several unresolved nodes of its sulfur metabolism were elucidated with the help of RNA transcript sequencing analysis. A model was proposed for the oxidation of the inorganic sulfur compound tetrathionate. In a second paper, this species’ transcriptional response to growth at low temperature was explored and revealed that At. ferrivorans increases expression of only very few known cold-stress genes, underlining its strong adaptation to cold environments. Another set of studies focused on the environmentally friendly metal- winning technology of bioleaching. One of the most important iron-oxidizers Function and Adaptation of Acidophiles in Natural and Applied in many biomining operations is Leptospirillum ferriphilum. Despite its Communities significance, only a draft genome sequence was available for its type strain. Doctoral Dissertation, Department of Biology and Environmental Sciences, Therefore, in the third paper of this thesis we published a high quality, closed Linnaeus University, Kalmar, 2018 genome sequence of this strain for future use as a reference, revealing a previously unidentified nitrogen fixation system and improving annotation of ISBN: 978-91-88761-94-1 (print), 978-91-88761-95-8 (pdf) genes relevant in biomining environments. In addition, RNA transcript and Published by: Linnaeus University Press, 351 95 Växjö protein patterns during L. ferriphilum’s growth on ferrous iron and in Printed by: DanagårdLiTHO, 2018 bioleaching culture were used to identify key traits that aid its survival in extremely acidic, metal-rich environments. The biomining of copper from chalcopyrite is plagued by a slow dissolution rate, which can reportedly be circumvented by low redox potentials. As conventional redox control is impossible in heap leaching, paper four explored the possibility of using differentially efficient ironoxidizers to influence this parameter. The facultative heterotrophic Sulfobacillus thermosulfidooxidans was identified as maintaining a redox potential of ~550 mV vs Ag/AgCl, favorable for chalcopyrite dissolution, Abstract Christel, Stephan (2018). Function and Adaptation of Acidophiles in Natural and Applied Communities, Linnaeus University Dissertations No 328/2018, ISBN: 978-91-88761-94-1 (print), 978-91-88761-95-8 (pdf). Written in English. Acidophiles are organisms that have evolved to grow optimally at high concentrations of protons. Members of this group are found in all three domains of life, although most of them belong to the Archaea and Bacteria. As their energy demand is often met chemolithotrophically by the oxidation of basic ions 2+ and molecules such as Fe , H2, and sulfur compounds, they are often found in environments marked by the natural or anthropogenic exposure of sulfide minerals. Nonetheless, organoheterotrophic growth is also common, especially at higher temperatures. Beside their remarkable resistance to proton attack, acidophiles are resistant to a multitude of other environmental factors, including toxic heavy metals, high temperatures, and oxidative stress. This allows them to thrive in environments with high metal concentrations and makes them ideal for application in so-called biomining technologies. The first study of this thesis investigated the iron-oxidizer Acidithiobacillus ferrivorans that is highly relevant for boreal biomining. Several unresolved nodes of its sulfur metabolism were elucidated with the help of RNA transcript sequencing analysis. A model was proposed for the oxidation of the inorganic sulfur compound tetrathionate. In a second paper, this species’ transcriptional response to growth at low temperature was explored and revealed that At. ferrivorans increases expression of only very few known cold-stress genes, underlining its strong adaptation to cold environments. Another set of studies focused on the environmentally friendly metal- winning technology of bioleaching. One of the most important iron-oxidizers Function and Adaptation of Acidophiles in Natural and Applied in many biomining operations is Leptospirillum ferriphilum. Despite its Communities significance, only a draft genome sequence was available for its type strain. Doctoral Dissertation, Department of Biology and Environmental Sciences, Therefore, in the third paper of this thesis we published a high quality, closed Linnaeus University, Kalmar, 2018 genome sequence of this strain for future use as a reference, revealing a previously unidentified nitrogen fixation system and improving annotation of ISBN: 978-91-88761-94-1 (print), 978-91-88761-95-8 (pdf) genes relevant in biomining environments. In addition, RNA transcript and Published by: Linnaeus University Press, 351 95 Växjö protein patterns during L. ferriphilum’s growth on ferrous iron and in Printed by: DanagårdLiTHO, 2018 bioleaching culture were used to identify key traits that aid its survival in extremely acidic, metal-rich environments. The biomining of copper from chalcopyrite is plagued by a slow dissolution rate, which can reportedly be circumvented by low redox potentials. As conventional redox control is impossible in heap leaching, paper four explored the possibility of using differentially efficient ironoxidizers to influence this parameter. The facultative heterotrophic Sulfobacillus thermosulfidooxidans was identified as maintaining a redox potential of ~550 mV vs Ag/AgCl, favorable for chalcopyrite dissolution, while L. ferriphilum caused the potential to raise far above this critical value. RNA transcript analysis was used to identify genomic features that may contribute to this behavior. Lastly, six fields in Northern Sweden were examined for the presence of acid sulfate soils in the fifth paper. The study revealed three acid sulfate soils. The presence of acidophiles that likely catalyze the production of acid in the soil was confirmed by community 16S gene amplicon analysis. One site that was flooded in a remediation attempt and is therefore anoxic still exhibited similar bacteria, however, these now likely grow via ferric iron reduction. This process consumes protons and could explain the observed rise in pH at this site. This thesis examines acidophiles in pure culture, as well as natural and designed communities. Key metabolic traits involved in the adaptation to their habitats were elucidated, and their application in mining operations was discussed. Special attention was paid to acidophiles in chalcopyrite bioleaching and in cold environments, including environmental acid sulfate soils in Northern Sweden.

Keywords: Acidophiles; Biomining; Psychrophiles; Adaptation; Acid Sulfate Soil; Redox Control while L. ferriphilum caused the potential to raise far above this critical value. RNA transcript analysis was used to identify genomic features that may contribute to this behavior. Lastly, six fields in Northern Sweden were examined for the presence of acid sulfate soils in the fifth paper. The study revealed three acid sulfate soils. The presence of acidophiles that likely catalyze the production of acid in the soil was confirmed by community 16S gene amplicon analysis. One site that was flooded in a remediation attempt and is therefore anoxic still exhibited similar bacteria, however, these now likely grow via ferric iron reduction. This process consumes protons and could explain the observed rise in pH at this site. This thesis examines acidophiles in pure culture, as well as natural and designed communities. Key metabolic traits involved in the adaptation to their habitats were elucidated, and their application in mining operations was discussed. Special attention was paid to acidophiles in chalcopyrite bioleaching and in cold environments, including environmental acid sulfate soils in Northern Sweden.

Keywords: Acidophiles; Biomining; Psychrophiles; Adaptation; Acid Sulfate Soil; Redox Control ”An expert is a person who has found out by his own painful experience all the mistakes that one can make in a very narrow field.”

Niels Bohr

Cover photo: Extremely acidic water of Rio Tinto (Spain), stained deeply red by vast concentrations of dissolved iron. Rights: Wikimedia Commons ”An expert is a person who has found out by his own painful experience all the mistakes that one can make in a very narrow field.”

Niels Bohr

Cover photo: Extremely acidic water of Rio Tinto (Spain), stained deeply red by vast concentrations of dissolved iron. Rights: Wikimedia Commons Table of Contents

List of Publications 7 Includedpublications...... 7 Author’scontributions...... 8 Publications not discussed in this thesis ...... 9

Abbreviations 11

Introduction 13

Acidophiles 17 Diversity of acidophilic prokaryotes ...... 18 Bacteria...... 18 Archaea...... 24 Energy and carbon metabolism ...... 28 Iron...... 29 Inorganic sulfur compounds ...... 31 Hydrogen...... 34 Carbon...... 34 Challenges and adaptations of life in acid ...... 36 pH...... 36 Heavymetals...... 37 Oxidativestress...... 39 Polyextremophiles...... 41 Temperature: Psychro- and thermoacidophiles ...... 41 Environmental and ecological implications of acidophiles ...... 44 Acidrockandminedrainage...... 44

5 Table of Contents

List of Publications 7 Includedpublications...... 7 Author’scontributions...... 8 Publications not discussed in this thesis ...... 9

Abbreviations 11

Introduction 13

5 Acidsulfatesoils...... 48 Volcanic and geothermal environments ...... 49 Applicationsofacidophiles...... 52 Biomining...... 52 Bioprospecting and genetic engineering ...... 57

Aims of this Thesis 59 List of Publications Methodology 61 Experimentsandsampling...... 61 Sequencing...... 63 Included publications Multi”-omics”analysis...... 63 The results of the following published or submitted manuscripts contributed to the content of this thesis. In the main text, they will be referred to by their roman Summary of Results 65 numerals as indicated here. All papers are reprinted with the respective publishers’ Papers I-IV: Systems biology of biomining organisms ...... 67 permission. Paper V: Acid sulfate soil in Sweden ...... 70

Conclusions 73 I Christel S, Fridlund J, Buetti-Dinh A, Buck M, Watkin EL, Dopson M. 2016. RNA transcript sequencing reveals inorganic sulfur compound Acknowledgements 75 oxidation pathways in the acidophile Acidithiobacillus ferrivorans. FEMS References 79 Microbiology Letters. doi:10.1093/femsle/fnw057.

II Christel S, Fridlund J, Watkin EL, Dopson M. 2016. Acidithiobacillus ferrivorans SS3 presents little RNA transcript response related to cold stress during growth at 8 °C suggesting it is a eurypsychrophile. Extremophiles. doi:10.1007/s00792-016-0882-2.

III Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin IV, Sand W, Wilmes P, Poetsch A, Dopson M. 2017. Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilumT. Applied and Environmental Microbiology. doi:10.1128/AEM.02091-17.

6 7 Acidsulfatesoils...... 48 Volcanic and geothermal environments ...... 49 Applicationsofacidophiles...... 52 Biomining...... 52 Bioprospecting and genetic engineering ...... 57

6 7 IV Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin Publications not discussed in this thesis IV, Sand W, Wilmes P, Poetsch A, Dopson M. Low-rate iron oxidation by In addition to the work conducted towards this thesis, several more projects and Sulfobacillus thermosulfidooxidans maintains a favorable redox potential collaborations were completed, and resulted in the following publications: for chalcopyrite bioleaching. Manuscript submitted

Ni G, Christel S, Roman P, Wong ZL, Bijmans MF, Dopson M. 2016. V Christel S, Yu C, Wu X, Josefsson S, Sohlenius G, Åström M, Dopson ◦ M. Comparison of Boreal Acid Sulfate Soil Microbial Communities in Electricity generation from an inorganic sulfur compound containing mining Oxidative and Reductive Environments. Manuscript submitted waste water by acidophilic microorganisms. Research in Microbiology. doi:10.1016/j.resmic.2016.04.010.

Broman E, Jawad A, Wu X, Christel S, Ni G, Lopez-Fernandez M, Sund- ◦ kvist JE, Dopson M. 2017. Low temperature, autotrophic microbial denitri- fication using thiosulfate or thiocyanate as electron donor. Biodegradation. doi:10.1007/s10532-017-9796-7. Author’s contributions

The author of this thesis has contributed to the discussed publications in the Högfors-Rönnholm E, Christel S, Dalhem K, Lillhonga T, Engblom S, ◦ following manner: Osterholm P, Dopson M. 2017. Chemical and microbiological evaluation of novel chemical treatment methods for acid sulfate soils. Science of the Total Environment. doi:10.1016/j.scitotenv.2017.12.287. I Sampling, Sequence analysis, Data interpretation, Manuscript

Högfors-Rönnholm E, Christel S, Engblom S, Dopson M. 2018. Indirect II Sampling, Sequence analysis, Data interpretation, Manuscript ◦ DNA extraction method suitable for acidic soil with high clay content. MethodsX. doi:10.1016/j.mex.2018.02.005. III Experiments, Sampling, Data interpretation, Manuscript

Bellenberg S, Buetti-Dinh A, Galli V, Ilie O, Herold M, Christel S, Boretska IV Experiments, Sampling, Data interpretation, Manuscript ◦ M, Pivkin IV, Wilmes P, Sand W, Vera M, Dopson M. 2018. Automated microscopical analysis of metal sulfide colonization by acidophilic microorgan- V Sequence analysis, Data interpretation, Manuscript isms. Applied and Environmental Microbiology. doi:10.1128/aem.01835-18.

8 9 IV Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin Publications not discussed in this thesis IV, Sand W, Wilmes P, Poetsch A, Dopson M. Low-rate iron oxidation by In addition to the work conducted towards this thesis, several more projects and Sulfobacillus thermosulfidooxidans maintains a favorable redox potential collaborations were completed, and resulted in the following publications: for chalcopyrite bioleaching. Manuscript submitted

8 9 Abbreviations

Aa. Acidianus (archaeal genus) Ac. Acidiphilium (bacterial genus) Acb. Alicyclobacillus (bacterial genus) Acc. Acidocella (bacterial genus) Acd. Acidicaldus (bacterial genus) Acx. Acidithrix (bacterial genus) Ah. Acidihalobacter (bacterial genus) Am. Acidimicrobium (bacterial genus) AMD acid mine drainage Ap. Acidiplasma (archaeal genus) ARD acid rock drainage ASS acid sulfate soil At. Acidithiobacillus (bacterial genus) ATP adenosine triphosphate

CIP cold induced protein CSP cold shock protein

Ds. Desulfosporosinus (bacterial genus)

EPS extracellular polymeric substances

Fp. Ferroplasma (archaeal genus) Fv. Ferrovum (bacterial genus) Fx. Ferrithrix (bacterial genus)

H. Hydrogenobaculum (bacterial genus)

ISC inorganic sulfur compound

11 Abbreviations

CIP cold induced protein CSP cold shock protein

Ds. Desulfosporosinus (bacterial genus)

EPS extracellular polymeric substances

Fp. Ferroplasma (archaeal genus) Fv. Ferrovum (bacterial genus) Fx. Ferrithrix (bacterial genus)

H. Hydrogenobaculum (bacterial genus)

ISC inorganic sulfur compound

11 L. Leptospirillum (bacterial genus)

M. Methylacidiphilum (bacterial genus) Ms. Metallosphaera (archaeal genus)

NADH nicotinamide adenine dinucleotide NGS next generation sequencing

ORP oxidation/reduction potential Introduction OTU operational taxonomic unit From the peaks of the Himalaya (Sanyal et al. 2018), to the very depths of the P. Picrophilus (archaeal genus) ocean’s Marianna trench (Peoples et al. 2018), Earth is saturated with life. While PASS potential acid sulfate soil some of it evolved to exceedingly complex multicellular organisms, the vast majority PMF proton motive force of life forms are not quite as complicated. These organisms have proliferated in the form of single cells since the very beginning of life on Earth, billions of years ago. ROS reactive oxygen species Their growth is powered by the harvest of energy released by chemical reactions RuBisCo ribulose bisphosphate carboxylase (Demirel & Sandler 2002) and due to their great adaptability, time allowed bacterial, archaeal, and eukaryotic life to expand into extreme environments in every possible S. Sulfobacillus (bacterial genus) direction (Madigan & Martinko 2006). Just as in the rich compost of ones backyard, Sb. Sulfolobus (archaeal genus) microbes colonize the sediments of frozen lakes in Antarctica (Sapp et al. 2018), Sl. Stygiolobus (archaeal genus) float in the dark cracks of deep bed rock 500 meter below the Baltic Sea (Wu et al. STR stirred tank reactor 2017), multiply in the boiling temperatures of Icelandic geysers (Gaisin et al. 2017), TCA Tricarboxylic acid cycle and swim through the salty waters of the Dead Sea (Bodaker et al. 2010). Not even Tp. Thermoplasma (archaeal genus) the hard vacuum of outer space appears to be beyond the limits of what single-celled life can endure, some of it being able to tolerate the complete lack of pressure and extreme solar radiation for extended periods of time (Moissl-Eichinger et al. 2016). With this in mind, there is little doubt that life on earth will continue, in one form or another, long after humankind eventually disappears. Due to the extreme nature of the environments they have adapted to inhabit, some of these life forms are referred to as extremophiles (Durvasula & Rao 2018). Members of this group are found in all three domains of life, but are more common in the prokaryotic world of Bacteria and Archaea (Figure 1). While species resistant to a multitude of extreme conditions exist, most are adapted to cope with fewer, often related stressful environmental factors. These include high temperature (Urbieta et al. 2015), high salinity (Bowers & Wiegel 2011), or high radiation levels (Ragon et al. 2011). Beside their resilience to conditions that would kill most other organisms,

12 13 L. Leptospirillum (bacterial genus)

M. Methylacidiphilum (bacterial genus) Ms. Metallosphaera (archaeal genus)

NADH nicotinamide adenine dinucleotide NGS next generation sequencing

12 13 Introduction

Figure 1: Phylogenetic tree adapted from Dalmaso et al. (2015), illustrating the presence of extremophiles within all three domains and life, along with the resistant characteristic appearing in at least one species of each genera indicated by color. extremophiles have in common that their metabolism is often simple and streamlined (Sabath et al. 2013; Saha et al. 2014) to reduce the amount of energy necessary to maintain cellular functions. The combination of these factors makes them interesting subjects in the study of the origin of life on earth (Rampelotto 2013). In the early days of earth, conditions were harsh, hot, and acidic (Ushikubo et al. 2008); shaped by high concentrations of sulfur compounds and carbon dioxide originating from volcanic activity (Nisbet & Sleep 2001). Complex organic molecules usable to gain energy were scarce (Westall et al. 2011). Environments like this exist to this day (Figure 2), for example in naturally exposed deposits of sulfide minerals, such as Figure 2: Acidophiles often inhabit sulfur-rich, acidic environments, such as Rio Tinto in the Iberian Pyritic Belt in Southern Spain (A), and the Norris Geyser Basin of Yellowstone National in the Iberic Pyritic Belt (Spain), in the acidic pools of Yellowstone National Park Park in Wyoming, USA (B). Photo rights: Flickr Creative Commons (USA), but also in man-made environments heavily influenced by mining activity (Simate & Ndlovu 2014). Here, the first acidophiles, acid-loving organisms, were

14 15 Introduction

14 15 Introduction isolated in the early 20th century (Temple & Colmer 1951; Waksman & Joffe 1922). Their capability to grow at low pH, using only basic ions and molecules such as iron and sulfur compounds to gain the energy needed to multiply, have recently also put them in the spotlight of investigations on how life on earth originated (Holmes 2017). However, regardless of the possibility of acidophiles being among the first life forms on Earth, organisms classified as such were, and are, immensely important in the geochemical cycles of our planet (Druschel et al. 2004) and profoundly influence the world that we live in. Acidophiles

Acidophiles are defined as organisms capable of sustaining cellular functions and growth at pH lower than 5. Consequently, microbes with even lower optima, i.e. pH<3, are called extreme acidophiles (Johnson 2007). Habitats providing such high concentrations of protons are relatively scarce (Quatrini & Johnson 2018), but originate from various sources. In nature, acidic environments are often connected to volcanic activity (Armienta et al. 2000; Varekamp 2008) or naturally exposed sulfide minerals that are oxidized to sulfuric acid by atmospheric oxygen (Furniss et al. 1999; Kwong et al. 2009). Since the beginning of the anthropocene, human activities also contribute significantly to these environments, for example by careless dumping of coal spoils and sulfidic ore tailings (see section Acid rock and mine drainage) or drainage of wetlands and their underlying sulfidic sediments (see section Acid sulfate soils). Just as extremophiles in general, acidophiles consist of members of all three domains of life. Among acidophilic eukaryotes, the most prominent are microalgae, protists, and fungi, that all contribute significantly to the diversity in acidic habitats (Baker et al. 2004). However, as most acidophilic species are enriched within the Bacteria and Archaea (Quatrini & Johnson 2016), this thesis will concentrate on members of these domains.

16 17 Introduction isolated in the early 20th century (Temple & Colmer 1951; Waksman & Joffe 1922). Their capability to grow at low pH, using only basic ions and molecules such as iron and sulfur compounds to gain the energy needed to multiply, have recently also put them in the spotlight of investigations on how life on earth originated (Holmes 2017). However, regardless of the possibility of acidophiles being among the first life forms on Earth, organisms classified as such were, and are, immensely important in the geochemical cycles of our planet (Druschel et al. 2004) and profoundly influence the world that we live in. Acidophiles

16 17 Acidophiles Diversity of acidophilic prokaryotes

Diversity of acidophilic prokaryotes

The recognized diversity in acidophilic Bacteria and Archaea has steadily increased since the discovery of the first acidophile, Acidithiobacillus thiooxidans, by Waksman & Joffe (1922). Yet, with the beginning of the new millennium, next generation sequencing (NGS) has greatly accelerated the identification of acidophilic organisms. While in the past microbial diversity was heavily biased towards what species were able to grow under laboratory conditions, today, cultivation has become largely unnecessary to identify new microbial species, as genome sequences can be assembled directly from environmental samples by metagenomics (Cardenas et al. 2010; Cowan et al. 2015). Further, advances in phylogenetics, such as 16S rRNA gene typing or more recently, multi-locus sequencing analysis have rigorously chal- lenged and improved taxonomic classification of microbial species and clades, as well as their placement in the universal tree of life (Hug et al. 2016; Nuñez et al. 2017).

Bacteria

Despite the challenging nature of their environment, acidophiles exhibit a high degree of phylogenetic diversity. Thousands of isolated strains are capable of growing at low pH, although within the Bacteria, each of them belongs to one of only six phyla: Proteobacteria, Nitrospirae, Firmicutes, Actinobacteria, Aquificae, or Verrucomicrobia (Figure 3; Dopson 2016). Considering the low species saturation of many known bacterial phyla, it is easily conceivable that this list may have to be extended in the future. Within their phyla, extreme acidophiles often cluster seperately, mostly on the genus level, as e.g. the genera Acidithiobacillus or Leptospirillum. This is albeit not always the case, and extremely acidophilic species can be intermixed in otherwise neutrophilic clades, e.g. within the Alicyclobacilli (Ciuffreda et al. 2015). Apart from the common resistance against low pH in acidophile clades, great heterogeneity often occurs in other metabolic aspects. This includes preference of electron donors and acceptors, carbon source, and other chemical and physical parameters (see section Energy and carbon Figure 3: Unrooted phylogenetic tree of 16S rRNA gene sequences from selected acidophilic metabolism and Challenges and adaptations of life in acid). A full account of the Bacteria, collected from SILVA (Quast et al. 2013). Phylogeny was inferred using FastTree (Price currently known bacterial acidophilic diversity is therefore beyond the scope of this et al. 2010) and the tree drawn by iTOL (Letunic & Bork 2016). Bootstraps < 0.5 are omitted. thesis. Nevertheless, ecologically and technologically important acidophiles will be explored in more detail in the following sections.

18 19 Acidophiles Diversity of acidophilic prokaryotes

Diversity of acidophilic prokaryotes

Bacteria

18 19 Acidophiles Diversity of acidophilic prokaryotes

Proteobacteria (Harrison 1984; Liu et al. 2011). Growth of Acidiphilium spp. is not limited to One of the most important acidophilic clades is the genus Acidithiobacillus within mildly acidic niches like for many other organoheterotrophic acidophiles (Jones the Proteobacteria class Acidithiobacillia (Williams & Kelly 2013) that contains et al. 2013), but ranges from near neutral pH 6 to extremely acidic pH 1, with the first acidophile ever isolated, At. thiooxidans. This species was originally reported optima around 3.5 at temperatures between 25-40 °C (Delabary et al. 2017; described as Thiobacillus thiooxidans, but has since then been reclassified (Kelly Kishimoto et al. 1995). This variability makes them the most frequently encountered & Wood 2000). While still intensely investigated, seven distinct species are organoheterotrophs in a wide range of acidic environments (Hamamura et al. 2005; currently described within the genus, including many of the most prominently Kay et al. 2013; Wichlacz et al. 1986). studied acidophiles, namely the mentioned At. thiooxidans plus At. ferooxidans, The species split from Acidiphilium genus to form the novel Acidocella include At. caldus, At. ferrivorans, At. ferridurans, At. ferriphilus, and At. albertensis Acidocella facilis, and Acc. aminolytica. More recently, two further species were (Nuñez et al. 2017). All of these species are gram-negative, rod-shaped autotrophs added to the group, Acc. aluminiidurans and Acc. aromatica (Jones et al. 2013; that are capable of oxidizing elemental sulfur and other inorganic sulfur compounds Kimoto et al. 2010). While on the 16S rRNA gene level they differ sufficiently to (ISCs) coupled to the reduction of oxygen for energy generation. Those that are justify the formation of a new genus, and each species maintains a specialized gene named to include a derivative of the latin ferrum (i.e. ferri/ferro) additionally set, Acidocella spp. are metabolically similar to their sister genus Acidiphilium, and have the capability to aerobically oxidize ferrous iron (Fe2+), and even use ferric inhabit the same temperature range albeit with a slightly higher pH niche (Jones et iron (Fe3+) as an electron acceptor for anaerobic growth. Some Acidithiobacillus al. 2013). spp. can also use elemental hydrogen as a energy source (Drobner et al. 1990). Lastly of note within the Proteobacteria is Acidicaldus organivorans, the sole All Acidithiobacilli are extreme acidophiles, and exhibit optimal growth at pH member of its genus. This species exhibits the highest optimal growth temperature values between 2-2.5 under mesophilic temperature conditions of 30-45 °C (Kelly of the phylum’s acidophiles, 50-55 °C at a pH of 2.5-3. It is capable of aerobic & Wood 2000). As an exception, At. ferrivorans has been described to be greatly sulfur oxidation, but as most thermophilic organisms, Acd. organivorans grows best tolerant to colder environments (Christel et al. 2016b; Hallberg et al. 2010). This organoheterotrophically. Of particular interest is that Acd. organivorans obtains characteristic is rather uncommon among acidophiles, reflected in this species being highest cell densities while degrading aromatic and phenolic compounds, substrates the only Acidithiobacillus found in boreal climates or at high altitudes (see section also used by Acc. aromatica, but otherwise rarely found to be used by acidophiles Temperature: Psychro- and thermoacidophiles). (Johnson et al. 2006). The genus Acidiphilium lies within the α-Proteobacteria, and is the second largest clade of acidophiles in the Proteobacteria phylum, despite being split by Nitrospirae the reclassification of two members to form the novel sister genus Acidocella Also of large importance within the bacterial acidophiles are members of the (Kishimoto et al. 1995). The genus’ remaining species include Acidiphilium Nitrospirae genus Leptospirillum, which are characterized by their spiral shape. acidophilum, Ac. cryptum, Ac. angustum, and Ac. rubrum. Acidiphilum spp. are Similar to the Acidithiobacilli, they are gram-negative, autotrophic, and most strains gram-negative, motile rods that gain their energy exclusively from oxidizing organic possess the capability to fix nitrogen (Christel et al. 2017; Parro & Moreno-Paz substrates or in some cases ISCs, using both oxygen and ferric iron as terminal 2004). In addition, they prefer similar although slightly higher temperature ranges electron acceptors. Ac. acidophilum is the only member of this genus able to aquire of around 30-45 °C (Schrenk et al. 1998). Leptospirilli do generally have lower carbon autotrophically (Coupland & Johnson 2008; Guay & Silver 1975). This pH optima between 1-1.6 compared to the Acidithiobacilli (Hippe 2000) and gain species was in fact previously assigned to the Thiobacilli (now Acidithiobacilli), their energy exclusively by the aerobic oxidation of iron (Coram & Rawlings 2002). as it was isolated from a culture previously considered purely consisting of At. Their iron oxidation systems are highly effective, allowing them to out-compete ferrooxidans, in which both species proliferated in an indistinguishable symbiosis most other iron-oxidizers (Rawlings et al. 1999). Therefore, Leptospirillum spp.

20 21 Acidophiles Diversity of acidophilic prokaryotes

20 21 Acidophiles Diversity of acidophilic prokaryotes often constitute a large portion of the microbial population in environments with mesophile, and thermophile species, although most grow optimally in moderately exceedingly low pH and high metal concentrations (see sections Acid rock and thermophilic conditions (Ciuffreda et al. 2015). While many Alicyclobacillus spp. mine drainage and Biomining). Three recognized species are comprised in the are obligate organoheterotrophs, some exhibit metabolic properties similar to the genus; Leptospirillum ferrooxidans, L. ferriphilum, and L. rubarum. In addition, Sulfobacilli. For example, Acb. aeris, Acb. ferrooxydans, and Acb. contaminans two candidate species have been identified by metagenomic approaches, and were are all capable of iron and ISC oxidation, despite growing faster on organic substrates preliminary named ’L. ferrodiazotrophum’, and ’Leptospirillum sp. group IV UBA (Goto et al. 2007; Guo et al. 2009; Jiang et al. 2008). BS’ (Goltsman et al. 2013). Actinobacteria Firmicutes The acidophile genera of the phylum Actinobacteria are not as well explored, al- The genus Sulfobacillus lies within the phylum Firmicutes, and contains five though several of them have been recognized (Figure 3). Acidimicrobium ferrooxi- identified species, namely Sulfobacillus acidophilus, S. sibiricus, S. benefacians, S. dans is the sole member of its genus (Clark & Norris 1996). It is capable of efficient thermosulfidooxidans, and S. thermotolerans. All of these organisms are moderately autotrophic growth using ferrous iron or molecular hydrogen, but not ISCs, as a en- thermophilic, preferring growth temperatures of 45-55 °C, and a pH between 1.5- ergy source as well as organoheterotropically in presence of yeast extract. Its optimal 2.5 (Golovacheva & Karavaiko 1978). Slower growth occurs across vastly larger growth temperature is 48 °C at pH 2 (Clum et al. 2009). temperature and pH ranges. Sulfobacillus spp. are immobile, gram-positive rods, Another genus of interest is Acidithiomicrobium, which to date has no named able to grow aerobically on iron, elemental sulfur, and ISCs, while using organic members. Strain P2 that is suggested to be its type species is both a moderate compounds as a carbon source (Watling et al. 2008). In the case of S. acidophilus, S. thermophile and an obligate autotroph, a combination that is in the acidophilic thermosulfidooxidans, and S. benefaciens,H2 can also serve as a source of electrons Bacteria otherwise only found in At. caldus. Strain P2 oxidizes elemental sulfur (Hedrich & Johnson 2013) and all Sulfobacillus spp. can utilize ferric iron as an and ferrous iron at pH<3 with a growth optimum at 55 °C (Norris et al. 2011). electron acceptor in times of limited oxygen availability (Watling et al. 2008). In A last example organism within the Actinobacteria is Ferrithrix thermotoler- addition, all members of this genus are facultative autotrophs, capable of acquiring ans, likewise a sole representative of its genus. Unlike the two previous genera, this carbon solely from CO2, although growth in this mode is slow and limited to a species is unable to utilize atmospheric CO2 and requires organic carbon sources. smaller range of high-energy electron donors (Watling et al. 2008). Also of special Its energy demand can be met by the oxidation of ferrous iron or organic substrates interest is that Sulfobacilli form endospores during times of high stress, such as coupled to the reduction of oxygen or ferric iron. Fx. thermotolerans prefers a low e.g. exceedingly low pH (Berkeley & Ali 1994). This makes the species of this pH of ~1.8 and exhibits its shortest generation time at 43 °C (Johnson et al. 2009). genus extraordinarily resilient to any range of temporarily unfavorable conditions and combined with their unusual variability in terms of substrates, temperature, and Aquificae and Verrucomicrobia pH, contributes to the wide environmental distribution of this genus. The last two bacterial phyla containing acidophiles are each comprised of only one A closely related genus is Alicyclobacillus, two members of which were origi- thermophilic acidophile that has been recognized. Hydrogenobaculum acidophilum nally assigned to the Sulfobacilli, Alicyclobacillus disulfidooxidans and Acb. toler- belongs to the phylum Aquificae and grows optimally at temperatures of 65 °C. ans. The Alicyclobacilli are a large clade of currently 22 gram-positive endospore It is an obligate aerobic autotroph, gaining energy from the oxidation of H2 in formers, which are the most common cause of food spoilage in the fruit juice indus- presence of elemental sulfur. At 3-4, its pH optimum is slightly higher than the try (Silva & Gibbs 2004). Alicyclobacilli proliferate at diverse pH values, the most definition of an extreme acidophile (Stohr et al. 2001). The second species is extreme being from 0.5-6 for Acb. disulfidooxidans (Karavaiko et al. 2005). Simi- Methylacidiphilum infernorum, which is named for its original isolation from the larily, the genus inhabits large temperature ranges and is comprised of cold-adapted, ”Hell’s Gate” geothermal site in Tikitere, New Zealand. In addition to its for

22 23 Acidophiles Diversity of acidophilic prokaryotes often constitute a large portion of the microbial population in environments with mesophile, and thermophile species, although most grow optimally in moderately exceedingly low pH and high metal concentrations (see sections Acid rock and thermophilic conditions (Ciuffreda et al. 2015). While many Alicyclobacillus spp. mine drainage and Biomining). Three recognized species are comprised in the are obligate organoheterotrophs, some exhibit metabolic properties similar to the genus; Leptospirillum ferrooxidans, L. ferriphilum, and L. rubarum. In addition, Sulfobacilli. For example, Acb. aeris, Acb. ferrooxydans, and Acb. contaminans two candidate species have been identified by metagenomic approaches, and were are all capable of iron and ISC oxidation, despite growing faster on organic substrates preliminary named ’L. ferrodiazotrophum’, and ’Leptospirillum sp. group IV UBA (Goto et al. 2007; Guo et al. 2009; Jiang et al. 2008). BS’ (Goltsman et al. 2013). Actinobacteria Firmicutes The acidophile genera of the phylum Actinobacteria are not as well explored, al- The genus Sulfobacillus lies within the phylum Firmicutes, and contains five though several of them have been recognized (Figure 3). Acidimicrobium ferrooxi- identified species, namely Sulfobacillus acidophilus, S. sibiricus, S. benefacians, S. dans is the sole member of its genus (Clark & Norris 1996). It is capable of efficient thermosulfidooxidans, and S. thermotolerans. All of these organisms are moderately autotrophic growth using ferrous iron or molecular hydrogen, but not ISCs, as a en- thermophilic, preferring growth temperatures of 45-55 °C, and a pH between 1.5- ergy source as well as organoheterotropically in presence of yeast extract. Its optimal 2.5 (Golovacheva & Karavaiko 1978). Slower growth occurs across vastly larger growth temperature is 48 °C at pH 2 (Clum et al. 2009). temperature and pH ranges. Sulfobacillus spp. are immobile, gram-positive rods, Another genus of interest is Acidithiomicrobium, which to date has no named able to grow aerobically on iron, elemental sulfur, and ISCs, while using organic members. Strain P2 that is suggested to be its type species is both a moderate compounds as a carbon source (Watling et al. 2008). In the case of S. acidophilus, S. thermophile and an obligate autotroph, a combination that is in the acidophilic thermosulfidooxidans, and S. benefaciens,H2 can also serve as a source of electrons Bacteria otherwise only found in At. caldus. Strain P2 oxidizes elemental sulfur (Hedrich & Johnson 2013) and all Sulfobacillus spp. can utilize ferric iron as an and ferrous iron at pH<3 with a growth optimum at 55 °C (Norris et al. 2011). electron acceptor in times of limited oxygen availability (Watling et al. 2008). In A last example organism within the Actinobacteria is Ferrithrix thermotoler- addition, all members of this genus are facultative autotrophs, capable of acquiring ans, likewise a sole representative of its genus. Unlike the two previous genera, this carbon solely from CO2, although growth in this mode is slow and limited to a species is unable to utilize atmospheric CO2 and requires organic carbon sources. smaller range of high-energy electron donors (Watling et al. 2008). Also of special Its energy demand can be met by the oxidation of ferrous iron or organic substrates interest is that Sulfobacilli form endospores during times of high stress, such as coupled to the reduction of oxygen or ferric iron. Fx. thermotolerans prefers a low e.g. exceedingly low pH (Berkeley & Ali 1994). This makes the species of this pH of ~1.8 and exhibits its shortest generation time at 43 °C (Johnson et al. 2009). genus extraordinarily resilient to any range of temporarily unfavorable conditions and combined with their unusual variability in terms of substrates, temperature, and Aquificae and Verrucomicrobia pH, contributes to the wide environmental distribution of this genus. The last two bacterial phyla containing acidophiles are each comprised of only one A closely related genus is Alicyclobacillus, two members of which were origi- thermophilic acidophile that has been recognized. Hydrogenobaculum acidophilum nally assigned to the Sulfobacilli, Alicyclobacillus disulfidooxidans and Acb. toler- belongs to the phylum Aquificae and grows optimally at temperatures of 65 °C. ans. The Alicyclobacilli are a large clade of currently 22 gram-positive endospore It is an obligate aerobic autotroph, gaining energy from the oxidation of H2 in formers, which are the most common cause of food spoilage in the fruit juice indus- presence of elemental sulfur. At 3-4, its pH optimum is slightly higher than the try (Silva & Gibbs 2004). Alicyclobacilli proliferate at diverse pH values, the most definition of an extreme acidophile (Stohr et al. 2001). The second species is extreme being from 0.5-6 for Acb. disulfidooxidans (Karavaiko et al. 2005). Simi- Methylacidiphilum infernorum, which is named for its original isolation from the larily, the genus inhabits large temperature ranges and is comprised of cold-adapted, ”Hell’s Gate” geothermal site in Tikitere, New Zealand. In addition to its for

22 23 Acidophiles Diversity of acidophilic prokaryotes acidophiles unusually high growth temperature of 60 °C, this species is also the only acidophilic methanotroph (Dunfield et al. 2007).

Archaea

While low and intermediate temperature environments are often dominated by Bacteria, high temperatures are usually the realm of Archaea. Some microorganisms of this domain exhibit optimal growth close to, or even beyond, the boiling point of water and many of the world’s most pH-tolerant life forms are Archaea (Figure 4; Baker-Austin et al. 2010; Futterer et al. 2004). This is often attributed to special adaptations that differentiate them from the Bacteria, such as e.g. their highly impermeable cell membranes (Baker-Austin & Dopson 2007; Macalady et al. 2004). Currently recognized archaeal acidophiles are found in two of the domain’s orders, the Sulfobales and Thermoplasmatales, belonging to the phyla Crenarchaeota and Euryarchaeota, respectively (Golyshina et al. 2016). While the Crenarchaeota acidophiles are exclusively thermo- and hyperthermophiles, the Euryarchaetoa prefer lower temperatures within meso- to moderate thermophile boundaries. Members of both clades are frequently isolated from environments shaped by volcanic or geothermal activity, such as Yellowstone National Park (USA), Pozzuoli (Italy), or Krisuvik (Iceland), but also from marine geothermal fields.

Crenarchaeota The genus Sulfolobus within the Crenarchaeotes is one of the best explored acidophilic archaeal clades. It includes eight named species (Quehenberger et al. 2017), Figure 4: Unrooted phylogenetic tree of 16S rRNA gene sequences from selected acidophilic as well as a myriad of unidentified strains. All Sulfolobus spp. are thermo- or hy- Archaea, collected from SILVA (Quast et al. 2013). Phylogeny was inferred using FastTree (Price perthermophiles, exhibiting temperature optima from 65 °C (Sulfolobus metallicus), et al. 2010) and the tree drawn by iTOL (Letunic & Bork 2016). Bootstraps < 0.5 are omitted. over 75 °C (Sb. acidocaldarius), up to 85 °C (remaining species). Sb. yangmingensis exhibits an even higher preferred growth temperature of 95 °C, albeit at a pH of 4 for supplemental energy and as a carbon source, with the exception of the obligate (Ren-Long et al. 1999), compared to the optimal pH between 2 and 3 of the remain- chemolithoautotroph Sb. metallicus, that is solely capable of the oxidation of ferrous ing members of the genus. Most but not all Sulfolobi inhabit solfataric environments iron and ISCs and the fixation of inorganic CO2 (Huber & Stetter 1991). rich in sulfur and use the available ISCs as an energy source to reduce oxygen, e.g. A genus closely related to Sulfolobus is Metallosphaera, a versatile group of Sb. metallicus, Sb. shibatae, or Sb. tokadaii. Similar to many acidophilic Bacteria, aerobes that oxidize ferrous iron, ISCs, molecular hydrogen, and organic substrates molecular hydrogen can also serve as an electron donor for these species (Huber while fixing both organic and inorganic carbon (Auernik & Kelly 2010). The et al. 1992). Nonetheless, Sulfolobus spp. additionally require organic molecules five recognized species of the genus are Metallosphaera sedula, Ms. prunae, Ms.

24 25 Acidophiles Diversity of acidophilic prokaryotes acidophiles unusually high growth temperature of 60 °C, this species is also the only acidophilic methanotroph (Dunfield et al. 2007).

Archaea

24 25 Acidophiles Diversity of acidophilic prokaryotes hakonensis, Ms. cuprina, and Ms. tengchongensis, all of which exhibit optimal tetraetherlipid membranes. Ferroplasma acidophilum and ’Fp. acidarmanus’ are growth at temperatures of 65-75 °C in a rather large pH range of 1-6 (Peng et al. facultative anaerobes, oxidizing ferrous iron together with organic compounds and 2014). using oxygen or ferric iron as electron acceptors (Dopson et al. 2004). Similarly, Although most of the discussed acidophilic Archaea are aerobic, anaerobic Acidiplasma cupricumulans and Ap. aeolicum are capable of anaerobic growth lifestyles are also common in this domain. An example of this is the genus Acidianus, using ferric iron, although cell growth is considerably faster while aerobically consisting of six recognized facultative anaerobes, Acidianus brierleyi, Aa. infernus, oxidizing ferrous iron or organic substrates (Golyshina et al. 2009). Acidiplasma Aa. ambivalens, Aa. manzaensis, Aa. tengchongensis, and Aa. sulfidivorans. spp. prefer slightly higher temperatures around 45-55 °C, compared to 35-45 °C of A seventh candidate was suggested and preliminarily named ’Aa. copahuensis’ Ferroplasma. Both genera are solely capable of assimilating organic carbon sources. (Giaveno et al. 2012). While they are capable of aerobic sulfur and hydrogen Lastly, the genus Picrophilus exhibits the lowest pH optima of any known life oxidation to gain energy, in the absence of molecular oxygen these organisms also form (Golyshina et al. 2016). Both members of the genus, Picrophilus torridus have the capacity to direct electrons to elemental sulfur or ferric iron (Giaveno et and P. oshimae, tolerate protons between pH 0-3.5, growing optimally at pH al. 2012; Plumb et al. 2007). Greatest cell yields were observed at temperatures 0.7 (Schleper et al. 1996). Perhaps relatedly, these species are the only known between 75 and 90 °C, at pH values of 1-3. organisms within the acidophilic Euryarchaeota that maintain a S-layer (Klingl Obligate anaerobes are rare within the acidophilic Crenarchaeota and only one 2014). Picrophilus spp. are obligate aerobes, and exclusively oxidize organic species has been described. Stygiolobus azoricus is the sole member of its genus substrates at 60 °C. P. torridus does also have an extremely small genome (1.54 and it grows exclusively by the oxidation of hydrogen coupled to elemental sulfur Mbp), comparable in size to Tp. acidophilum (Futterer et al. 2004). reduction (Segerer et al. 1991).

Euryarchaeota All acidophilic Euryarchaeota belong to the Thermoplasmatales. This clade includes the most extreme acidophiles known and currently consists of only three families and five genera, some of which will be explored in the following section. The genus Thermoplasma contains two described members, Thermoplasma acidophilum and Tp. volcanium. Both species grow optimally at pH ~2, but are able to tolerate pH values as low as 0.5 at temperatures of 60 °C (Segerer et al. 1988). Their energy metabolism involves the oxidation of complex organic molecules coupled to the reduction of oxygen. In addition, Thermoplasma spp. are capable of facultative anaerobic growth using elemental sulfur as an electron acceptor. Interestingly, both members of the genus do not possess a cell wall and are shrouded only by a single triple-layer tetraether membrane (Langworthy 1982). Additionally, with 1.56 Mbp, Tp. acidophilum exhibits one of the smallest genomes ever sequenced for a free living organism (Ruepp et al. 2000). The genera Acidiplasma and Ferroplasma show very similar properties. Their growth range lies between pH 0-4, with an optimum of pH 1-1.5. Similar to Thermoplasma, no members of these genera form a cell wall, but rely solely on

26 27 Acidophiles Diversity of acidophilic prokaryotes hakonensis, Ms. cuprina, and Ms. tengchongensis, all of which exhibit optimal tetraetherlipid membranes. Ferroplasma acidophilum and ’Fp. acidarmanus’ are growth at temperatures of 65-75 °C in a rather large pH range of 1-6 (Peng et al. facultative anaerobes, oxidizing ferrous iron together with organic compounds and 2014). using oxygen or ferric iron as electron acceptors (Dopson et al. 2004). Similarly, Although most of the discussed acidophilic Archaea are aerobic, anaerobic Acidiplasma cupricumulans and Ap. aeolicum are capable of anaerobic growth lifestyles are also common in this domain. An example of this is the genus Acidianus, using ferric iron, although cell growth is considerably faster while aerobically consisting of six recognized facultative anaerobes, Acidianus brierleyi, Aa. infernus, oxidizing ferrous iron or organic substrates (Golyshina et al. 2009). Acidiplasma Aa. ambivalens, Aa. manzaensis, Aa. tengchongensis, and Aa. sulfidivorans. spp. prefer slightly higher temperatures around 45-55 °C, compared to 35-45 °C of A seventh candidate was suggested and preliminarily named ’Aa. copahuensis’ Ferroplasma. Both genera are solely capable of assimilating organic carbon sources. (Giaveno et al. 2012). While they are capable of aerobic sulfur and hydrogen Lastly, the genus Picrophilus exhibits the lowest pH optima of any known life oxidation to gain energy, in the absence of molecular oxygen these organisms also form (Golyshina et al. 2016). Both members of the genus, Picrophilus torridus have the capacity to direct electrons to elemental sulfur or ferric iron (Giaveno et and P. oshimae, tolerate protons between pH 0-3.5, growing optimally at pH al. 2012; Plumb et al. 2007). Greatest cell yields were observed at temperatures 0.7 (Schleper et al. 1996). Perhaps relatedly, these species are the only known between 75 and 90 °C, at pH values of 1-3. organisms within the acidophilic Euryarchaeota that maintain a S-layer (Klingl Obligate anaerobes are rare within the acidophilic Crenarchaeota and only one 2014). Picrophilus spp. are obligate aerobes, and exclusively oxidize organic species has been described. Stygiolobus azoricus is the sole member of its genus substrates at 60 °C. P. torridus does also have an extremely small genome (1.54 and it grows exclusively by the oxidation of hydrogen coupled to elemental sulfur Mbp), comparable in size to Tp. acidophilum (Futterer et al. 2004). reduction (Segerer et al. 1991).

26 27 Acidophiles Energy and carbon metabolism

Energy and carbon metabolism

As described in the previous sections, acidophiles meet their energy demand 2+ exclusively chemotrophically, by oxidizing ferrous iron (Fe ), reduced ISCs, H2, and/or organic carbon compounds (Quatrini & Johnson 2018; Figure 5A). Electrons extracted from these molecules or ions are transferred to terminal electron acceptors, 3+ which for acidophiles so far appear to be limited to O2, ferric iron (Fe ), and ISCs including sulfate. The amount of energy available from the oxidation of an electron donor and the reduction of an electron acceptor can be estimated using their respective standard reduction potentials as two halfs of a galvanic cell (Figure 5B). The voltage between the two half cells is proportional to the free energy released by combining the two reactions, which can be harvested as the so called proton motive force (PMF) to obtain ATP, or by the generation of NADH that is used as reducing power in numerous reactions within the cell. PMF is produced by coupling the transport of electrons from the substrate to the terminal electron acceptor with the dislocation of protons from the cytoplasm. Thus, a potential is created at the outer side of the cell membrane which in turn can be utilized by transmembral ATPases. These proteins allow the flow of protons back into the cytoplasm and use their potential for the generation of ATP from ADP. The second cellular energy currency, NADH, is generated by the direct exergonic electron transfer from the substrate to NAD+, e.g. during glycolysis in organotrophic organisms or by Figure 5: Illustration of key metabolic traits of acidophiles (A), adapted from Quatrini & Johnson NADH-dehydrogenases in lithotrophs. In litothrophic acidophiles, this process is (2018) and standard reduction potentials E0 (at pH 0) and E0´ (at pH 7) of common substrates and complicated by both the pH dependency of many substrate’s reduction potentials electron carriers of acidophiles (B). As by convention, the couples are depicted as ”oxidized state / reduced state”. Due to the cytoplasmic location at neutral pH and the associated strong negativity of and the presence of a large pH gradient across the cell membrane (Ingledew 1982). the NAD+/NADH potential (-0.32V), NADH generation needs to occur by reverse upphill electron At low pH, most substrates do not contain enough reducing power (i.e. their standard transport that consumes PMF for many substrates at acidic pH (as illustratated e.g. for ferrous iron), compared to regular downhill transport for terminal electron acceptor reduction. reduction potential is too positive) to transfer electrons directly to NAD+, which is located in the cytoplasm at close to neutral pH (Figure 5B). Therefore, in the so Iron called reverse or uphill electron transport (named having the traditional redox tower in mind; the vertical depiction of a standard reduction potential scale with negative The oxidation of ferrous iron to gain energy is not exclusive to acidophiles, potentials at the top and positive potentials at the bottom), PMF generated during the even though the acidity of their environment greatly increases the viability of the reduction of a terminal electron acceptor has to be consumed to assist the endergonic process compared to pH neutral niches. Due to the very high standard reduction generation of NADH under these conditions (Ingledew 1982; Nitschke & Bonnefoy potential of the Fe3+/Fe2+ redox couple, oxygen is its only feasible oxidant to 2016). Despite these limitations, acidophiles have evolved mechanisms to exploit a obtain sufficient energy to support active cell metabolism (Figure 5B). However, in large range of chemical reactions and manage to acquire sufficient energy to thrive aerobic conditions and circumneutral pH, ferrous iron is spontaneously oxidized and in some of the harshest environments on the planet. subsequently precipitated as ferric hydroxide. Only at low pH, Fe2+ ions are stable

28 29 Acidophiles Energy and carbon metabolism

Energy and carbon metabolism

28 29 Acidophiles Energy and carbon metabolism despite the presence of oxygen and are readily available for microbial oxidation. hypothesis that this method for energy generation evolved independently multiple Additionally, the standard reduction potential of oxygen rises with decreasing pH, times in different organisms (Nitschke & Bonnefoy 2016). broadening the potential difference and increasing the amount of energy available Most iron-oxidizing acidophiles are also capable of iron reduction under for harvest (Nitschke & Bonnefoy 2016). Consequently, only few neutrophiles have anaerobic conditions, generally coupled to the oxidation of ISCs but also hydrogen evolved systems for energy acquisition via iron oxidation, while they are ubiquitous (Hedrich & Johnson 2013). This co-occurrence led to the assumption that both iron in acidophilic clades and have there evolved to great efficiency, e.g. in the strong pathways at least in part share the same proteins (Corbett & Ingledew 1987; Pronk et iron-oxidizers of the Leptospirillum genus. al. 1991). However, more recently it was discovered that e.g. rusticyanin expressed Biological oxidation of Fe2+ ions takes place at the outer membrane, averting in both conditions in At. ferriphilus was not functional during anaerobic growth by the import of iron species into the cell, where they can generate harmful radical iron reduction (Ohmura et al. 2002). In addition, an extensive lag phase between compounds within the cytoplasm (see section Oxidative stress). In most bacterial aerobic and anaerobic growth reported in At. ferrooxidans suggests enzymatically acidophiles, such as the model iron-oxidizer At. ferrooxidans, the initial electron distinct pathways (Ohmura et al. 2002). Despite continued efforts to elucidate the extraction is conducted by a designated cytochrome c (Quatrini et al. 2009; Talla reduction mechanism via proteomics and transcriptomics (Kucera et al. 2012; Osorio et al. 2014). In Archaea, no evidence for the presence of cytochrome c has been et al. 2013), crucial elements remain elusive and no consensus model has yet been found to date and consequently, the blue copper protein sulfocyanin is proposed reported (Nitschke & Bonnefoy 2016). to be responsible for the extraction of electrons from Fe2+, e.g. in Ferroplasma spp. (Castelle et al. 2015). This type of protein is also present in Bacteria in the Inorganic sulfur compounds form of rusticyanin, although interestingly, members of the bacterial Sulfobacilli ISCs in acidic environments occur in diverse speciation, ranging in sulfur express archaeal sulfocyanin rather than rusticyanin. Beside being involved in oxidation states from -II (hydrogen sulfide, H S) to +VI (sulfate, SO 2–). Almost electron extraction in Archaea and electron transfer in Bacteria, blue copper proteins 2 4 all sulfur species can be used as electron donors by acidophiles, e.g. H S, elemental are suggested to be responsible for the bifurcation of electrons between the uphill 2 sulfur (S0), thiosulfate (S O 2–), and tetrathionate (S O 2–). The end product of and downhill electron transport (Holmes & Bonnefoy 2007), directing electrons to 2 3 4 6 microbial sulfur oxidation is sulfuric acid. The presence of ISCs in the environment either PMF or NADH production, respectively. Alternatives to these proteins also is therefore often the cause of severe acidification and in turn, many sulfur-oxidizers exist, e.g. iso-rusticyanin b, and iron oxidase Iro in At. ferrivorans (Talla et al. are acidophiles (Dopson & Johnson 2012). With the exception of hydrogen sulfide, 2014). The downhill pathway ultimately transfers electrons to an oxidase of aa3 or the energy generation from reduced ISCs shares the same limitations as ferrous cbb3-type, from which oxygen is reduced as a terminal electron acceptor. Directing iron under acidic conditions, in that they require reverse electron transfer for the electrons to the opposite uphill pathway includes transfer via the bc1 complex and the production of NADH, although with less energetic investment (Figure 5B). Even quinone pool towards NADH generation by respective dehydrogenases (Nitschke & the H S standard reduction potential is too high to directly reduce NAD+. However, Bonnefoy 2016; Quatrini et al. 2009). Interestingly, and likely due to their capability 2 in contrast to other ISCs it is low enough to inject electrons directly into the to reduce NAD+ during growth on organic substrates, these proteins are often not quinone pool, from which they can be used by various cellular reactions (Nitschke detected in facultative organotrophic iron-oxidizers such as the Ferroplasma genus & Bonnefoy 2016). Reduced ISCs can net up to eight electrons per sulfur atom, (Bonnefoy & Holmes 2012). Other Archaea, i.e. Sulfolobus spp. and Ms. sedula, compared to only one per Fe2+. This makes them attractive energy sources, justifying exhibit a radically different iron oxidation pathway, consisting of a single haem- extensive enzymatic investment. Nonetheless, most acidophiles capable of doing so copper terminal oxidase in conjunction with cytochrome b, that is suggested to preferentially oxidize iron when fed both substrates (Kupka et al. 2009; Ponce et al. provide both up- and downhill pathway functionalities (Bonnefoy & Holmes 2012). 2012). The mechanisms underlying this regulation are still elusive and appear to be The large inter-species variety of iron oxidation systems provides evidence for the

30 31 Acidophiles Energy and carbon metabolism despite the presence of oxygen and are readily available for microbial oxidation. hypothesis that this method for energy generation evolved independently multiple Additionally, the standard reduction potential of oxygen rises with decreasing pH, times in different organisms (Nitschke & Bonnefoy 2016). broadening the potential difference and increasing the amount of energy available Most iron-oxidizing acidophiles are also capable of iron reduction under for harvest (Nitschke & Bonnefoy 2016). Consequently, only few neutrophiles have anaerobic conditions, generally coupled to the oxidation of ISCs but also hydrogen evolved systems for energy acquisition via iron oxidation, while they are ubiquitous (Hedrich & Johnson 2013). This co-occurrence led to the assumption that both iron in acidophilic clades and have there evolved to great efficiency, e.g. in the strong pathways at least in part share the same proteins (Corbett & Ingledew 1987; Pronk et iron-oxidizers of the Leptospirillum genus. al. 1991). However, more recently it was discovered that e.g. rusticyanin expressed Biological oxidation of Fe2+ ions takes place at the outer membrane, averting in both conditions in At. ferriphilus was not functional during anaerobic growth by the import of iron species into the cell, where they can generate harmful radical iron reduction (Ohmura et al. 2002). In addition, an extensive lag phase between compounds within the cytoplasm (see section Oxidative stress). In most bacterial aerobic and anaerobic growth reported in At. ferrooxidans suggests enzymatically acidophiles, such as the model iron-oxidizer At. ferrooxidans, the initial electron distinct pathways (Ohmura et al. 2002). Despite continued efforts to elucidate the extraction is conducted by a designated cytochrome c (Quatrini et al. 2009; Talla reduction mechanism via proteomics and transcriptomics (Kucera et al. 2012; Osorio et al. 2014). In Archaea, no evidence for the presence of cytochrome c has been et al. 2013), crucial elements remain elusive and no consensus model has yet been found to date and consequently, the blue copper protein sulfocyanin is proposed reported (Nitschke & Bonnefoy 2016). to be responsible for the extraction of electrons from Fe2+, e.g. in Ferroplasma spp. (Castelle et al. 2015). This type of protein is also present in Bacteria in the Inorganic sulfur compounds form of rusticyanin, although interestingly, members of the bacterial Sulfobacilli ISCs in acidic environments occur in diverse speciation, ranging in sulfur express archaeal sulfocyanin rather than rusticyanin. Beside being involved in oxidation states from -II (hydrogen sulfide, H S) to +VI (sulfate, SO 2–). Almost electron extraction in Archaea and electron transfer in Bacteria, blue copper proteins 2 4 all sulfur species can be used as electron donors by acidophiles, e.g. H S, elemental are suggested to be responsible for the bifurcation of electrons between the uphill 2 sulfur (S0), thiosulfate (S O 2–), and tetrathionate (S O 2–). The end product of and downhill electron transport (Holmes & Bonnefoy 2007), directing electrons to 2 3 4 6 microbial sulfur oxidation is sulfuric acid. The presence of ISCs in the environment either PMF or NADH production, respectively. Alternatives to these proteins also is therefore often the cause of severe acidification and in turn, many sulfur-oxidizers exist, e.g. iso-rusticyanin b, and iron oxidase Iro in At. ferrivorans (Talla et al. are acidophiles (Dopson & Johnson 2012). With the exception of hydrogen sulfide, 2014). The downhill pathway ultimately transfers electrons to an oxidase of aa3 or the energy generation from reduced ISCs shares the same limitations as ferrous cbb3-type, from which oxygen is reduced as a terminal electron acceptor. Directing iron under acidic conditions, in that they require reverse electron transfer for the electrons to the opposite uphill pathway includes transfer via the bc1 complex and the production of NADH, although with less energetic investment (Figure 5B). Even quinone pool towards NADH generation by respective dehydrogenases (Nitschke & the H S standard reduction potential is too high to directly reduce NAD+. However, Bonnefoy 2016; Quatrini et al. 2009). Interestingly, and likely due to their capability 2 in contrast to other ISCs it is low enough to inject electrons directly into the to reduce NAD+ during growth on organic substrates, these proteins are often not quinone pool, from which they can be used by various cellular reactions (Nitschke detected in facultative organotrophic iron-oxidizers such as the Ferroplasma genus & Bonnefoy 2016). Reduced ISCs can net up to eight electrons per sulfur atom, (Bonnefoy & Holmes 2012). Other Archaea, i.e. Sulfolobus spp. and Ms. sedula, compared to only one per Fe2+. This makes them attractive energy sources, justifying exhibit a radically different iron oxidation pathway, consisting of a single haem- extensive enzymatic investment. Nonetheless, most acidophiles capable of doing so copper terminal oxidase in conjunction with cytochrome b, that is suggested to preferentially oxidize iron when fed both substrates (Kupka et al. 2009; Ponce et al. provide both up- and downhill pathway functionalities (Bonnefoy & Holmes 2012). 2012). The mechanisms underlying this regulation are still elusive and appear to be The large inter-species variety of iron oxidation systems provides evidence for the

30 31 Acidophiles Energy and carbon metabolism

into the quinone pool and releasing elemental sulfur, which can then be degraded by sulfur oxygenase reductase Sor. Additionally, the polysulfides tetrathionate and thiosulfate can be hydrolized by tetrathionate hydrolase TetH, and the Sox complex, respectively. Both reactions ultimately result in sulfate and a further reduced quinone pool. Alternatively, thiosulfate can be oxidized by cytochrome c, which transfers

electrons directly to oxygen via an aa3-type cytochrome oxidase and increases the PMF in the process. Other acidophiles often use similar pathways, but as previously mentioned, due to the large number of intermediates and abiotic side reactions, the diversity of sulfur oxidation systems is very high. However, in contrast to iron oxidation, it appears there is no inherent difference in sulfur oxidation systems between Archaea and Bacteria (Nitschke & Bonnefoy 2016). The reduction of ISCs is also frequently observed in prokaryotes, although more commonly in neutrophiles. This is due to the high toxicity of the end product of this process, hydrogen sulfide. While this compound occurs prevalently in ionic form and can be precipitated as metal sulfide in pH neutral conditions, at pH<5 the more

toxic H2S is formed (Koschorreck 2008). Therefore, utilization of sulfur compounds as terminal electron acceptors is scarce in acidic environments (Ňancucheo et al. Figure 6: Model of microbial ISCs metabolism based on Kletzin (unpublished), Dopson & Johnson (2012), and Nitschke & Bonnefoy (2016), including enzymes catalyzing the respective 2016) and only a few acidophiles capable of doing so have been isolated. Currently reactions. Dashed arrows indicate disproportionation, faint lines indicate spontaneous abiotic the process appears to be more prevalent within the archaeal domain, with the reactions. 1) polysulfide reductase; 2) sulfide:quinone oxidoreductase (Sqr) or sulfide:cytochrome c oxidoreductase; 3) sulfur reductase; 4) heterodisulfide reductase (Hdr); 5) sulfite:acceptor exception of sulfate reduction, which has only been observed in Bacteria (Dopson oxidoreductase; 6) ATP sulfurylase; 7) reverse ATP sulfurylase (Sat) or adenylylsulfate:phosphate & Johnson 2012). Crenarchaeota such as Aa. ambivalens, Aa. brierleyi, and adenylyltransferase; 8) adenylylsulfate reductase; 9) sulfite reductase; 10) tetrathionate reductase; 11) thiosulfate:quinone oxidoreductase (Tqo, DoxDA) or thiosulfate dehydrogenase (Tsd); 12) Sl. azoricus evolved to use electrons obtained from molecular hydrogen to reduce Sox complex; 13) sulfur oxygenase reductase (Sor); 14) thiosulfate reductastase; 15) tetrathionate hydrolase (TetH); 16) O-acetylserin or O-phosphoserine sulfhydrolases; 17) cysteine desulfurase elemental sulfur, using a quinone-mediated pathway including a hydrogenase and sulfur reductase (Laska et al. 2003; Segerer et al. 1991). The same life-style can be adapted by the Proteobacteria At. ferrooxidans and At. ferriphilum (Ohmura largely unexplored in organisms other than At. ferrooxidans (Ponce et al. 2012). et al. 2002; Osorio et al. 2013). Other sulfur-reducing organisms extract electrons Due to the high amount of different compounds and intermediates, microbial from organic substrates, e.g. the Archaea ’Caldisphaera draconis’ and ’Acidilobus sulfur metabolism is immensely complex (Figure 6). Its study is further complicated sulfurireducens’. As previously mentioned, no known Archaea are capable of sulfate by the typically acidic conditions at which the respective processes are catalyzed, reduction at low pH and Bacteria known to have this capacity are pH-tolerant rather as many ISCs are highly unstable at low pH and tend to react with each other or than extreme acidophiles. The Firmicutes genus Desulfosporosinus accomodates spontaneously disproportionate (Suzuki 1999). Sulfur oxidation pathway models several such moderately acidophilic species, i.e. Desulfosporosinus acidiphilus, for several acidophiles have been proposed, including e.g. the obligate sulfur- and Ds. acididurans, which were isolated from acidic river sediments (Alazard oxidizer At. caldus (Mangold et al. 2011). In this species, ISCs are suggested to be et al. 2010; Sánchez-Andrea et al. 2014b). Other acid tolerant sulfate-reducing transported into the periplasm by outer membrane protein Omp40. There, hydrogen Bacteria include members of the genera Desulfurella, Thermodesulfobium, and sulfide can be oxidized by sulfide quinone reductase Sqr, injecting electrons directly Syntrophobacter (Sanchez-Andrea et al. 2011).

32 33 Acidophiles Energy and carbon metabolism

Acidophilic sulfur-reduction has long been thought to be rare at best, mostly mospheric carbon dioxide (Calvin 1962) and includes the possibility to concentrate owing to the toxicity of its main product. More recently, metagenomics of acidic CO2 in so called carboxysomes to increase fixation efficiency (Shively et al. 1998). environments as well as novel isolation strategies have started to uncover more RuBisCo is found in a large variety of bacterial acidophiles, such as Acidithiobacilli acidophilic sulfur-reducing diversity, and will be valuable tools in increasing the or Leptospirilli (Esparza et al. 2010). Other species fix carbon by reversing their understanding of this ecological niche (Ňancucheo et al. 2016; Pimenov et al. 2015; TCA cycle, e.g. the hydrogen-oxidizer H. acidophilum, or even express multiple Pinto et al. 2016). pathways, like L. ferriphilum (Christel et al. 2017). Acidophilic Archaea utilize an entirely different system in which glyoxylate is produced as the fixation product by Hydrogen the variants of the 3-hydroxypropionate cycle (Strauss & Fuchs 1993). Obligate lithoheterotrophic organisms are exceedingly rare and consequently, Molecular hydrogen is a common substrate for microbial growth and can there are no known heterotrophic acidophiles that are not also facultative or obligate be used to reduce oxygen or alternative electron acceptors, with high energetic organotrophs. Even though some may prefer to utilize CO2 as a carbon source (e.g. yields (Figure 5B; Schwartz et al. 2013). Its oxidation to 2 H+ releases two Ac. acidiphilum), most organotrophs have no need for inorganic carbon fixation electrons in a single reversible step, catalyzed by the enzyme hydrogenase (Frey systems as they are capable of diverting intermediates of their carbon based energy 2002). In acidic environments, H can originate from the proton induced dilution 2 metabolism towards usage as cell building blocks. Acidophilic representatives of base metals and minerals, and is therefore widely available. The capability to of this group include many of the thermophilic Archaea, such as the genera grow by the oxidation of molecular hydrogen has long been known in acidophilic Ferroplasma, Sulfolobus, and Metallosphaera, but also the bacterial Acidiphilium Archaea, including members of the genera Sulfolobus, Acidianus, Metallosphaera, spp. and Am. ferrooxidans. Similar to their neutrophilic counterparts, acidophilic and Stygiolobus (Brock et al. 1972; Huber et al. 1989; Segerer et al. 1986). organotrophs likely use glycolysis (S. thermosulfidooxidans; Zakharchuk et al. Until recently, At. ferrooxidans and H. acidophilum were the only confirmed 1994), Entner-Doudoroff (Ac. cryptum; Shuttleworth et al. 1985), or Pentose- acidophilic bacterial hydrogen-oxidizers (Drobner et al. 1990; Shima & Suzuki Phosphate pathways (Sb. solfataricus; (She et al. 2001)), followed by often 1993). Hedrich & Johnson (2013) consequently tested 38 bacterial strains for incomplete (”horse-shoe”) TCA cycles to oxidize organic matter (Cardenas et al. hydrogen-enabled growth and reported positive results for members of the genera 2010; Wood et al. 2004), and generate ATP via substrate-level phosphorylation and Acidithiobacillus, Sulfobacillus, and Acidimicrobium, which combined hydrogen NADH mediated PMF. oxidation with oxygen, Fe3+, or ISC reduction. Although some species have been well studied, little attention has been paid to acidophile carbon metabolism (Johnson & Hallberg 2009). More research is Carbon necessary to extend our understanding of their mechanisms, as the degradation of Carbon is a substantial cell constituent for all life forms, that needs to be taken carbon compounds is an important ecological process in acidic environments. At up as CO2 by autotrophs or in the form of organic molecules by heterotrophs. The low pH, sacid groups that are abundant in organic matter exist predominantly in fixation of carbon dioxide in acidophiles proceeds similarly as in neutrophiles, al- their protonated, unpolar form and are therefore capable of crossing cell membranes though it is complicated by the low availability of soluble carbonate and bicarbonate and cause acidification of the cytoplasm (see section pH). Therefore, the presence ions at low pH. Several autotrophic carbon fixation pathways have been identified in of organo- and heterotrophic community members capable of removing these microorganisms, of which the Calvin-Benson-Bassham cycle is the most prevalent substrates is favorable for other acidophiles. In turn, organoheterotrophs are able to in acidophilic Bacteria and elsewhere (Johnson & Hallberg 2009). This pathway symbiotically metabolize low weight carbon compounds assembled and expunged is carried by the enzyme RuBisCo that catalyzes the formation of glucose from at- by the primary producing autotrophs (Quatrini & Johnson 2018).

34 35 Acidophiles Energy and carbon metabolism

34 35 Acidophiles Challenges and adaptations of life in acid

Challenges and adaptations of life in acid likely achieved by import of relatively inert metal cations such as K+, as acidophiles exhibit a large abundance of genes coding for putative cation transporters on their While tolerance against stress caused by vast concentrations of protons is the genomes (Baker-Austin & Dopson 2007; Buetti-Dinh et al. 2016). Lastly, influx of determining trait of acidophiles, it is far from the only factor that has severe impli- protons into the cell is decreased by employing specialized porins with reduced pore cations on life in acidic environments. Low pH directly influences the dissolution of size (Amaro et al. 1991). many substances that can affect microorganisms, especially heavy metals, but also Protons that successfully cross into the cell are often sequestered by molecules other compounds that in high concentrations inhibit cellular functions. Additionally, that exhibit high acid dissociation constants, and are abundant in the cytoplasm. reactive oxygen species (ROS) are readily formed in many acidic environments and These include e.g. phosphate, pyruvate, and glutamate, but also the amino acids are a source of severe oxidative damage to living matter. Therefore, acidophiles lysine and histidine that bear alkaline side chains (Castanie-Cornet et al. 1999). have developed an arsenal of adaptations to cope with these parameters. Further, acid-generating compounds such as organic acids can be oxidized or reduced in order to raise their dissociation constants (Futterer et al. 2004), although pH it is debatable if this a function of pH homeostasis or merely a byproduct of pH values of acidophile habitats vary, but generally have a lower limit around organoheterotrophic energy and carbon metabolism (Baker-Austin & Dopson 2007). pH 0, below which the physical conditions are too harsh to allow for any microbes Despite these adaptations, acidity will inevitably increase even in acidophile to multiply (see section Diversity of acidophilic prokaryotes). Most extreme cells, i.e. by proton-driven ATPases. As ATP generation is an omni-present acidophiles prefer growth conditions between pH 1.5-3 and therefore, possess a process that imports large quantities of protons through the membrane, these need multitude of DNA and protein repair systems to deal with acid related damage to to be constantly pumped out of the cell. This can be achieved by the electron cell structures (Crossman et al. 2004; Ram et al. 2005). However, just like in other transport chain, which couples substrate oxidation to proton export (see section organisms, their basic intracellular reactions and functions, such as the PMF, require Energy and carbon metabolism), or by other energy dependent primary transport near neutral conditions. Due to the logarithmic definition of pH, this means that systems. Additionally, acidophile genomes show numerous secondary transport acidophiles have to maintain a ~10,000-fold proton gradient (∆pH) across their systems capable of extruding protons from the cytoplasm. Nevertheless, active cell membrane. The preservation of this gradient, termed pH homeostasis, can be energy generation by substrate oxidation is an integral process for long-term pH achieved via three simple principles: (i) restricting proton influx, (ii) buffering of homeostasis and therefore, survival in acidophiles. intracellular protons, and (iii) proton efflux (Baker-Austin & Dopson 2007). The cell wall and membrane are the barriers between the inside and outside Heavy metals of a cell, and the focus point of the ∆pH. To prevent the overly rapid influx of While many metals are essential for various functions of living cells, any such protons, acidophiles have developed cell membranes that are not as susceptible to element can become toxic at high concentrations. Their effects on living cells are acid hydrolysis, and more impermeable to protons than the ones of their neutrophilic diverse and often characteristic for the specific metal (Dopson et al. 2003), e.g. the counterparts. Archaeal membrane lipids bound by ether linkages are highly resistant blocking of distinct receptors and transport systems on the outside of microbial cells. to acid attack (Macalady et al. 2004), but also bacterial acidophile membranes have Most metal mediated toxicity is localized within the cell and includes coordinate enhanced resistance, e.g. by a increased abundance of hopanoids at low pH (Jones bonding with functional groups of enzymes, displacement of essential trace metals, et al. 2012a). In addition to structural changes, acidophiles also exhibit a reverse, and oxidative damage to cell components (see section Oxidative stress). At neutral inside-positive, membrane potential. While neutrophiles use an inside-negative pH, metals are mostly covalently bound or part of insoluble salt crystals, and their potential to increase their PMF, acidophiles need to amass positive charges in the cell environmental concentration are accordingly low. At decreased pH however, their to repel the influx of protons and prevent rapid acidification of the cytoplasm. This is

36 37 Acidophiles Challenges and adaptations of life in acid

36 37 Acidophiles Challenges and adaptations of life in acid dissolution is greatly enhanced (Dold 2017; Reddy et al. 1995). Consequently, acidic ubiquitous occurrence of metal resistance systems, acidophiles exhibit up to 1000- waters leach large amounts of metal ions from the surrounding environment and fold higher tolerance to various metals compared to neutrophiles. Recently, this the indigenous acidophiles are exposed to significantly higher metal concentrations disparity has been attributed to the nature of acidic environments themselves, as compared to neutrophiles (Blowes et al. 2003; Kadnikov et al. 2016). In fact, many well as to the defining property of acidophiles, their high pH tolerance (Dopson et acidophiles even promote the dissolution of metals (see section Acid rock and mine al. 2014). Unlike neutrophiles, acidophiles maintain an inside-positive membrane drainage) and due to the nature of their environments, frequently encounter e.g. iron, potential in order to repel proton influx (see section pH). Since most metal ions are copper, arsenic, aluminum, nickel, and zinc (Dopson et al. 2003). Nevertheless, both also positively charged, this adaptation simultaneously increases metal tolerance. acidophiles and neutrophiles employ similar tools to resist the detrimental effects of But also the acidic environment could play a role in decreasing the reactive portion heavy metals (Dopson & Holmes 2014). This, together with the fact that many of metal ions. Both protons and sulfate are among the species with the highest such systems are genetically encoded in potentially transposable genomic islands, concentration in natural low-pH environments, due to the aerobic oxidation of ISCs supports the hypothesis that metal resistance is a property heavily facilitated by to sulfuric acid (see section Acid rock and mine drainage). Any charged binding horizontal gene transfer (Navarro et al. 2013). sites upon the surface of a cell therefore attracts not only metal cations, but also Genetically traceable adaptations to heavy metal resistance are various, but significantly more abundant protons. At the same time, large concentrations of similarly to resistances to other toxic compounds, can be divided into the categories negative sulfate ions shield the metals positive charge and therefore decrease their (i) efflux, (ii) inactivation, and (iii) conversion. Many organisms exhibit metal efflux reactivity. The observed high metal tolerance in acidophiles may therefore be the pumps, which can be specific to a certain metal, such as P-type copper ATPases effect of both biotic and abiotic mechanisms (Dopson et al. 2014). (Huang et al. 2016; Rensing et al. 2000) or transport a range of similar metals, e.g. the RND system (Nies 1999; Nikaido 2018). Yet, these pumps are energy dependent Oxidative stress and therefore, metabolically expensive. To avoid the consumption of ATP or ∆pH, Oxidative chemical agents are ubiquitous in microbial habitats, and pose a microbes can also inactivate toxic metal ions by complexation, i.e. by inorganic threat to cell function via the bulk oxidation of vital cell components such as DNA, polyphosphates (Orell et al. 2012). The shielding of the metal’s electronegativity by RNA, proteins, lipids, and cofactors (Fridovich 1978; Jones et al. 2012b). Often, such ligands greatly decreases its reactivity as a toxic agent within the cytoplasm this oxidative stress is embodied by ROS that can originate from various sources, until it can be expunged by more cost-efficient transporters (Remonsellez et al. but also derive from H O produced during microbial aerobic respiration (Ferrer 2006). A similar protective function is the formation of biofilms, in which abundant 2 2 et al. 2016). This presents a problem especially in iron-oxidizing acidophiles, extracellular polymeric substances (EPS) complex metal ions, as well as establish a as due to the low amount of energy gained by the reduction of a single oxygen diffusion barrier that reduces the portion of metals coming in contact with microbial molecule by Fe2+, oxygen consumption has to be increased to meet the cellular cells (Harrison et al. 2007). Lastly, toxic metal ions can be converted to less toxic energy demand (Rawlings 2005). Further, high metal concentrations in acidic species via enzymatic reactions. One such example is the reduction of mercury in environments, in particular of copper, iron, or zinc, among others, play a major role Sb. solfataricus, transforming the highly toxic Hg2+ ion to elemental Hg0, which in free radical generation due to their involvement in the Fenton and Haber-Weiss subsequently diffuses out of the cell due to its high volatility (Schelert et al. 2004). reactions (Equations 1 and 2, respectively; Ferrer et al. 2016). Comparing the number of metal resistance components in acidophile and neutrophile genomes does not reveal a clear prevalence of such systems in either M2+ 2H O H O + HO• + HOO• (1) group, with the exception of some extremely resistant acidophilic organisms; highly 2 2 −− −→ 2 enriched with efflux pumps, chaperones, and RND transporters for a vast range of HOO• + H O H O + O + HO• (2) 2 2 −−→ 2 2 metals, e.g. At. ferrooxidans, L. ferriphilum, and others. However, despite the

38 39 Acidophiles Challenges and adaptations of life in acid dissolution is greatly enhanced (Dold 2017; Reddy et al. 1995). Consequently, acidic ubiquitous occurrence of metal resistance systems, acidophiles exhibit up to 1000- waters leach large amounts of metal ions from the surrounding environment and fold higher tolerance to various metals compared to neutrophiles. Recently, this the indigenous acidophiles are exposed to significantly higher metal concentrations disparity has been attributed to the nature of acidic environments themselves, as compared to neutrophiles (Blowes et al. 2003; Kadnikov et al. 2016). In fact, many well as to the defining property of acidophiles, their high pH tolerance (Dopson et acidophiles even promote the dissolution of metals (see section Acid rock and mine al. 2014). Unlike neutrophiles, acidophiles maintain an inside-positive membrane drainage) and due to the nature of their environments, frequently encounter e.g. iron, potential in order to repel proton influx (see section pH). Since most metal ions are copper, arsenic, aluminum, nickel, and zinc (Dopson et al. 2003). Nevertheless, both also positively charged, this adaptation simultaneously increases metal tolerance. acidophiles and neutrophiles employ similar tools to resist the detrimental effects of But also the acidic environment could play a role in decreasing the reactive portion heavy metals (Dopson & Holmes 2014). This, together with the fact that many of metal ions. Both protons and sulfate are among the species with the highest such systems are genetically encoded in potentially transposable genomic islands, concentration in natural low-pH environments, due to the aerobic oxidation of ISCs supports the hypothesis that metal resistance is a property heavily facilitated by to sulfuric acid (see section Acid rock and mine drainage). Any charged binding horizontal gene transfer (Navarro et al. 2013). sites upon the surface of a cell therefore attracts not only metal cations, but also Genetically traceable adaptations to heavy metal resistance are various, but significantly more abundant protons. At the same time, large concentrations of similarly to resistances to other toxic compounds, can be divided into the categories negative sulfate ions shield the metals positive charge and therefore decrease their (i) efflux, (ii) inactivation, and (iii) conversion. Many organisms exhibit metal efflux reactivity. The observed high metal tolerance in acidophiles may therefore be the pumps, which can be specific to a certain metal, such as P-type copper ATPases effect of both biotic and abiotic mechanisms (Dopson et al. 2014). (Huang et al. 2016; Rensing et al. 2000) or transport a range of similar metals, e.g. the RND system (Nies 1999; Nikaido 2018). Yet, these pumps are energy dependent Oxidative stress and therefore, metabolically expensive. To avoid the consumption of ATP or ∆pH, Oxidative chemical agents are ubiquitous in microbial habitats, and pose a microbes can also inactivate toxic metal ions by complexation, i.e. by inorganic threat to cell function via the bulk oxidation of vital cell components such as DNA, polyphosphates (Orell et al. 2012). The shielding of the metal’s electronegativity by RNA, proteins, lipids, and cofactors (Fridovich 1978; Jones et al. 2012b). Often, such ligands greatly decreases its reactivity as a toxic agent within the cytoplasm this oxidative stress is embodied by ROS that can originate from various sources, until it can be expunged by more cost-efficient transporters (Remonsellez et al. but also derive from H O produced during microbial aerobic respiration (Ferrer 2006). A similar protective function is the formation of biofilms, in which abundant 2 2 et al. 2016). This presents a problem especially in iron-oxidizing acidophiles, extracellular polymeric substances (EPS) complex metal ions, as well as establish a as due to the low amount of energy gained by the reduction of a single oxygen diffusion barrier that reduces the portion of metals coming in contact with microbial molecule by Fe2+, oxygen consumption has to be increased to meet the cellular cells (Harrison et al. 2007). Lastly, toxic metal ions can be converted to less toxic energy demand (Rawlings 2005). Further, high metal concentrations in acidic species via enzymatic reactions. One such example is the reduction of mercury in environments, in particular of copper, iron, or zinc, among others, play a major role Sb. solfataricus, transforming the highly toxic Hg2+ ion to elemental Hg0, which in free radical generation due to their involvement in the Fenton and Haber-Weiss subsequently diffuses out of the cell due to its high volatility (Schelert et al. 2004). reactions (Equations 1 and 2, respectively; Ferrer et al. 2016). Comparing the number of metal resistance components in acidophile and neutrophile genomes does not reveal a clear prevalence of such systems in either M2+ 2H O H O + HO• + HOO• (1) group, with the exception of some extremely resistant acidophilic organisms; highly 2 2 −− −→ 2 enriched with efflux pumps, chaperones, and RND transporters for a vast range of HOO• + H O H O + O + HO• (2) 2 2 −−→ 2 2 metals, e.g. At. ferrooxidans, L. ferriphilum, and others. However, despite the

38 39 Acidophiles Polyextremophiles

These reactions can also occur directly on the surface of metal sulfides, such Polyextremophiles as pyrite (Schoonen et al. 2006), which is one of the main sources of natural acidic As described in section Challenges and adaptations of life in acid, acidophiles environments inhabited by acidophiles (see section Acid rock and mine drainage). are highly resistant to low pH, high heavy metal concentrations, and oxidative stress. Therefore, these microbes are commonly exposed to high levels of oxidative stress Many acidic environments expose microbes to additional stressing conditions and and require mechanisms to protect their cell components in order to survive. are therefore likely to be inhabited by so-called polyextremophiles. These include The most simple among these protection systems are organic antioxidants such organisms enduring e.g. temperatures drastically deviating from the commonly as glutathione, which in some acidophiles maintain a reducing environment by preferred mesophilic 30-40 °C, sun-induced UV radiation, or osmotic stress by high scavenging and neutralizing free radicals, and conduct basic repairs of oxidative salt concentrations. Tolerating such a large range of detrimental factors requires an damage (Fahey 2001). In many other acidophiles however, this task is fulfilled by extraordinarily effective set of adaptations and rigorous streamlining of unessential CoA disulfide reductase. Upon sensing increased levels of oxidants, acidophiles systems, often resulting in highly specialized organisms exclusively found in their may also enhance expression of more complex enzymatic tools, e.g. superoxide respective niche. dismutase, peroxidase, and thioredoxin, all of which are commonly found on the genome of acidophiles (Cardenas et al. 2012). These proteins neutralize radicals Temperature: Psychro- and thermoacidophiles like HOO•, reduce ROOR´ groups in by oxidative stress affected macromolecules, and repair disulfide bridges in oxidized proteins, respectively. Interestingly, the Water temperature is likely the most variable parameter in acidic environments, oxidative stress protein catalase that is ubiquitous in neutrophiles has been detected ranging from close to freezing in e.g. acidic mine streams in Scandinavia (Liljeqvist only in a minority of acidophile genomes (Cardenas et al. 2012). It is unclear et al. 2015) to above boiling in pressurized sulfidic deep-sea hydrothermal vents (see how H2O2 detoxification is achieved in these organisms, although a role similar to section Volcanic and geothermal environments). Microbes capable of multiplying at catalase has been suggested for the more common rubrerythrin (Maaty et al. 2009). low temperatures are termed psychrophiles, while so-called thermophiles proliferate Nevertheless, the low abundance of catalase analogues in acidophiles suggests at high temperatures. these microbes preferentially deal with oxidative stress by increasing their repair Traditionally, organisms growing at low temperatures were divided into true capabilities rather than scavenge radicals and H2O2. psychrophiles and psychrotolerants. Their classification is based on the condition at which optimal, i.e. fastest, growth is observed. Psychrophiles exhibit fastest growth at temperatures below 10 °C, while psychrotolerants survive at these temperatures but grow more rapidly in near mesophilic conditions (De Maayer et al. 2014). This designation does not account for the lower speed of metabolic reactions at low temperatures, which inherently slows down growth and therefore, does not describe the degree of adaptation to cold conditions. Instead, the prefixes eury- and steno- have been suggested (i.e. stenopsychrophile/eurypsychrophile), to describe psychrophiles exhibiting growth in wide and narrow temperature spectra, respectively (Bakermans & Nealson 2004; Cavicchioli 2016). Low temperatures pose various challenges to microorganisms, often related to reduced molecular motility. These include decreased substrate and intermediate diffusion, excessive protein rigidity that can slow down reaction kinetics, as well as over-stabilization of DNA and RNA secondary structures (Casanueva et al. 2010;

40 41 Acidophiles Polyextremophiles

D’Amico et al. 2006). Additionally, low temperatures facilate the dissolution of even higher temperatures. Heat resistance in microorganisms is mostly the result gaseous O2, and ice formation can concentrate ions in ice-free pockets, processes that of adaptations that increase stability of cell components, such as stabilization of additionally lead to osmotic stress (Watkin & Zammit 2016). A major response to nucleic acids, enzymes and membranes, or the production of chaperones that aid cold conditions that is employed by organisms of all three domains is the expression correct protein folding (Stetter 1999). Additionally, key cellular functions such as of cold shock proteins (CSPs) and cold induced proteins (CIPs) (Barria et al. DNA replication and repair are modified, to ensure their unimpaired performance at 2013; Phadtare 2004). Both groups overlap and consist of proteins of various higher temperatures (Chien et al. 1976). function, some only remotely related to cold adaptation. A large portion however A large proportion of chemolithotrophic and acidophilic Bacteria are moderate exhibits nucleic acid binding motifs and is involved in the melting of rigid DNA thermophiles growing at temperatures between 40-60 °C, including e.g. the in and RNA structures of the chromosome, transcriptome, and ribosomes (Phadtare & previous sections described Am. ferrooxidans and At. caldus (Dopson 2016). Severinov 2010). Further, desaturases present in cold-adapted microbes can increase Higher temperatures are in contrast dominated by the Archaea, with the above membrane fluidity at low temperatures and therefore aid e.g. nutrient diffusion mentioned exceptions of H. acidophilum and M. infernorum. Thermophilic and (Liljeqvist 2012). hyperthermophilic Archaea are especially resistant to heat, proposedly due to their No stenopsychrophilic (i.e. exclusively psychrophilic) acidophiles have been particularly stable, ether-linked cell membranes, compared to bacterial ester-linked identified to date, despite the considerable diversity in many cold acidic environ- membranes (Albers et al. 2000). Therefore, prevalently crenarchaeal acidophiles ments (Kay et al. 2013; Liljeqvist et al. 2015). These habitats often contain vari- such as Sulfolobus, Metallosphaera, and Acidianus spp. are able to tolerate ous bacterial species, yet also described eurypsychrophilic acidophiles that actively temperatures beyond 70 °C. These species dominate exceedingly hot environments multiply at temperatures of 0-10 °C are still rare (Liljeqvist et al. 2015). One of the such as geothermal springs (Golyshina et al. 2016; Ward et al. 2017), where they most prominent example of such organisms is the Proteobacterium At. ferrivorans oxidize various substrates including Fe2+, ISCs, and organic carbon. This makes (Christel et al. 2016b; Hallberg et al. 2010). This species is commonly found in them especially interesting for application in biomining operations, such as the high- cold acidic mining waste streams where it fixes CO2 and catalyzes the oxidation of temperature bioleaching of copper minerals (see section Biomining). iron and sulfur compounds, contributing substantially to the present biomass (Lil- jeqvist et al. 2013). Another cold-tolerant acidophile is a representative of the Be- taproteobacteriales order Ferrovales, ’Ferrovum myxofaciens’, first identified in the low-temperature environment of an abandoned welsh copper mine (Kay et al. 2013). This species is obligately aerobic and gains its energy exclusively by the oxidation of ferrous iron, while fixing inorganic carbon dioxide (Johnson et al. 2014). Lastly of interest is the heterotrophic Actinobacterium ’Acidithrix ferrooxidans’, which despite being reported to actively oxidize iron at temperatures below 10 °C, has not been found to grow in these conditions (Jones & Johnson 2015). Although they are often initially cold, the chemical reactions catalyzed by acidophiles can significantly heat up their environment (see section Acid rock and mine drainage and Biomining), creating conditions for moderately thermophilic and thermophilic Bacteria and Archaea. Other acidic environments derived from volcanic and geothermal activity, and are inherently hot (see section Volcanic and geothermal environments). These habitats attract organisms with growth optima at

42 43 Acidophiles Polyextremophiles

42 43 Acidophiles Environmental and ecological implications of acidophiles

Environmental and ecological implications of acidophiles Acid rock and mine drainage

Acid rock drainage (ARD) is the result of natural processes that lead to the release of acid and heavy metals into the environment. It occurs in areas where sulfidic minerals are exposed to the atmosphere and therefore, come in contact with water and oxygen (Dold 2017; Warren 2011). The oxidation of some of these minerals, i.e. pyrite (FeS2, ”fool’s gold”), is exothermic and proceeds spontaneously without the need of additional energy (Santos et al. 2016). The reaction’s end product is ferrous iron and sulfuric acid (Equation 3).

7 2+ 2 + FeS + O + H O Fe + 2 SO − + 2H (3) 2 2 2 2 −−→ 4

If not sufficiently buffered by the presence of accompanying minerals such as lime, silicates, or carbonates (Dold 2017; Parbhakar-Fox & Lottermoser 2015; Sherlock Figure 7: Schematic illustration of the two mineral dissolution pathways, adapted from Schippers et al. 1995), the protons released by the dissolution reaction can lead to a strong & Sand (1999) acidification, which affects the surrounding environment and provides the right conditions for more severe follow-up reactions, as well as the growth of a wide range In addition to metals, ISCs are released in various forms. Depending on the nature of of acidophiles. These microbes promote the most harmful aspect of ARD, the release the mineral, two different dissolution mechanisms are described (Figure 7; Schippers of toxic heavy metals. As described in section Energy and carbon metabolism, & Sand 1999). Acid insoluble minerals, such as pyrite, molybdenite, or tungstenite, many of the acidophilic Bacteria and Archaea gain energy by the oxidation of are subject to the thiosulfate pathway, in which thiosulfate is the first free sulfur iron and/or sulfur compounds. ARD sites provide an ideal environment for such species (Luther 1987; Moses et al. 1987). Most other metal sulfides are acid soluble microorganisms, in which they transform the ferrous iron released from the mineral and behave differently. Minerals such as chalcopyrite, arsenopyrite, or sphalerite to ferric iron, a strong oxidant. Ferric ions are able to attack the sulfur moiety of more are attacked by a combination of ferric iron and protons, and release more reduced stable sulfide minerals (Vera et al. 2013), which can contain various heavy metals, sulfur compounds according to the polysulfide mechanism (Dutrizac 1974). While such as arsenic, copper, zinc, cobalt, etc. Similar to the oxidation by oxygen, this thiosulfate can be oxidized both biologically and abiotically by ferric iron, many process breaks the mineral’s chemical bond and releases its components as ions. products of the polysulfide pathway (e.g. S0) are accessible only to microbial During the reaction, for each consumed ferric ion, one or more ferrous ions are oxidation. In producing more sulfuric acid, both these processes increase the net produced that the present acidophiles can in turn oxidize again, initiating a chain release of protons and further decrease the pH of the receiving water body. As a reaction and potentiating the release of acid and metals (Equation 4). result, the biological diversity in the environment decreases significantly (Baker & Banfield 2003; Kuang et al. 2013), as organisms not capable of tolerating elevated MFeS + 2 Fe3+ + 2H+ M2+ + 3 Fe2+ + 2S0 + H O (4) 2 −−→ 2 proton and metal concentrations rapidly decline. As all the mentioned reactions are exothermic, ARD waters can also heat-up substantially. Decreased pH and large

44 45 Acidophiles Environmental and ecological implications of acidophiles

Environmental and ecological implications of acidophiles Acid rock and mine drainage

7 2+ 2 + FeS + O + H O Fe + 2 SO − + 2H (3) 2 2 2 2 −−→ 4

44 45 Acidophiles Environmental and ecological implications of acidophiles variability with respect to temperature, metal concentration and carbon content is on often drive the development of more extreme microbial communities, including e.g. the other hand responsible for a large diversity of acidophiles typically encountered thermophilic Archaea in addition to bacterial acidophiles (Baker & Banfield 2003). in these environments. Microbes identified from ARD include Bacteria and Archaea Due to their size, complexity, and inaccessibility, remediation of such sites with a wide range of preferred temperatures and substrates, including psychrophiles proves extremely difficult. Nevertheless, strategies are available to minimize their such as At. ferrovorans or Fv. myxofaciens (see section Temperature: Psychro- and effects, and regulations increasing the liability of mining companies are being thermoacidophiles), as well as meso- and moderately thermophilic Acidithiobacilli, passed globally (Parbhakar-Fox & Lottermoser 2015). Today, the design of new Leptospirilli, and Acidimicrobia (Baker & Banfield 2003). mining operations has to include careful considerations for prevention and treatment While it may be presumptuous to call a natural process occurring since millions of potentially dangerous mining waste. These can be divided in two different of years an environmental problem, ARD is an undesired reality in many places approaches; (i) prevention and/or neutralization of acid at the site of generation or around the globe and decreases water quality for human consumption (Verburg et (ii) treatment of the AMD stream or affected water body. One popular method to al. 2009). A prime example for this is the Rio Tinto, a river originating in the prevent and neutralize acid generation is the application of lime, which is mixed mountains of Andalusia, Spain. For ~100 km, it winds through the Iberic Pyrite with water, and spread over the affected ore body. The buffering capacity of lime Belt, a 250 km long geological entity consisting mainly of iron and copper sulfides raises the pH, preventing further dissolution of the minerals, and ideally inactivating (Amils 2016). Here, the river has likely been subjected to ARD for millions of years acidophile organisms catalyzing the process. However, this treatment does often not (Leistel et al. 1997). The Iberic Pyrite Belt is however also heavily influenced by last, and has to be repeated frequently (Caraballo et al. 2009). Longer lasting, widely human mining activities, which began there as early as 3000 BC (Davis Jr. et al. adapted methods include the covering of tailings with soil, flooding, or underground 2000). Effluents from active and closed mines, along with their tailings that were storage of the acid generating material. This way, contact with atmospheric oxygen discarded on the slopes bordering the river, contribute to the acidity and heavy metal is limited and the aerobic chemical reactions necessary for AMD production are load of the environment in the same way as naturally exposed minerals. Therefore, greatly diminished (Moncur et al. 2015; Peppas et al. 2000). in this area Rio Tinto can reach pH values ~2, and the water contains up to 20 g/L If the treatment of the source of acidity is not feasible, or does not prove dissolved iron (Gonzalez-Toril et al. 2003), staining the water deeply red (Figure 2, effective, the actual AMD streams have to be treated by neutralizing acidity and and front cover of this thesis). removing heavy metals, reduced ISCs, and sulfate. Stand-alone applications such Today, the environmental impact of this acid mine drainage (AMD) (Hoffert as liming, aeration and precipitation, and many others are available to remediate 1947; Younger 2017) globally dwarfs that of natural ARD (Quatrini & Johnson single or multiple parameters of AMD (Verburg et al. 2009). Depending on the 2018), and contaminates regions previously unaffected by its underground mineral waste’s streams volume, though, these methods can be costly. More recently, content (Blowes et al. 2003). Many examples more extreme than Rio Tinto exist, scientists have investigated biological ways for mining waste water treatment, with the Iron Mountain Mine close to Redding, California (USA) as an infamous including sulfate-reducing Bacteria and biosorption (Choi 2015; Sánchez-Andrea et front-runner. Iron Mountain was operated above and below ground from 1860 until al. 2014a). Additionally, increasingly complex methods involving combinations of 1963 and exploited for a vast range of metals (Jacobs & Testa 2014). Despite the biological, physical, and chemical technologies are under development to remediate mine’s closure, to this day the oxidative dissolution of pyrite exposed in the shafts waste streams and gain valuable resources at the same time, e.g. so-called microbial leads to the discharge of AMD exhibiting pH values as low as -3.6 (Nordstrom et fuel cells, which utilize electric currents produced by microbes to precipitate heavy al. 2000); the most acidic waters ever measured in the environment. Additionally, metals (Ni et al. 2016). the waste stream exhibits strongly increased temperatures of up to 50 °C, heated by the exothermic mineral dissolution reactions (Druschel et al. 2004). The more drastic pH and temperature conditions of many AMD sites compared to AMD

46 47 Acidophiles Environmental and ecological implications of acidophiles variability with respect to temperature, metal concentration and carbon content is on often drive the development of more extreme microbial communities, including e.g. the other hand responsible for a large diversity of acidophiles typically encountered thermophilic Archaea in addition to bacterial acidophiles (Baker & Banfield 2003). in these environments. Microbes identified from ARD include Bacteria and Archaea Due to their size, complexity, and inaccessibility, remediation of such sites with a wide range of preferred temperatures and substrates, including psychrophiles proves extremely difficult. Nevertheless, strategies are available to minimize their such as At. ferrovorans or Fv. myxofaciens (see section Temperature: Psychro- and effects, and regulations increasing the liability of mining companies are being thermoacidophiles), as well as meso- and moderately thermophilic Acidithiobacilli, passed globally (Parbhakar-Fox & Lottermoser 2015). Today, the design of new Leptospirilli, and Acidimicrobia (Baker & Banfield 2003). mining operations has to include careful considerations for prevention and treatment While it may be presumptuous to call a natural process occurring since millions of potentially dangerous mining waste. These can be divided in two different of years an environmental problem, ARD is an undesired reality in many places approaches; (i) prevention and/or neutralization of acid at the site of generation or around the globe and decreases water quality for human consumption (Verburg et (ii) treatment of the AMD stream or affected water body. One popular method to al. 2009). A prime example for this is the Rio Tinto, a river originating in the prevent and neutralize acid generation is the application of lime, which is mixed mountains of Andalusia, Spain. For ~100 km, it winds through the Iberic Pyrite with water, and spread over the affected ore body. The buffering capacity of lime Belt, a 250 km long geological entity consisting mainly of iron and copper sulfides raises the pH, preventing further dissolution of the minerals, and ideally inactivating (Amils 2016). Here, the river has likely been subjected to ARD for millions of years acidophile organisms catalyzing the process. However, this treatment does often not (Leistel et al. 1997). The Iberic Pyrite Belt is however also heavily influenced by last, and has to be repeated frequently (Caraballo et al. 2009). Longer lasting, widely human mining activities, which began there as early as 3000 BC (Davis Jr. et al. adapted methods include the covering of tailings with soil, flooding, or underground 2000). Effluents from active and closed mines, along with their tailings that were storage of the acid generating material. This way, contact with atmospheric oxygen discarded on the slopes bordering the river, contribute to the acidity and heavy metal is limited and the aerobic chemical reactions necessary for AMD production are load of the environment in the same way as naturally exposed minerals. Therefore, greatly diminished (Moncur et al. 2015; Peppas et al. 2000). in this area Rio Tinto can reach pH values ~2, and the water contains up to 20 g/L If the treatment of the source of acidity is not feasible, or does not prove dissolved iron (Gonzalez-Toril et al. 2003), staining the water deeply red (Figure 2, effective, the actual AMD streams have to be treated by neutralizing acidity and and front cover of this thesis). removing heavy metals, reduced ISCs, and sulfate. Stand-alone applications such Today, the environmental impact of this acid mine drainage (AMD) (Hoffert as liming, aeration and precipitation, and many others are available to remediate 1947; Younger 2017) globally dwarfs that of natural ARD (Quatrini & Johnson single or multiple parameters of AMD (Verburg et al. 2009). Depending on the 2018), and contaminates regions previously unaffected by its underground mineral waste’s streams volume, though, these methods can be costly. More recently, content (Blowes et al. 2003). Many examples more extreme than Rio Tinto exist, scientists have investigated biological ways for mining waste water treatment, with the Iron Mountain Mine close to Redding, California (USA) as an infamous including sulfate-reducing Bacteria and biosorption (Choi 2015; Sánchez-Andrea et front-runner. Iron Mountain was operated above and below ground from 1860 until al. 2014a). Additionally, increasingly complex methods involving combinations of 1963 and exploited for a vast range of metals (Jacobs & Testa 2014). Despite the biological, physical, and chemical technologies are under development to remediate mine’s closure, to this day the oxidative dissolution of pyrite exposed in the shafts waste streams and gain valuable resources at the same time, e.g. so-called microbial leads to the discharge of AMD exhibiting pH values as low as -3.6 (Nordstrom et fuel cells, which utilize electric currents produced by microbes to precipitate heavy al. 2000); the most acidic waters ever measured in the environment. Additionally, metals (Ni et al. 2016). the waste stream exhibits strongly increased temperatures of up to 50 °C, heated by the exothermic mineral dissolution reactions (Druschel et al. 2004). The more drastic pH and temperature conditions of many AMD sites compared to AMD

46 47 Acidophiles Environmental and ecological implications of acidophiles

Acid sulfate soils to the large portion of organic matter in soil, organoheterotrophs are also abundant. Factors that influence ASS community composition include temperature, pH, and Acid sulfate soils (ASSs) form by the same chemical principles as ARD, in relatedly, state of oxidation. Members of the Acidithiobacilli were among the that they are caused by the oxidation of sulfide bearing minerals, although they earliest acidophiles to be isolated from these environments (Arkesteyn 1980). More are not usually connected to large ore bodies. Instead, ASSs originate from past recently, 16S rRNA gene amplicon sequencing allowed the identification of many sea sediments that contain fine grains of metal sulfides such as pyrite, formed via more organisms including acidophilic Acidobacteria, but also a large abundance the microbially catalyzed reaction of sulfate from sea water with iron oxides in of neutrophilic Halanaerobiales and Xanthomonadaceae, i.e. Rhodanobacter spp. the anaerobic sediment zones (Bloomfleld & Coulter 1974). Such deposits are (Hogfors-Ronnholm et al. 2017; Wu et al. 2015). termed potential acid sulfate soils (PASSs). They occupy only small areas, but are Remediation of ASS is challenging and attempts are currently limited to sites commonplace around the world, and include many low-lying coastal regions on all of high economic value, such as agricultural or construction land (Michael 2013). continents except Antarctica (Michael 2013). PASSs do not pose an environmental Strategies applied to prevent further acid release into the environment follow the problem, as long as they are submerged and not in contact with air. However, same principles as in AMD treatment, consisting of addition of buffers such as land uplift over the millenia after the last ice age has exposed many PASSs to lime or carbonate (Baldwin & Fraser 2009) and the depletion of oxygen by re- atmospheric oxygen (Boman et al. 2010; Cook et al. 2000), causing the oxidation of flooding or augmentation with organic matter (Michael et al. 2015; Minh 1998). metal sulfides, assisted by microorganisms as described in section Acid rock and More recently, iron-oxidizing microbes that catalyze the sulfide mineral dissolution mine drainage (Wu et al. 2013). Human activity such as drainage of wetlands have been targeted direclty, e.g. by the addition of acetate-releasing peat, although or large excavations have also significantly expanded the areas affected by ASS so far only with limited effects (Hogfors-Ronnholm et al. 2017). (Minh et al. 1997). The increased occurrence of droughts, suggested to be caused by anthropogenic climate change, also contributes to seasonal acid release from soils (Fitzpatrick et al. 2017). Volcanic and geothermal environments The sulfuric acid generated by the oxidation of the sulfide minerals causes a Continental fault lines or other sites with reduced thickness or fragmentation of severely decreased pH in the oxidized soil layers, even though due to the buffering the planetary crust do not only manifest as majestic volcanoes, but also allow deep capacity of its constituents, the drop is not as drastic as in e.g. ARD. Typical ground water to come in contact with magma and hot bedrock. This leads to the ASS exhibit pH values between 3-5, but can reach as low as pH 2, and mobilize formation of superheated water and steam, which can subsequently be brought to significant amounts of metals (Nordmyr et al. 2008). The leaching of toxic heavy the surface by geothermal activity, resulting in so called fumaroles. Fumarole fields metals poses the most severe environmental impact of ASSs, causing damage to in areas with high sulfide mineral content often emit sulfurous gases along with the aquatic environments including fish kills (Powell & Martens 2005), damage to superheated water, and are consequently called solfatara. Volcanic gases such as agriculture (Bronswijk et al. 1995), and threatening human health (Hinwood et H2S and SO2 are highly reactive, and can produce elemental sulfur when reacting al. 2006). Another effect of ASSs is the deoxygenation of surface waters, which with each other (Dopson & Johnson 2012). In the presence of sulfur-oxidizing 2+ 3+ is caused by the aerobic oxidation of large amounts of leached Fe to Fe , and Bacteria or Archaea, formed S0 is biologically oxidized to sulfuric acid. Further, severely affects aquatic life (Sullivan et al. 2002). the dissolution of SO2 in water produces sulfurous acid (H2SO3). Both processes Studies examining the composition of microbial community within ASSs are can lead to strong environmental acidification, with pH values reaching lower than still scarce, but large microbial diversity has already been reported ASS. Affected 1 at some sites (Schleper et al. 1995; Segerer et al. 1986). This, together with the soils can encompass both neutrophilic and acidophilic Bacteria and Archaea (Ling springs’ high temperature of up to 100 °C, makes solfatara ideal habitats particularly et al. 2015), often overlapping with organisms found in ARD (Wu et al. 2013). Due for (hyper-) thermophilic acidophile Archaea, but also some Bacteria with lower

48 49 Acidophiles Environmental and ecological implications of acidophiles

48 49 Acidophiles Environmental and ecological implications of acidophiles temperature optima (Hedrich & Schippers 2016). (Cardenas et al. 2015; Huber & Stetter 1989). Numerous studies have even Acidic geothermal environments are found globally, e.g. in Iceland, Italy, suggested that cellular life could have originated in this highly diverse niches, as Hawaii, China, Australia, and many others. The arguably most prominent solfatara the steep chemical and thermal gradients provide a constant energy reservoir, and field is the Norris Geysir Basin within Yellowstone National Park, Wyoming (USA; support the synthesis of organic compounds out of inorganic precursors (Colı́n- Figure 2B). The pools of this basin have average temperatures between 65 and Garcı́a 2016). 90 °C, and contain high amounts of organic carbon. Their pH is moderate, and is generally measured around 4. Nearby basins are more acidic, such as the Green Dragon Springs, and the Roaring Mountain, which both exhibit pH<3, although their temperatures are lower, with 65-75, and ~40 °C, respectively (Montana State Geothermal Site Database, accessed July 2018). The Yellowstone solfatara fields have been subject to intense study for more than 50 years, and many acidophilic microbes were isolated from its pools, e.g. the first sulfur-oxidizing Archaea Aa. brierleyi (Brierley 2008) and the very first acidophilic hyperthermophile, Sb. acidocaldarius (Darland & Brock 1971). Due to the basins’ diverse conditions, a wide range of microbial diversity is found in the area, including aerobic and anaerobic chemolithotrophic Bacteria and Archaea (Johnson et al. 2003; Kozubal et al. 2012), but also an abundance of organoheterotrophic community members (Beam et al. 2013). Another type of geothermal sites are marine hydrothermal vents on the sea floor, exhibiting the same chemical reactions as terrestrial fumaroles and solfatara. Due to the depth induced pressure, the water temperatures of these so-called ”Black smokers” can can exceed 350 °C at the point of exit (Nakamura & Takai 2014), but cool rapidly to ~120 °C at the deposit walls of the vent (Damm 2013). There, some of the most hyperthermophilic organisms known can be found, such as the neutrophilic Archaea ’Strain 121’ (Kashefi & Lovley 2003) or the acid-tolerant Pyrolobus fumarii (Blochl et al. 1997). More moderate niches are commonly found only short distances from the exhaust and exhibit temperatures between 50-80 °C and pH values of 3-6 (Antranikian et al. 2017; Desbruyères et al. 2001). pH, redox conditions, and chemical concentrations in hydrothermal vents are strongly dependent on their flow rate and temperature, which can create highly stratified environments. Consequently, a wide range of novel acid-tolerant and moderately acidophile biodiversity has been isolated from these niches, e.g. members of the ’Deep-sea hydrothermal vent Euryarchaeota 2’ (Flores et al. 2012) that includes Aciduliprofundum boonei (Reysenbach et al. 2006), but also extreme acidophiles, i.e. the previously mentioned salt-tolerant acidophile Acidihalobacter prosperus

50 51 Acidophiles Environmental and ecological implications of acidophiles temperature optima (Hedrich & Schippers 2016). (Cardenas et al. 2015; Huber & Stetter 1989). Numerous studies have even Acidic geothermal environments are found globally, e.g. in Iceland, Italy, suggested that cellular life could have originated in this highly diverse niches, as Hawaii, China, Australia, and many others. The arguably most prominent solfatara the steep chemical and thermal gradients provide a constant energy reservoir, and field is the Norris Geysir Basin within Yellowstone National Park, Wyoming (USA; support the synthesis of organic compounds out of inorganic precursors (Colı́n- Figure 2B). The pools of this basin have average temperatures between 65 and Garcı́a 2016). 90 °C, and contain high amounts of organic carbon. Their pH is moderate, and is generally measured around 4. Nearby basins are more acidic, such as the Green Dragon Springs, and the Roaring Mountain, which both exhibit pH<3, although their temperatures are lower, with 65-75, and ~40 °C, respectively (Montana State Geothermal Site Database, accessed July 2018). The Yellowstone solfatara fields have been subject to intense study for more than 50 years, and many acidophilic microbes were isolated from its pools, e.g. the first sulfur-oxidizing Archaea Aa. brierleyi (Brierley 2008) and the very first acidophilic hyperthermophile, Sb. acidocaldarius (Darland & Brock 1971). Due to the basins’ diverse conditions, a wide range of microbial diversity is found in the area, including aerobic and anaerobic chemolithotrophic Bacteria and Archaea (Johnson et al. 2003; Kozubal et al. 2012), but also an abundance of organoheterotrophic community members (Beam et al. 2013). Another type of geothermal sites are marine hydrothermal vents on the sea floor, exhibiting the same chemical reactions as terrestrial fumaroles and solfatara. Due to the depth induced pressure, the water temperatures of these so-called ”Black smokers” can can exceed 350 °C at the point of exit (Nakamura & Takai 2014), but cool rapidly to ~120 °C at the deposit walls of the vent (Damm 2013). There, some of the most hyperthermophilic organisms known can be found, such as the neutrophilic Archaea ’Strain 121’ (Kashefi & Lovley 2003) or the acid-tolerant Pyrolobus fumarii (Blochl et al. 1997). More moderate niches are commonly found only short distances from the exhaust and exhibit temperatures between 50-80 °C and pH values of 3-6 (Antranikian et al. 2017; Desbruyères et al. 2001). pH, redox conditions, and chemical concentrations in hydrothermal vents are strongly dependent on their flow rate and temperature, which can create highly stratified environments. Consequently, a wide range of novel acid-tolerant and moderately acidophile biodiversity has been isolated from these niches, e.g. members of the ’Deep-sea hydrothermal vent Euryarchaeota 2’ (Flores et al. 2012) that includes Aciduliprofundum boonei (Reysenbach et al. 2006), but also extreme acidophiles, i.e. the previously mentioned salt-tolerant acidophile Acidihalobacter prosperus

50 51 Acidophiles Applications of acidophiles

Applications of acidophiles Biomining

Biomining, or biohydrometallurgy, describes the intentional application of acidophiles for the oxidation of insoluble metal sulfides to water soluble salts. The dissolution of the sulfide minerals occurs according to the mechanism described by Equation 4 in section Acid rock and mine drainage. The role of acidophilic microorganism lies mainly in the regeneration of the oxidant, ferric iron. In practice, this process can be used for two purposes; (i) to gain metals of interest by bringing them in solution in a process referred to as bioleaching (Vera et al. 2013), or for (ii) biooxidation, the removal of gangue materials that hinder access to more valuable metals contained in the mineral structure (Brierley & Brierley 2013). Biomining operations are employed in two different ways; dumps or heaps and stirred tank reactors (STRs). Ore dumps presented the earliest form of mostly un- intentional biomining and consisted of low grade sulfidic ore discarded from mines because it was considered of too low grade for further processing. Exposed to water Figure 8: Schematic illustration of a typical chalcopyrite heap-bioleaching operation, including and air, oxidation of the mineral commenced naturally and led to acid mine drainage. solvent extraction and electro winning steps From this uncontrolled discharge, humans gained metals such as copper already in ancient times (Ehrlich 2001). More recently, dumps were optimized into engineered erate thermophilic Bacteria such as Acidithiobacilli, Leptospirilli, and Sulfobacilli, heaps, which are stacked upon lining pads to prevent loss of leaching liquor and its but also thermo- and hyperthermophilic organisms such as Archaea of the Sulfobales associated environmental damage (Petersen 2016). Additionally, heaps are venti- or Thermoplasmatales order. Therefore, as temperatures in the heap rise, microbes lated with air from the bottom, and irrigated with diluted sulfuric acid from the top with a corresponding growth optimum are continuously added, and can maintain the (Figure 8). This greatly accelerated the mineral dissolution process, and made the processes necessary for the mineral dissolution (see section Diversity of acidophilic collection of metals more efficient (Demergasso et al. 2017). As bioleaching reac- prokaryotes and Temperature: Psychro- and thermoacidophiles). Nevertheless, mi- tions are exothermic, their acceleration leads to an increase in heap temperature (Ha- crobial communities that develop in biomining operations are only partly in control linen et al. 2012). This is in part promoted by operators, as all reactions in the heap of engineers, as many acidophile niches overlap, and sterilization of the mineral is will increase in speed and more mineral will be dissolved in the same time. How- not feasible. Therefore, ore type, temperature, and aeration are the major factors ever, continued optimization also presented the challenge of overheating. The overly influencing the microbial diversity. Fast-growing mesophilic iron-oxidizers like At. rapid increase of heap temperatures can cause devastating losses in the microbial ferrooxidans are typically the first to colonize bioleaching heaps, while moderately community, leading to extended periods of inactivity, or even complete sterilization thermophilic iron- and sulfur-oxidizers such as L. ferriphilum and At. caldus typ- of parts of the heap (Leahy et al. 2007; Shiers et al. 2017). In addition to the natural ically dominate soon after (Demergasso et al. 2005; Halinen et al. 2012). In later occurring microorganisms, most heaps are therefore inoculated with a mixture of stages, heaps are characterized by high metal concentrations and temperatures, pro- acidophiles exhibiting growth optima at different temperatures to prevent the loss viding conditions for more thermophilic Bacteria and Archaea, e.g. Sulfobacillus of microbial activity. These strategic biomining organisms include meso- and mod- spp. or members of the genus Ferroplasma (Demergasso et al. 2005; Pradhan et al. 2008).

52 53 Acidophiles Applications of acidophiles

Applications of acidophiles Biomining

52 53 Acidophiles Applications of acidophiles

Today, heaps come in different sizes, but can cover several square kilometers solution to accelerate the process. Additionally, once constructed, a heap has to be of land, and reach heights of up to 100 meters when stacked (Domic 2007). Due maintained for an unascertained amount of time, as the release of acid and heavy to the enormous volumes, control of a heap is difficult and implied costs limit metals is near impossible to stop, and would otherwise devastate the surrounding its manipulation to inoculation with biomining microorganisms (Watling 2016), environment (see section Acid rock and mine drainage). This long-term commit- irrigation and aeration, and to a lesser extend, grain size of the mineral (Watling ment by mining companies necessary for profitable biomining has so far prevented 2006). even more wide-spread utilization of its potential. In order to control the oxidation process even better, STRs were developed To reduce the time necessary for completion of a bioleaching operation, (Rawlings & Johnson 2007). Closed reactors allow for the adjustment of a researchers and engineers have extensively investigated both physical and biological multitude of parameters such as temperature, pH, oxidation/reduction potential aspects of biomining, and our knowledge of the process increases steadily. Their (ORP), metal concentrations, and even the use of catalysts (Mahmoud et al. 2017). results can directly help mining companies, e.g. by modeling biomining on a specific This significantly reduces the time needed for complete metal extraction from ore body to assess microbial functionality, strategies, and profitability of planned several months or years in a heap, to days or weeks in STRs (Hedrich et al. operations (Govender et al. 2014; Petersen 2010), aid operation of bioleaching heaps 2018). Accordingly, metal concentrations rise more rapidly within such a reactor (Demergasso et al. 2010), or devise methods for remediation of acid mine drainage and generally reach higher levels as in bioheaps, so the utilization of specially (Verburg et al. 2009). Nonetheless, to date, significant problems remain unsolved, adapted microbial communities is required (Dopson & Holmes 2014). Additionally, and more studies are needed to fully understand the interaction of physical, chemical, reactors accommodate mineral batches several orders of magnitude smaller than and biological parameters in biomining operations. heaps, making it necessary to concentrate the ore prior to processing, e.g. via flotation. This and the higher operating costs of STRs compared to heaps make Chalcopyrite bioleaching them profitable only for the most valuable metals, such as silver, gold, uranium, or Few metals have played such profound a role in human development as copper. As cobalt (Brierley & Brierley 2013; Hong et al. 2016). the primary metal in electric circuits, its demand has increased ever since the onset In today’s age of environmental awareness and search for sustainable indus- of industrialization, and prices skyrocketed particularly after the year 2000 (Alonso- trial processes, bioleaching has become of special interest. In particular, heap- Ayuso et al. 2014). To meet this ever growing demand, biomining has long been bioleaching is commonly considered to provide a more environmentally friendly employed in many parts of the world, i.e. in China, Australia, South Africa, and most alternative to traditional metal extraction by cyanide leaching or pyrometallurgy, prominently in Chile (Domic 2007; Watling 2006); countries in which enriched ore as it largely avoids the toxic emissions associated with those methods (Rawlings bodies are located. Today, >15% of the global copper production can be attributed

2002). But bioleaching has also steadily gained interest in the more profit-oriented to bioleaching of secondary copper minerals such as chalcite, Cu2S (Brierley & industrial sector. As high-grade ore bodies become depleted globally, biomining Brierley 2013; Sandstrom et al. 2005). The world’s most abundant primary copper operations are capable of metal recovery from ores far less concentrated than those mineral, chalcopyrite (CuFeS2), is notoriously difficult to leach and suffers from an previously considered profitable, reopening metal sources long discarded by mining extensive lag phase and slow dissolution rates in large-scale operations (Riekkola- companies for exploitation (Petersen 2016). No process is without its drawbacks Vanhanen 2013). While this may be attributed to the semiconductor properties of however, and in biomining one such is presented by its extensive duration, and the chalcopyrite itself (Crundwell 2015), most researchers believe passivating layers long lag phase between construction of the heap and first recovery of metals. This to be responsible, formed on the mineral surface under specific conditions during phase is often attributed to the time needed for acidophile organisms to colonize the the bioleaching process (Panda et al. 2015). The exact nature of the passivating material. As each and every mine has a unique composition of minerals, contain- layer is albeit still under debate, and conclusive evidence has yet to be collected ing varying concentrations of in part toxic heavy metals, there can be no universal (Khoshkhoo et al. 2014a; Wang et al. 2016). Nonetheless, strategies of delaying or

54 55 Acidophiles Applications of acidophiles

54 55 Acidophiles Applications of acidophiles circumventing passivation have been derived. These include bioleaching of finer the Sotkamo mine in Talvivaara, Finland (Riekkola-Vanhanen 2013). In these grains or at high temperature using thermo-acidophiles (Ma et al. 2017; Saitoh et conditions, a specialized microbial community developed, containing acidophilic al. 2017). Especially the ORP in chalcopyrite bioleaching has received significant psychrophiles capable of iron- and sulfur oxidation (Halinen et al. 2012), such attention (Khoshkhoo et al. 2014c; Third et al. 2002), and is suggested to be the as At. ferrivorans (see section Temperature: Psychro- and thermoacidophiles). most important factor influencing leaching kinetics (Khoshkhoo et al. 2014b and Both community composition and gene transcription of involved organisms have references therein). It is commonly observed that dissolution of CuFeS2 sharply been studied extensively (Kupka et al. 2009; Liljeqvist et al. 2015), and allowed declines after ORP exceeds a certain threshold. Accordingly, a mechanism was insights into features and adaptations necessary for low temperature bioleaching proposed in which at low redox potentials and presence of Fe2+ and Cu+ ions, (Christel et al. 2016a,b; Dopson et al. 2007). The knowledge obtained by studying chalcopyrite is transformed to chalcite (Hiroyoshi et al. 2013). This mineral is more the Sotkamo mine and similar sites could prove highly valuable in the design of readily oxidized by ferric iron than the original chalcopyrite, which could explain additional high latitude, boreal biomining operations that could unlock ore bodies the enhanced leaching rates. previously inaccessible to biomining. All proposed solutions to increase efficiency of chalcopyrite bioleaching have their own difficulties and drawbacks, and options for manipulation of heap attributes Bioprospecting and genetic engineering can range from cumbersome and expensive to downright impossible. Therefore, Beside their application in the metal industry, acidophiles are also of interest more cost-efficient methods are under investigation. While definite results have yet for other industrial branches. The advances of NGS have recently re-kindled to be reported, promising approaches have been tested successfully in laboratory the search for genetic elements and enzymes that can be used for commercial scale experiments, i.e. redox controlled leaching by the inoculation of selected mi- processes, an effort that is now called bioprospecting. Microbial catalysts play croorganisms (Masaki et al. 2018). Eventually overcoming the problems associated important roles in many industries, but often fail to withstand extreme conditions. with chalcopyrite bioleaching would be a tremendous milestone, and might irrevo- Therefore, enzymes produced by extremophiles, termed extremozymes, have been cably shift the favor towards this sustainable technology. paid increasing attention (Elleuche et al. 2014). Many extracellular proteins produced by acidophiles are attractive due to their capability to catalyze industrially Biomining in boreal climates important reactions at low pH and often high temperatures (Raddadi et al. 2015). As mentioned in section Chalcopyrite bioleaching, most of the world’s biomining Several commercial branches already utilize acidophiles or their enzymes. For operations are located in China, Australia, South Africa, and Chile. Here, the instance, acid-stable pectinases have greatly increased yields in the fruit juice climate is usually warm and temperature differences between the seasons small, industry (Sharma et al. 2016) and xylanases with low pH optima aid the breakdown providing optimal conditions for establishing a microbial bioleaching community. of hemicellulose in acidic bread dough (Shah et al. 2006). Further, acidophiles are Although it has to be recognized that a large portion of the world’s richest ore under investigation by scientists of the medical field, due to their potential use for bodies are located in these countries, their convenient climate may also be one producing therapeutic agents or as delivery vectors for pharmaceuticals that can not of the reasons for their pioneer role in the large-scale application of biomining. withstand the acidic conditions of the gastrointestinal tract (Fuhrmann & Leroux Suitable ore bodies are albeit not limited to warm climates, and also occur in areas 2013; Wang et al. 2009). near the arctic circle, e.g. in Northern Sweden and Finland (Riekkola-Vanhanen By introducing desired functions into the robust ”chassis” of these microorgan- 2010; Stromberg & Banwart 1994). Due to the harsh temperature changes in these isms, genetic engineering could make acidophiles even more useful. These could latitudes, operation of open-air bioleaching heaps proves difficult, especially during include e.g. additional genes for metal resistance or carbon fixation for biomining the start-up phase where the energy released by exothermic reactions is not yet organisms (Gumulya et al. 2018), but also systems for the production of biofuels sufficient to heat the heap. To date, only one such facility has been established,

56 57 Acidophiles Applications of acidophiles circumventing passivation have been derived. These include bioleaching of finer the Sotkamo mine in Talvivaara, Finland (Riekkola-Vanhanen 2013). In these grains or at high temperature using thermo-acidophiles (Ma et al. 2017; Saitoh et conditions, a specialized microbial community developed, containing acidophilic al. 2017). Especially the ORP in chalcopyrite bioleaching has received significant psychrophiles capable of iron- and sulfur oxidation (Halinen et al. 2012), such attention (Khoshkhoo et al. 2014c; Third et al. 2002), and is suggested to be the as At. ferrivorans (see section Temperature: Psychro- and thermoacidophiles). most important factor influencing leaching kinetics (Khoshkhoo et al. 2014b and Both community composition and gene transcription of involved organisms have references therein). It is commonly observed that dissolution of CuFeS2 sharply been studied extensively (Kupka et al. 2009; Liljeqvist et al. 2015), and allowed declines after ORP exceeds a certain threshold. Accordingly, a mechanism was insights into features and adaptations necessary for low temperature bioleaching proposed in which at low redox potentials and presence of Fe2+ and Cu+ ions, (Christel et al. 2016a,b; Dopson et al. 2007). The knowledge obtained by studying chalcopyrite is transformed to chalcite (Hiroyoshi et al. 2013). This mineral is more the Sotkamo mine and similar sites could prove highly valuable in the design of readily oxidized by ferric iron than the original chalcopyrite, which could explain additional high latitude, boreal biomining operations that could unlock ore bodies the enhanced leaching rates. previously inaccessible to biomining. All proposed solutions to increase efficiency of chalcopyrite bioleaching have their own difficulties and drawbacks, and options for manipulation of heap attributes Bioprospecting and genetic engineering can range from cumbersome and expensive to downright impossible. Therefore, Beside their application in the metal industry, acidophiles are also of interest more cost-efficient methods are under investigation. While definite results have yet for other industrial branches. The advances of NGS have recently re-kindled to be reported, promising approaches have been tested successfully in laboratory the search for genetic elements and enzymes that can be used for commercial scale experiments, i.e. redox controlled leaching by the inoculation of selected mi- processes, an effort that is now called bioprospecting. Microbial catalysts play croorganisms (Masaki et al. 2018). Eventually overcoming the problems associated important roles in many industries, but often fail to withstand extreme conditions. with chalcopyrite bioleaching would be a tremendous milestone, and might irrevo- Therefore, enzymes produced by extremophiles, termed extremozymes, have been cably shift the favor towards this sustainable technology. paid increasing attention (Elleuche et al. 2014). Many extracellular proteins produced by acidophiles are attractive due to their capability to catalyze industrially Biomining in boreal climates important reactions at low pH and often high temperatures (Raddadi et al. 2015). As mentioned in section Chalcopyrite bioleaching, most of the world’s biomining Several commercial branches already utilize acidophiles or their enzymes. For operations are located in China, Australia, South Africa, and Chile. Here, the instance, acid-stable pectinases have greatly increased yields in the fruit juice climate is usually warm and temperature differences between the seasons small, industry (Sharma et al. 2016) and xylanases with low pH optima aid the breakdown providing optimal conditions for establishing a microbial bioleaching community. of hemicellulose in acidic bread dough (Shah et al. 2006). Further, acidophiles are Although it has to be recognized that a large portion of the world’s richest ore under investigation by scientists of the medical field, due to their potential use for bodies are located in these countries, their convenient climate may also be one producing therapeutic agents or as delivery vectors for pharmaceuticals that can not of the reasons for their pioneer role in the large-scale application of biomining. withstand the acidic conditions of the gastrointestinal tract (Fuhrmann & Leroux Suitable ore bodies are albeit not limited to warm climates, and also occur in areas 2013; Wang et al. 2009). near the arctic circle, e.g. in Northern Sweden and Finland (Riekkola-Vanhanen By introducing desired functions into the robust ”chassis” of these microorgan- 2010; Stromberg & Banwart 1994). Due to the harsh temperature changes in these isms, genetic engineering could make acidophiles even more useful. These could latitudes, operation of open-air bioleaching heaps proves difficult, especially during include e.g. additional genes for metal resistance or carbon fixation for biomining the start-up phase where the energy released by exothermic reactions is not yet organisms (Gumulya et al. 2018), but also systems for the production of biofuels sufficient to heat the heap. To date, only one such facility has been established,

56 57 Acidophiles

(Kernan et al. 2015) or the mentioned therapeutic application of acidophiles in the stomach. The development of molecular tools necessary for the manipulation of the genome has however been lagging behind in acidophiles compared to neutrophilic organisms (Gumulya et al. 2018). This has often been related to attributes of their acidic habitats that disrupt engineering mechanisms, e.g. by inactivating antibiotics used for selection of mutated clones. In addition, their low genetic stability (Grogan et al. 2001) enhances acidophiles’ natural adaptability to adverse conditions, but makes them poor subjects of genetic manipulation. Nonetheless, progress in this Aims of this Thesis field is continuously being made. Tools for DNA delivery, selection, and regulated The thesis at hand investigates functions and adaptations of acidophilic mi- expression are becoming increasingly available for model species such as At. fer- croorganisms in natural environments, as well as during their application as bio- rooxidans, At. caldus, and Sulfolobus spp. (Gumulya et al. 2018), opening the field mining organisms. The results obtained by the included studies are intended to aid of synthetic biology now also for acidophiles. in the development of strategies for ARD/AMD remediation, increase the feasibility of the environmentally sustainable technology of bioleaching, and serve as a reference for future research.

Three main points of focus were chosen:

• Clarification of sulfur oxidation pathways and identification of cold adaptation systems in the acidophile At. ferrivorans (Papers I, II). This species is of large interest as it significantly contributes to acidophilic biomass in ARD/AMD, in particular in cold, boreal climates. Additionally, as the only eurypsychrophilic acidophiles capable of both iron and sulfur oxidation, it has great potential to be applied in boreal biomining operations.

• Expansion of knowledge related to chalcopyrite heap bioleaching (III, IV). The microbially promoted dissolution of chalcopyrite significantly contributes to the global copper production, but is still plagued by slow dissolution rates. Acidophiles involved in the process are intensely studied, and prominently among them L. ferriphilum, At. caldus, and S. thermosulfidooxidans. Laboratory scale bioleaching experiments were conducted to understand microbial interactions within a model community of these three biomining organisms. Further, a complete genome sequence was obtained in order to characterize the L. ferriphilum type-strain using genomic, transcriptomic, and proteomic approaches.

58 59 Acidophiles

Three main points of focus were chosen:

58 59 Aims of this Thesis

• Investigation of ASSs in Northern Sweden (V). Field samples were taken aimed at identifying acidophilic community members and making predictions about their potential environmental impact and prospects of successful remediation.

Methodology

All studies in this thesis required either experimental work in the laboratory or field sampling. Each project also included extensive nucleic acid and to a lesser extent, protein sequencing (Table 1). Sequence data analysis and interpretation were performed as final steps before preparing the manuscripts. A methodology in brief can be found in the following sections. For more detailed descriptions of the used materials and methods the reader is referred to the respective papers.

Experiments and sampling

At. ferrivorans sulfur oxidation and cold adaptation (I, II) were assessed via two tetrathionate-fed continuous cultivation experiments, run for several weeks at 8 and 20 °C, respectively. The two growth conditions were sampled in duplicate and rapidly cooled to ensure isolation of a representative transcriptome. Cells were then pelleted by centrifugation and lysed using Tri-reagent (Ambion) according to the manufacturer’s recommendations. The lysate was cleaned by addition of bromo-chloropropane, followed by centrifugation with retention of the supernatant. Genomic DNA was removed applying the Turbo DNA-free Kit (Ambion). L. ferriphilum (III) was maintained both in batch mode and by continuous cultivation, using ferrous sulfate as electron donor at 37 °C. The Genomic-tip 100/G extraction kit (Quiagen) was applied to obtain uninterrupted genomic DNA suitable for ultra long-read genome sequencing from a single sample from the batch culture. Samples for RNA plus protein isolation were taken in triplicate from the continuous cultures. They were rapidly cooled and cells were harvested by centrifugation. Extraction of RNA and proteins was initiated by cell lysis using cryo-milling and bead beating, followed by application of the Allprep isolation kit (Quiagen). Bioleaching experiments (III, IV) were conducted in shaking flasks using chal-

60 61 Aims of this Thesis

Methodology

Experiments and sampling

60 61 Methodology Sequencing

copyrite concentrate. Quadruplet cultures inoculated singly or with combinations of I Organism Paper 1: Table III II niomna omnt il sampling Field community Environmental V IV the biomining model species At. caldus, L. ferriphilum, and S. thermosulfidooxidans were incubated at 38 °C. Experiments were analyzed for pH, ORP, Fe2+, dissolved

umr feprmna odtosadsqecn ehooisue ntesuisicue nti thesis. this in included studies the in used technologies sequencing and conditions experimental of Summary and elemental sulfur, as well as total iron and copper until 14 days after onset of t ferrivorans At. .ferriphilum L. ferrivorans At. t caldus At. .thermosulfidooxidans S. ferriphilum L. microbial activity. Thereafter, planktonic cells were harvested from the medium by centrifugation and RNA and proteins were extracted as described above for L. ferriphilum. The investigated ASSs (V) were sampled from six sites in Västerbotten county (Sweden). The soil profile’s pH was measured, samples of the different horizons subsequently dried, and analyzed for heavy metal and sulfur content by inductive coupled plasma mass and optical emission spectrometry, respectively. Bacterial soil communities were analyzed by separation of intact cells from the soil and subsequent utvto usrt eprtr °)Sequencing S (°C) Temperature Substrate Continuous Cultivation ac Fe Batch S Continuous ac CuFeS CuFeS Batch Batch Fe Continuous extraction of metagenomic DNA using the PowerSoil DNA Isolation Kit (MoBio). V3-V5 regions of the 16S rRNA gene were amplified and uniquely labeled for sequencing.

4 4 Sequencing O O 2+ 2+ 6 6 2– 2–

2 2 All nucleic acid sequencing conducted during the work on this thesis was performed by the Science for Life Laboratory (SciLife; Stockholm, Sweden), including ,20 8, 20 37 37 37 37 library preparations. A complete, circular genome sequence of L. ferriphilum (III) was obtained using two Pacific BioSciences (PacBio) single-molecule real-time sequencing cells. 16S rRNA gene amplicon sequencing for community analysis of ASS (V) was performed using the Illumina MiSeq v3 platform. Total and depleted RNA transcript sequences (I, II, III, IV) were obtained using an Illumina HiSeq2500 in high output mode. Protein sequences (III) were obtained by collaborators using Transcriptome Transcriptome Transcriptome Proteome Transcriptome, Proteome Transcriptome, Genome 6 RAgene rRNA 16S an EASY-nLC 1000 liquid chromatography system (Thermo Scientific), and a Q- Exactive HF mass spectrometer (Thermo Scientific).

Multi ”-omics” analysis

Various bioinformatic tools were used to analyze sequencing data. The L. ferriphilum genomic reads obtained from PacBio ultra long-read sequencing (III) were assembled at the sequencing facility by HGAP3. The resulting circular contig was annotated by Prokka (Seemann 2014) using the standard plus a custom database

62 63 Methodology Sequencing

4 4 Sequencing O O 2+ 2+ 6 6 2– 2–

Multi ”-omics” analysis

62 63 Methodology including genes from related biomining organisms from the Integrated Microbial Genomes system (Markowitz et al. 2012). 16S rRNA gene amplicons (V) were analyzed through the UPARSE pipeline (Edgar 2013) and operational taxonomic units (OTUs) annotated against the SILVA database (Quast et al. 2013). Data analysis was conducted in R using the phyloseq package (McMurdie & Holmes 2013). At. ferrivorans transcriptomic reads (I, II) were processed using its published genome sequence as a reference and the ”Tuxedo” pipeline (Trapnell et al. 2012), in- Summary of Results cluding differential expression analysis of the two temperature conditions (II). For The studies conducted in this thesis examined acidophiles in natural and (meta-) transcriptome analysis of L. ferriphilum (III) and the bioleaching experi- applied communities with the help of ”-omics” technologies (Figure 9). Genomic ments (IV), sequencing reads were mapped to the respective reference genomes, and metagenomic analysis allows for the identification of the genetic potential and differential expression analysis was performed using the DESeq2 package in R of an organism or community, respectively. Microorganisms in the laboratory or (Love et al. 2014). environment do however not always use their full arsenal of encoded genes and Protein identification for L. ferriphilum (III) was performed using the An- therefore, (meta-) transcriptomics and proteomics are useful tools to identify actively dromeda software (Cox et al. 2011). Perseus (Tyanova et al. 2016) aided in dif- transcribed and translated genes. In addition, microbial regulation and adaptation to ferential protein abundance assessment and statistical analysis. different conditions can be assessed by comparing expression patterns between them. Within the context of biomining, a special focus was set on the cold-adapted At. ferrivorans and the important iron-oxidizer L. ferriphilum in this thesis. As discussed in the previous sections, biomining is defined as the microbially assisted dissolution of sulfide minerals to gain or ease access to metals of interest. So far, in particular heap bioleaching operations are mostly localized in warm or moderately temperated regions. An organism that is able to catalyze the necessary processes also at low temperatures is At. ferrivorans. This bacterium is well established to be present in cold, acidic environments and is known to be capable of oxidizing both iron and ISCs. Aspects of its sulfur metabolism and adaptation to cold environments were therefore elucidated. A mineral that is prominently, albeit with great difficulty, exploited by bioleaching is chalcopyrite, the world’s most abundant remaining source of copper. One outstanding organism frequently found to represent large portions of microbial biomass in biomining operations is L. ferriphilum. To understand this species’ role in these environments even better, its type-strain’s complete genome sequence was acquired and analyzed, along with its transcription and translation in during bioleaching. Subsequently, it was tested how an acidophile community consisting of L. ferriphilum, S. thermosulfidooxidans, and At. caldus interacts and how manipula-

64 65 Methodology including genes from related biomining organisms from the Integrated Microbial Genomes system (Markowitz et al. 2012). 16S rRNA gene amplicons (V) were analyzed through the UPARSE pipeline (Edgar 2013) and operational taxonomic units (OTUs) annotated against the SILVA database (Quast et al. 2013). Data analysis was conducted in R using the phyloseq package (McMurdie & Holmes 2013). At. ferrivorans transcriptomic reads (I, II) were processed using its published genome sequence as a reference and the ”Tuxedo” pipeline (Trapnell et al. 2012), in- Summary of Results cluding differential expression analysis of the two temperature conditions (II). For The studies conducted in this thesis examined acidophiles in natural and (meta-) transcriptome analysis of L. ferriphilum (III) and the bioleaching experi- applied communities with the help of ”-omics” technologies (Figure 9). Genomic ments (IV), sequencing reads were mapped to the respective reference genomes, and metagenomic analysis allows for the identification of the genetic potential and differential expression analysis was performed using the DESeq2 package in R of an organism or community, respectively. Microorganisms in the laboratory or (Love et al. 2014). environment do however not always use their full arsenal of encoded genes and Protein identification for L. ferriphilum (III) was performed using the An- therefore, (meta-) transcriptomics and proteomics are useful tools to identify actively dromeda software (Cox et al. 2011). Perseus (Tyanova et al. 2016) aided in dif- transcribed and translated genes. In addition, microbial regulation and adaptation to ferential protein abundance assessment and statistical analysis. different conditions can be assessed by comparing expression patterns between them. Within the context of biomining, a special focus was set on the cold-adapted At. ferrivorans and the important iron-oxidizer L. ferriphilum in this thesis. As discussed in the previous sections, biomining is defined as the microbially assisted dissolution of sulfide minerals to gain or ease access to metals of interest. So far, in particular heap bioleaching operations are mostly localized in warm or moderately temperated regions. An organism that is able to catalyze the necessary processes also at low temperatures is At. ferrivorans. This bacterium is well established to be present in cold, acidic environments and is known to be capable of oxidizing both iron and ISCs. Aspects of its sulfur metabolism and adaptation to cold environments were therefore elucidated. A mineral that is prominently, albeit with great difficulty, exploited by bioleaching is chalcopyrite, the world’s most abundant remaining source of copper. One outstanding organism frequently found to represent large portions of microbial biomass in biomining operations is L. ferriphilum. To understand this species’ role in these environments even better, its type-strain’s complete genome sequence was acquired and analyzed, along with its transcription and translation in during bioleaching. Subsequently, it was tested how an acidophile community consisting of L. ferriphilum, S. thermosulfidooxidans, and At. caldus interacts and how manipula-

64 65 Summary of Results Papers I-IV: Systems biology of biomining organisms

tions of this defined community could result in improved leaching rates. The topic of ASS has largely been ignored in the boreal regions, although their detrimental effects on the environment are well established. Therefore, the state of several ASS sites in Northern Sweden and their microbial community were investigated in an attempt to raise awareness of the problem, and increase our understanding of the process in cold climates.

Papers I-IV: Systems biology of biomining organisms Physiology of cold-adapted Acidithiobacillus ferrivorans Previous genomic analysis of At. ferrivorans strain SS3 revealed genes coding for significant redundancy in the species’ oxidative sulfur metabolism (Liljeqvist et al. 2013; Talla et al. 2014). For instance, two systems for elemental sulfur oxidation are present (Hdr and Sor), two thiosulfate oxidation systems (Sox complex and DoxDA), and two copies of genes encoding sulfide oxidation (Sqr). In order to elucidate their actual utilization, RNA transcript analysis of these genes was conducted on cells grown on the biomining relevant substrate tetrathionate (I). According to the number of transcripts encoding the respective gene products, initial tetrathionate oxidation was conducted by TetH1. The formed thiosulfate was first believed to be oxidized by the truncated sox cluster, as it showed higher transcript counts compared to doxDA. Yet, closer analysis of the truncated genes revealed several important binding sites missing, raising in question the genes’ functionality, and making it more likely that in fact the doxDA-cluster facilitates thiosulfate oxidation. The significant expression of Sox in the analyzed strain could however indicate that environmental strains utilize functional homologues of both systems. Transcript counts attributed to elemental sulfur oxidation systems were dominated by the hdr-cluster, although in the tested conditions the sulfur originated from enzymatic reactions rather than extracellular, as the experimental culture was given soluble tetrathionate as a substrate. In biomining applications, elemental sulfur is available in the environment, and gene expression may vary. Lastly, transcripts related to sulfide oxidation by two copies of sqr were investigated and revealed the expression of both genes, albeit with a slight preference towards sqr2. In addition to sulfur related systems, genes predicted to be involved in ferrous iron oxidation were observed to be expressed, i.e. the rus-cluster and iro Figure 9: Summary of ”-omics” analysis conducted during the work on this thesis. Roman numerals refer to the papers as described in section Included publications iron oxidase. This could indicate a permanent ”ready-state” for iron oxidation in At. ferrivorans, and confirm a preference to ferrous iron for its energy acquisition, as

66 67 Summary of Results Papers I-IV: Systems biology of biomining organisms

66 67 Summary of Results Papers I-IV: Systems biology of biomining organisms previously reported for other Acidithiobacilli (Ponce et al. 2012). fixation, motility, and chemotaxis, but also more specific adaptations, such as Beside substrate utilization, previously unexplored molecular mechanisms pH homeostasis, metal resistance, and oxidative stress response. In addition, a responsible for At. ferrivoran’s adaptation to low temperatures were investigated; cluster of nif genes responsible for nitrogen fixation was identified that was not using differential expression analysis of growth at 8 versus 20 °C (II). A large previously reported in the type strain. Analysis of RNA transcript numbers and number of cold-tolerance systems were identified on the At. ferrivorans SS3 protein concentrations revealed the transcription and translation patterns during genome, including (i) DNA-binding CSPs, (ii) chaperones and helicases in particular growth on ferrous iron, although e.g. the function of nitrogen fixation could not be of the DEAD/DEAH type, (iii) cell wall and membrane modification proteins resolved, likely due to the availability of ammonium and therefore lacking need for (desaturases and hopanoid biosynthesis proteins), (iv) systems related to the import the expression of these genes in the medium. Interestingly however, although carbon and synthesis of compatible solutes (e.g. trehalose), and (v) oxidative stress response fixation is suggested to be conducted via the reverse TCA cycle in L. ferriphilum, that aids in tolerating increased oxygen solubility at low temperatures (catalase, RuBisCo exhibits similarly high protein concentrations. superoxide dismutase, and cobalamin synthesis). Despite this big arsenal and L. ferriphilumT transcription and translation in continuous culture was further At. ferrivorans’ previous classification as merely ”psychro-tolerant”, only 13.8% compared with chalcopyrite bioleaching cultures, in order to identify features and of the identified cold adaptation genes were significantly differentially expressed adaptations important for its application as a biomining organism. Unexpectedly (p 0.05) between the two conditions. Genes up-regulated during growth at 8 few genes exhibited significant differential expression or translation (p 0.05), ≤ ≤ °C included one CSP and a few genes associated with compatible solutes and underlining this species’ remarkable adaptation to acidic, metal-rich environments. membrane modification. Instead, genes related to energy metabolism and translation Among the observed alterations were strongly increased transcript numbers related were enhanced. This indicates that At. ferrivorans SS3 was not stressed by this to metal resistance in the bioleaching cultures. Protein concentrations belonging temperature, and underlines the futility of defining levels of psychrophily (i.e. to this group yet appeared to decrease, with the exception of several cus copper psychrophile vs psychrotolerant) according to arbitrary temperature caps rather than efflux proteins. Additionally, biofilm formation seemed to be strongly enhanced, ranges. Instead, we suggest to designate At. ferrivorans a eurypsychrophile. as observed by the increase of transcripts and proteins related to chemotaxis and In bioleaching operations in cold climates, iron and sulfur oxidation needs to motility in the planktonic cells of the bioleaching experiments. occur over a wide temperature scale, often rapidly changing from close to freezing to 25-30 °C. The data collected in these studies confirms and in part explains, the Community-controlled leaching of chalcopyrite affinity of At. ferrivorans to cold, sulfidic environments associated with the mining Chalcopyrite bioleaching suffers from slow dissolution rates, likely due to the forma- industry. The results are encouraging to conduct more research, but also to explore tion of passivating layers. It is long known that passivation is reduced at low redox this species’ potential in more applied industrial settings. potentials, but conventional redox control is impossible in bioleaching heaps. Mi- crobial redox control was first reported only recently (Masaki et al. 2018), and con- Physiology of iron-oxidizer Leptospirillum ferriphilum firmed by our results (IV). Chalcopyrite bioleaching experiments were conducted Despite the high relevance of L. ferriphilum in bioleaching environments, no using microbial communities consisting of combinations of L. ferriphilum, S. ther- complete genome sequence of its type strain DSM 14647 was available prior to mosulfidooxidans, and At. caldus, and exhibited large differences in ORP. All com- our study (III). A circular genome obtained by PacBio ultra long-read sequencing binations including L. ferriphilum showed redox potentials vastly exceeding 600 added ~160 kbp to the existing draft, and was therefore analyzed and published mV (vs Ag/AgCl), while in cultures only containing S. thermosulfidooxidans for as a reference for future research. Functional annotation confirmed and extended microbial iron oxidation ORP remained significantly below that value. Increased previously reported features of L. ferriphilumT’s extremely acidic life style. These ORP correlated negatively with the measured copper release from the chalcopyrite, included systems of general cellular function, e.g. energy conservation, carbon confirming that exclusion of aggressive iron-oxidizers such as L. ferriphilum, and

68 69 Summary of Results Papers I-IV: Systems biology of biomining organisms previously reported for other Acidithiobacilli (Ponce et al. 2012). fixation, motility, and chemotaxis, but also more specific adaptations, such as Beside substrate utilization, previously unexplored molecular mechanisms pH homeostasis, metal resistance, and oxidative stress response. In addition, a responsible for At. ferrivoran’s adaptation to low temperatures were investigated; cluster of nif genes responsible for nitrogen fixation was identified that was not using differential expression analysis of growth at 8 versus 20 °C (II). A large previously reported in the type strain. Analysis of RNA transcript numbers and number of cold-tolerance systems were identified on the At. ferrivorans SS3 protein concentrations revealed the transcription and translation patterns during genome, including (i) DNA-binding CSPs, (ii) chaperones and helicases in particular growth on ferrous iron, although e.g. the function of nitrogen fixation could not be of the DEAD/DEAH type, (iii) cell wall and membrane modification proteins resolved, likely due to the availability of ammonium and therefore lacking need for (desaturases and hopanoid biosynthesis proteins), (iv) systems related to the import the expression of these genes in the medium. Interestingly however, although carbon and synthesis of compatible solutes (e.g. trehalose), and (v) oxidative stress response fixation is suggested to be conducted via the reverse TCA cycle in L. ferriphilum, that aids in tolerating increased oxygen solubility at low temperatures (catalase, RuBisCo exhibits similarly high protein concentrations. superoxide dismutase, and cobalamin synthesis). Despite this big arsenal and L. ferriphilumT transcription and translation in continuous culture was further At. ferrivorans’ previous classification as merely ”psychro-tolerant”, only 13.8% compared with chalcopyrite bioleaching cultures, in order to identify features and of the identified cold adaptation genes were significantly differentially expressed adaptations important for its application as a biomining organism. Unexpectedly (p 0.05) between the two conditions. Genes up-regulated during growth at 8 few genes exhibited significant differential expression or translation (p 0.05), ≤ ≤ °C included one CSP and a few genes associated with compatible solutes and underlining this species’ remarkable adaptation to acidic, metal-rich environments. membrane modification. Instead, genes related to energy metabolism and translation Among the observed alterations were strongly increased transcript numbers related were enhanced. This indicates that At. ferrivorans SS3 was not stressed by this to metal resistance in the bioleaching cultures. Protein concentrations belonging temperature, and underlines the futility of defining levels of psychrophily (i.e. to this group yet appeared to decrease, with the exception of several cus copper psychrophile vs psychrotolerant) according to arbitrary temperature caps rather than efflux proteins. Additionally, biofilm formation seemed to be strongly enhanced, ranges. Instead, we suggest to designate At. ferrivorans a eurypsychrophile. as observed by the increase of transcripts and proteins related to chemotaxis and In bioleaching operations in cold climates, iron and sulfur oxidation needs to motility in the planktonic cells of the bioleaching experiments. occur over a wide temperature scale, often rapidly changing from close to freezing to 25-30 °C. The data collected in these studies confirms and in part explains, the Community-controlled leaching of chalcopyrite affinity of At. ferrivorans to cold, sulfidic environments associated with the mining Chalcopyrite bioleaching suffers from slow dissolution rates, likely due to the forma- industry. The results are encouraging to conduct more research, but also to explore tion of passivating layers. It is long known that passivation is reduced at low redox this species’ potential in more applied industrial settings. potentials, but conventional redox control is impossible in bioleaching heaps. Mi- crobial redox control was first reported only recently (Masaki et al. 2018), and con- Physiology of iron-oxidizer Leptospirillum ferriphilum firmed by our results (IV). Chalcopyrite bioleaching experiments were conducted Despite the high relevance of L. ferriphilum in bioleaching environments, no using microbial communities consisting of combinations of L. ferriphilum, S. ther- complete genome sequence of its type strain DSM 14647 was available prior to mosulfidooxidans, and At. caldus, and exhibited large differences in ORP. All com- our study (III). A circular genome obtained by PacBio ultra long-read sequencing binations including L. ferriphilum showed redox potentials vastly exceeding 600 added ~160 kbp to the existing draft, and was therefore analyzed and published mV (vs Ag/AgCl), while in cultures only containing S. thermosulfidooxidans for as a reference for future research. Functional annotation confirmed and extended microbial iron oxidation ORP remained significantly below that value. Increased previously reported features of L. ferriphilumT’s extremely acidic life style. These ORP correlated negatively with the measured copper release from the chalcopyrite, included systems of general cellular function, e.g. energy conservation, carbon confirming that exclusion of aggressive iron-oxidizers such as L. ferriphilum, and

68 69 Summary of Results Paper V: Acid sulfate soil in Sweden promotion of weaker iron-oxidizers such as S. thermosulfidooxidans could benefit a similar microbial community as the non-flooded ASSs, prominently including bioleaching operations. In order to identify features potentially responsible for the Acidimicrobiaceae and Xanthomonadales. This supports the hypothesis that this site difference in iron-oxidation efficiency of these two species, their transcriptional re- used to be oxidized prior to the flooding. Due to this environment now being anoxic, sponse to co-culture was analyzed. The data revealed no effect in L. ferriphilum, but many of the acidophilic community members possibly grow via ferric iron reduction, strong down-regulation of iron oxidation genes in S. thermosulfidooxidans during a process that is proton consuming and could be responsible for the increased pH. co-culture. This could suggest affinity differences in ferrous iron oxidation sys- The results of this study confirm the oxidation of PASS occurs in boreal climates, tems of the two microorganisms, and encourages future research in the identified and elucidates the microbial community members potentially involved in both acid gene’s products. Further differences were discovered by microscopical analysis, generation and remediation. which revealed a large proportion of S. thermosulfidooxidans cells to remain planktonic, while the majority of L. ferriphilum attaches to the mineral. The attachment of these cells by EPS could lead to localized concentration of ferric ions that additionally raise ORP and negatively affect copper release. The data collected during this study confirms that microbial inoculation strategies are a viable tool for redox control, and underlines the importance of developing methods to manage acidophilic communities in biomining operations.

Paper V: Acid sulfate soil in Sweden

Six sites in in the Västerbotten county in Northern Sweden were investigated for the presence of ASS (V). The measured geochemical parameters revealed three of the tested soils to be classical ASSs with decreased pH and sulfur concentration, as well as trace metal loss. One of these sites has been flooded for an extended period of time, and exhibited higher pH compared to the other two. The three remaining sites displayed heterogeneous soil profiles containing both clay and sandy material, but had overall higher pH and sulfur content and were therefore not considered ASS. Microbial alpha diversity indices of the soil profiles did not correlate with the measured parameters, indicating that e.g. decreased pH did not prevent the formation of a diverse soil community. Consequently, analysis of the present microorganisms was concentrated on the classic ASSs. 16S rRNA gene sequences isolated from these environments included mainly typical soil bacteria, but also several OTUs closely related to Halanaerobiales spp. that were originally isolated from the acidic Rio Tinto. Many other OTUs aligned closely to known acidophiles as well, e.g. the family Acidimicrobiales, or to iron-oxidizing bacteria such as Gallionellaceae or Ferrovaceae. In contrast to a studied ASS field in Finland, members of the Acidithiobacilli were not identified. The flooded ASS site exhibited

70 71 Summary of Results Paper V: Acid sulfate soil in Sweden promotion of weaker iron-oxidizers such as S. thermosulfidooxidans could benefit a similar microbial community as the non-flooded ASSs, prominently including bioleaching operations. In order to identify features potentially responsible for the Acidimicrobiaceae and Xanthomonadales. This supports the hypothesis that this site difference in iron-oxidation efficiency of these two species, their transcriptional re- used to be oxidized prior to the flooding. Due to this environment now being anoxic, sponse to co-culture was analyzed. The data revealed no effect in L. ferriphilum, but many of the acidophilic community members possibly grow via ferric iron reduction, strong down-regulation of iron oxidation genes in S. thermosulfidooxidans during a process that is proton consuming and could be responsible for the increased pH. co-culture. This could suggest affinity differences in ferrous iron oxidation sys- The results of this study confirm the oxidation of PASS occurs in boreal climates, tems of the two microorganisms, and encourages future research in the identified and elucidates the microbial community members potentially involved in both acid gene’s products. Further differences were discovered by microscopical analysis, generation and remediation. which revealed a large proportion of S. thermosulfidooxidans cells to remain planktonic, while the majority of L. ferriphilum attaches to the mineral. The attachment of these cells by EPS could lead to localized concentration of ferric ions that additionally raise ORP and negatively affect copper release. The data collected during this study confirms that microbial inoculation strategies are a viable tool for redox control, and underlines the importance of developing methods to manage acidophilic communities in biomining operations.

Paper V: Acid sulfate soil in Sweden

70 71 Conclusions

Acidophiles are remarkable life forms, surviving in some of the harshest environments on the planet. Vast concentrations of protons, toxic heavy metals, and aggressive oxidative agents are only some of the obstacles acidophilic Archaea and Bacteria have to overcome to multiply and colonize a habitat, and often these organisms persist by using energy from nothing but the most basic molecules. Humanity devised methods to utilize their unique metabolic properties only shortly after their discovery in the early 20th century and today, acidophiles are most widely used to leach metals from sulfidic minerals. Nevertheless, much can still be learned from these organisms. In recent years genomic, transcriptomic, and proteomic analysis of acidophiles has given unprecedented insights into the ecology and adaptation of these microbes. This, for instance, greatly deepened our understanding of the causes and progression of ARD, AMD, and ASS, allowing us to improve old, and develop new approaches for remediation. Countless studies have been conducted focusing on the application of acidophiles for biomining, and substantial advances have been made with respect to the organisms involved, which types of ore can be leached, and what parameters can be tweaked to increase the efficiency of industrial operations. Room for improvement however still remains, e.g. in the understanding and overcoming of chalcopyrite passivation in heap bioleaching. Also much is still to be done in the field of genetic engineering of acidophiles, where tools for manipulation of the genome have only recently become available. With ever increasing knowledge and many of yesterday’s now seemingly ”simple” questions answered, the problems of today are inevitably becoming more and more complex, and will not be solved by the effort of a single scientist or even group. With the broad accessibility to a wide range of analytic technologies, interdisciplinary efforts are a necessity, and will become even more so, with numerous experts interacting and working as a team toward a common goal. Such

73 Conclusions

73 efforts are already observable in this, and many other fields, and fortunately widely supported by funding bodies. Nevertheless, in the era of big data one should also never lose sight of the biochemical confirmation of predicted features and remember that a result is only as good as the used database.

Acknowledgements

If somebody had told me 15 years ago that with almost 30, I would still go to ”school”, I would have laughed for days. Although I was never sure what to do in my life, I definitely knew sure what I was not going to do. The University entrance diploma, for example. I would just go find a job and do some real work. Earn some money. Have a good life. Like other people. Then, somehow with a diploma in my pocket, I was never actually going to go to University. Maybe I would do some half-half, study a bit, but also get a job. Get some money. Like other people. After three years of ”accidental” Bachelor studies, I was never going to do a Master’s. It was high time to get that job. I guess thats point goes to me; I never actually did get a Master’s degree after all. On the other hand, thats only because my PhD position got in the way. You know, the one that I was never gonna do. Nevertheless, in the end I have to admit that everything worked out quite fine for me. I love to do research, and surely would miss it if I had to change my career. However, by now I guess it is time to appreciate that universal ”Never say never”, and just roll with it. Denn erstens kommt es anders, und zweitens als man denkt. A remarkable thing is that there are a lot of people that kept up with my non- sense in that turbulent time, and even supported and appreciated it. Without them, this thesis could not have been written. My first and foremost gratitude goes to my parents, Susanne and Frank, and my brother Manuel, which were ever patient, and supported whatever I decided to do.

Mama und Papa, vielen Dank, von ganzem Herzen, für eure Gedult und eure Un- terstützung. Obwohl ich nie eine Ahnung hatte was mal aus mir wird, habt ihr mich nie unter Druck gesetzt, und mich meinen eigenen Weg finden lassen. Und es hat ja auch ganz gut geklappt, bisher zumindest. Auch jetzt weiss ich noch nicht, wohin es mich mal verschlagen wird, aber habt keine Sorge. Die Welt ist klein, und ich werde

74 75 efforts are already observable in this, and many other fields, and fortunately widely supported by funding bodies. Nevertheless, in the era of big data one should also never lose sight of the biochemical confirmation of predicted features and remember that a result is only as good as the used database.

Acknowledgements

74 75 für immer euer Sohn sein. Ich liebe euch. met here in Kalmar and because of you there is not a single day where I don’t have a reason to smile. Nothing bad that life or work could throw at me these last years Next, I would like to thank the man that gave me, a German guy with a mohawk, had any power, compared to the love and support you show me. You calm me when a big mouth, and no idea when to shut up, a chance to work in his group. Mark, I I’m troubled, and make me laugh when I’m frustrated. I can do anything, because I am very grateful for your trust and support during the now almost seven years we know you will always be by my side. I am the luckiest guy in the world for it, and I know each other. As your student, help was at most one email away, and I hardly will never stop loving you. Thank you. can imagine a better supervisor. My group was going through large changes at times. People left, others arrived. I would like to thank in particular my office mates, Elias and Domenico, for discussions, fun, and everything in between. Margarita (yeah, you still count as ”group”, sorry), thanks for your Spanish temper, you made many a rainy day brighter, even if it was mostly from underground. Gaofeng, Laura, Stephanie, and Magnus, thank you for being great colleagues, and all the best to you. Many others have made my work and life in Kalmar a more than pleasant journey, and you have my sincere thanks and appreciation. I am not a fan of huge lists, however, and all you people know who you are anyway, so please forgive if I do not mention every single one of you. Feel hugged nonetheless, and take the opportunity for a physical hug as long as I am here. Some people have to be mentioned though. Alexis, Mireia, Carina, Michelle, Jo. Neither of you people is still in Kalmar, but you were my oldest friends here. Its not the same without you. Anu, little Håkan, big Håkan, Anna. You must be among the best nordic people ever. Neither of you I will ever forget. Older friends remain in Germany. Thomas, my sunshine, we know each other 13 years now and you still make my heart beat faster every time i see you. More than half of this time we are actually not living in the same town, and I think this makes our friendship pretty damn impressive. You are my best friend, and this will never change. Caro, thanks for taking care of him. He is not the youngest or prettiest anymore, and I don’t think he would do so well without you. Anna. Our friendship is special because we barely ever see each other. Our paths ran alongside only twice, but somehow we stayed in contact ever after you called me out for my dialect in the very first lecture of the ”Water Science” program. I have never seen a Bavarian that so much wanted to be a Swabian. Thank you for being able to talk dialect and be myself every now and then in the ”Pott” and in Göttingen, for ridiculous amounts of eaten pizza, and late-night discussions about literally anything. Lastly, I want to thank the most special person in my life. Xiaofen, we have

76 77 für immer euer Sohn sein. Ich liebe euch. met here in Kalmar and because of you there is not a single day where I don’t have a reason to smile. Nothing bad that life or work could throw at me these last years Next, I would like to thank the man that gave me, a German guy with a mohawk, had any power, compared to the love and support you show me. You calm me when a big mouth, and no idea when to shut up, a chance to work in his group. Mark, I I’m troubled, and make me laugh when I’m frustrated. I can do anything, because I am very grateful for your trust and support during the now almost seven years we know you will always be by my side. I am the luckiest guy in the world for it, and I know each other. As your student, help was at most one email away, and I hardly will never stop loving you. Thank you. can imagine a better supervisor. My group was going through large changes at times. People left, others arrived. I would like to thank in particular my office mates, Elias and Domenico, for discussions, fun, and everything in between. Margarita (yeah, you still count as ”group”, sorry), thanks for your Spanish temper, you made many a rainy day brighter, even if it was mostly from underground. Gaofeng, Laura, Stephanie, and Magnus, thank you for being great colleagues, and all the best to you. Many others have made my work and life in Kalmar a more than pleasant journey, and you have my sincere thanks and appreciation. I am not a fan of huge lists, however, and all you people know who you are anyway, so please forgive if I do not mention every single one of you. Feel hugged nonetheless, and take the opportunity for a physical hug as long as I am here. Some people have to be mentioned though. Alexis, Mireia, Carina, Michelle, Jo. Neither of you people is still in Kalmar, but you were my oldest friends here. Its not the same without you. Anu, little Håkan, big Håkan, Anna. You must be among the best nordic people ever. Neither of you I will ever forget. Older friends remain in Germany. Thomas, my sunshine, we know each other 13 years now and you still make my heart beat faster every time i see you. More than half of this time we are actually not living in the same town, and I think this makes our friendship pretty damn impressive. You are my best friend, and this will never change. Caro, thanks for taking care of him. He is not the youngest or prettiest anymore, and I don’t think he would do so well without you. Anna. Our friendship is special because we barely ever see each other. Our paths ran alongside only twice, but somehow we stayed in contact ever after you called me out for my dialect in the very first lecture of the ”Water Science” program. I have never seen a Bavarian that so much wanted to be a Swabian. Thank you for being able to talk dialect and be myself every now and then in the ”Pott” and in Göttingen, for ridiculous amounts of eaten pizza, and late-night discussions about literally anything. Lastly, I want to thank the most special person in my life. Xiaofen, we have

76 77 References

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Annual Review of Microbiology 55: 333–356. Journal of Aquatic Science and Technology 20: Ferrer, A, Orellana, O & Levicán, G (2016). Oxidative stress and metal tolerance in extreme acidophiles. Demergasso, CS, Galleguillos, PAP, Escudero, LVG, Zepeda, VJA, Castillo, D & Casamayor, EO (2005) In: Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Molecular characterization of microbial populations in a low-grade copper ore bioleaching test Academic Press Norfolk, VA, pp. 63–76. heap. Hydrometallurgy 80: 241–253. Fitzpatrick, R, Shand, P & Mosley, L (2017) Acid sulfate soil evolution models and pedogenic pathways Demergasso, C, Galleguillos, F, Soto, P, Serón, M & Iturriaga, V (2010) Microbial succession during a during drought and reflooding cycles in irrigated areas and adjacent natural wetlands. Geoderma heap bioleaching cycle of low grade copper sulfides. Hydrometallurgy 104: 382–390. 308: 270–290. Demergasso, C, Véliz, R, Galleguillos, PA, Marı́n, S, Acosta, M, Zepeda, VJ, Bekios, J & Zeballos, J Flores, GE, Wagner, ID, Liu, Y & Reysenbach, AL (2012) Distribution, abundance, and diversity patterns (2017) From knowledge to best practices in bioleaching. Solid State Phenomena 262: 285–289. of the thermoacidophilic Deep-Sea Hydrothermal Vent Euryarchaeota 2. Frontiers in Microbiology Demirel, Y & Sandler, SI (2002) Thermodynamics and bioenergetics. Biophysical Chemistry 97: 87–111. 3: 47. Desbruyères, D, Biscoito, M, Caprais, JC, Colaço, A, Comtet, T, Crassous, P, Fouquet, Y, Khripounoff, Frey, M (2002) Hydrogenases: hydrogen-activating enzymes. Chembiochem 3: 153–160. A, Bris, NL, Olu, K, et al. (2001) Variations in deep-sea hydrothermal vent communities on the Fridovich, I (1978) The biology of oxygen radicals. Science 201: 875–880. mid-atlantic ridge near the Azores plateau. Deep Sea Research Part I: Oceanographic Research Fuhrmann, G & Leroux, JC (2013) Improving the stability and activity of oral therapeutic enzymes - Papers 48: 1325–1346. recent advances and perspectives. Pharmaceutical Research 31: 1099–1105. Dold, B (2017) Acid rock drainage prediction: a critical review. Journal of Geochemical Exploration 172: Furniss, G, Hinman, NW, Doyle, GA & Runnells, DD (1999) Radiocarbon-dated ferricrete provides a 120–132. record of natural acid rock drainage and paleoclimatic changes. Environmental Geology 37: 102– Domic, EM (2007). A review of the development and current status of copper bioleaching operations in 106. Chile: 25 years of successful commercial implementation. In: Biomining. Ed. by DE Rawlings & Futterer, O, Angelov, A, Liesegang, H, Gottschalk, G, Schleper, C, Schepers, B, Dock, C, Antranikian, G DB Johnson. Berlin: Springer-Verlag, pp. 81–95. & Liebl, W (2004) Genome sequence of Picrophilus torridus and its implications for life around pH Dopson, M (2016). Physiological and phylogenetic diversity of acidophilic bacteria. In: Acidophiles - life 0. Proceedings of the National academy of Sciences of the United States of America 101: 9091–6. in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Academic Press. Gaisin, VA, Kalashnikov, AM, Grouzdev, DS, Sukhacheva, MV, Kuznetsov, BB & Gorlenko, VM (2017) Dopson, M, Baker-Austin, C, Hind, A, Bowman, JP & Bond, PL (2004) Characterization of Ferroplasma Chloroflexus islandicus sp. nov., a thermophilic filamentous anoxygenic phototrophic bacterium isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and from a geyser. International Journal of Systematic and Evolutionary Microbiology 67: 1381–1386. industrial bioleaching environments. Applied and Environmental Microbiology 70: 2079–88. Giaveno, MA, Urbieta, MS, Ulloa, JR, Toril, EG & Donati, ER (2012) Physiologic versatility and growth Dopson, M, Baker-Austin, C, Koppineedi, PR & Bond, PL (2003) Growth in sulfidic mineral environ- flexibility as the main characteristics of a novel thermoacidophilic Acidianus strain isolated from ments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology 149: 1959–70. Copahue geothermal area in Argentina. Microbial Ecology 65: 336–346. Dopson, M, Halinen, AK, Rahunen, N, Ozkaya, B, Sahinkaya, E, Kaksonen, AH, Lindstrom, EB & Golovacheva, RS & Karavaiko, GI (1978) A new genus of thermophilic spore-forming bacteria, Puhakka, JA (2007) Mineral and iron oxidation at low temperatures by pure and mixed cultures of Sulfobacillus. Microbiology (English translation of Mikrobiologiya) 47: 658–664. acidophilic microorganisms. Biotechnology and Bioengineering 97: 1205–15. Goltsman, DSA, Dasari, M, Thomas, BC, Shah, MB, VerBerkmoes, NC, Hettich, RL & Banfield, JF Dopson, M & Holmes, DS (2014) Metal resistance in acidophilic microorganisms and its significance for (2013) New group in the Leptospirillum clade: cultivation-independent community genomics, biotechnologies. Applied Microbiology and Biotechnology 98: 8133–44. proteomics, and transcriptomics of the new species Leptospirillum Group IV UBA BS. Applied Dopson, M, Ossandon, FJ, Lovgren, L & Holmes, DS (2014) Metal resistance or tolerance? Acidophiles and Environmental Microbiology 79: 5384–5393. confront high metal loads via both abiotic and biotic mechanisms. Frontiers in Microbiology 5: Golyshina, OV, Yakimov, MM, Lunsdorf, H, Ferrer, M, Nimtz, M, Timmis, KN, Wray, V, Tindall, BJ 157. & Golyshin, PN (2009) Acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family Dopson, M & Johnson, DB (2012) Biodiversity, metabolism and applications of acidophilic sulfur- Ferroplasmaceae isolated from a hydrothermal pool, and transfer of Ferroplasma cupricumulans metabolizing microorganisms. Environmental Microbiology 14: 2620–2631. to Acidiplasma cupricumulans comb. nov. International Journal of Systematic and Evolutionary Drobner, E, Huber, H & Stetter, KO (1990) Thiobacillus ferrooxidans, a facultative hydrogen oxidizer. Microbiology 59: 2815–2823. Applied and Environmental Microbiology 56: 2922–3. Golyshina, O, Ferrer, M & Golyshin, PN (2016). Diversity and physiologies of acidophilic archaea. In: Druschel, GK, Baker, BJ, Gihring, TM & Banfield, JF (2004) Acid mine drainage biogeochemistry at Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Iron Mountain, California. Geochemical Transactions 5: 13–32. Academic Press. Dunfield, PF, Yuryev, A, Senin, P, Smirnova, AV,Stott, MB, Hou, S, Ly, B, Saw, JH, Zhou, Z, Ren, Y, et al. Gonzalez-Toril, E, Llobet-Brossa, E, Casamayor, EO, Amann, R & Amils, R (2003) Microbial ecology (2007) Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. of an extreme acidic environment, the Tinto river. Applied and Environmental Microbiology 69: Nature 450: 879–82. 4853–65. Durvasula, R & Rao, D (2018) Extremophiles: From biology to biotechnology. CRC Press. 399 pp. Goto, K, Mochida, K, Kato, Y, Asahara, M, Fujita, R, An, SY, Kasai, H & Yokota, A (2007) Proposal of Dutrizac J.E.; MacDonald, R (1974) Ferric ion as a leaching medium. Minerals Science and Engineering six species of moderately thermophilic, acidophilic, endospore-forming bacteria: Alicyclobacillus 6: 59–95. contaminans sp. nov., Alicyclobacillus fastidiosus sp. nov., Alicyclobacillus kakegawensis sp. nov., Edgar, RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Alicyclobacillus macrosporangiidus sp. nov., Alicyclobacillus sacchari sp. nov. and Alicyclobacil- Methods 10: 996–8. lus shizuokensis sp. nov. International Journal of Systematic and Evolutionary Microbiology 57: Ehrlich, HL (2001) Past, present and future of biohydrometallurgy. Hydrometallurgy 59: 127–134. 1276–1285.

82 83 Davis Jr., R, Welty, AT, Borrego, J, Morales, JA, Pendon, JG & Ryan, JG (2000) Rio Tinto estuary (spain): Elleuche, S, Schroder, C, Sahm, K & Antranikian, G (2014) Extremozymes - biocatalysts with unique 5000 years of pollution. Environmental Geology 39: 1107–1116. properties from extremophilic microorganisms. Current Opinion in Biotechnology 29: 116–23. De Maayer, P, Anderson, D, Cary, C & Cowan, DA (2014) Some like it cold: understanding the survival Esparza, M, Cardenas, JP, Bowien, B, Jedlicki, E & Holmes, DS (2010) Genes and pathways for CO2 strategies of psychrophiles. EMBO Reports 15: 508–17. fixation in the obligate, chemolithoautotrophic acidophile, Acidithiobacillus ferrooxidans. BMC Delabary, GS, Souza Lima, AO de & Silva, MAC da (2017) Characterization of acidophilic bacteria Microbiology 10: 229. related to Acidiphilium cryptum from a coal-mining-impacted river of South Brazil. Brazilian Fahey, RC (2001) Novel thiols of prokaryotes. Annual Review of Microbiology 55: 333–356. Journal of Aquatic Science and Technology 20: Ferrer, A, Orellana, O & Levicán, G (2016). Oxidative stress and metal tolerance in extreme acidophiles. Demergasso, CS, Galleguillos, PAP, Escudero, LVG, Zepeda, VJA, Castillo, D & Casamayor, EO (2005) In: Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Molecular characterization of microbial populations in a low-grade copper ore bioleaching test Academic Press Norfolk, VA, pp. 63–76. heap. Hydrometallurgy 80: 241–253. Fitzpatrick, R, Shand, P & Mosley, L (2017) Acid sulfate soil evolution models and pedogenic pathways Demergasso, C, Galleguillos, F, Soto, P, Serón, M & Iturriaga, V (2010) Microbial succession during a during drought and reflooding cycles in irrigated areas and adjacent natural wetlands. Geoderma heap bioleaching cycle of low grade copper sulfides. Hydrometallurgy 104: 382–390. 308: 270–290. Demergasso, C, Véliz, R, Galleguillos, PA, Marı́n, S, Acosta, M, Zepeda, VJ, Bekios, J & Zeballos, J Flores, GE, Wagner, ID, Liu, Y & Reysenbach, AL (2012) Distribution, abundance, and diversity patterns (2017) From knowledge to best practices in bioleaching. Solid State Phenomena 262: 285–289. of the thermoacidophilic Deep-Sea Hydrothermal Vent Euryarchaeota 2. Frontiers in Microbiology Demirel, Y & Sandler, SI (2002) Thermodynamics and bioenergetics. Biophysical Chemistry 97: 87–111. 3: 47. Desbruyères, D, Biscoito, M, Caprais, JC, Colaço, A, Comtet, T, Crassous, P, Fouquet, Y, Khripounoff, Frey, M (2002) Hydrogenases: hydrogen-activating enzymes. Chembiochem 3: 153–160. A, Bris, NL, Olu, K, et al. (2001) Variations in deep-sea hydrothermal vent communities on the Fridovich, I (1978) The biology of oxygen radicals. Science 201: 875–880. mid-atlantic ridge near the Azores plateau. Deep Sea Research Part I: Oceanographic Research Fuhrmann, G & Leroux, JC (2013) Improving the stability and activity of oral therapeutic enzymes - Papers 48: 1325–1346. recent advances and perspectives. Pharmaceutical Research 31: 1099–1105. Dold, B (2017) Acid rock drainage prediction: a critical review. Journal of Geochemical Exploration 172: Furniss, G, Hinman, NW, Doyle, GA & Runnells, DD (1999) Radiocarbon-dated ferricrete provides a 120–132. record of natural acid rock drainage and paleoclimatic changes. Environmental Geology 37: 102– Domic, EM (2007). A review of the development and current status of copper bioleaching operations in 106. Chile: 25 years of successful commercial implementation. In: Biomining. Ed. by DE Rawlings & Futterer, O, Angelov, A, Liesegang, H, Gottschalk, G, Schleper, C, Schepers, B, Dock, C, Antranikian, G DB Johnson. Berlin: Springer-Verlag, pp. 81–95. & Liebl, W (2004) Genome sequence of Picrophilus torridus and its implications for life around pH Dopson, M (2016). Physiological and phylogenetic diversity of acidophilic bacteria. In: Acidophiles - life 0. Proceedings of the National academy of Sciences of the United States of America 101: 9091–6. in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Academic Press. Gaisin, VA, Kalashnikov, AM, Grouzdev, DS, Sukhacheva, MV, Kuznetsov, BB & Gorlenko, VM (2017) Dopson, M, Baker-Austin, C, Hind, A, Bowman, JP & Bond, PL (2004) Characterization of Ferroplasma Chloroflexus islandicus sp. nov., a thermophilic filamentous anoxygenic phototrophic bacterium isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and from a geyser. International Journal of Systematic and Evolutionary Microbiology 67: 1381–1386. industrial bioleaching environments. Applied and Environmental Microbiology 70: 2079–88. Giaveno, MA, Urbieta, MS, Ulloa, JR, Toril, EG & Donati, ER (2012) Physiologic versatility and growth Dopson, M, Baker-Austin, C, Koppineedi, PR & Bond, PL (2003) Growth in sulfidic mineral environ- flexibility as the main characteristics of a novel thermoacidophilic Acidianus strain isolated from ments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology 149: 1959–70. Copahue geothermal area in Argentina. Microbial Ecology 65: 336–346. Dopson, M, Halinen, AK, Rahunen, N, Ozkaya, B, Sahinkaya, E, Kaksonen, AH, Lindstrom, EB & Golovacheva, RS & Karavaiko, GI (1978) A new genus of thermophilic spore-forming bacteria, Puhakka, JA (2007) Mineral and iron oxidation at low temperatures by pure and mixed cultures of Sulfobacillus. Microbiology (English translation of Mikrobiologiya) 47: 658–664. acidophilic microorganisms. Biotechnology and Bioengineering 97: 1205–15. Goltsman, DSA, Dasari, M, Thomas, BC, Shah, MB, VerBerkmoes, NC, Hettich, RL & Banfield, JF Dopson, M & Holmes, DS (2014) Metal resistance in acidophilic microorganisms and its significance for (2013) New group in the Leptospirillum clade: cultivation-independent community genomics, biotechnologies. Applied Microbiology and Biotechnology 98: 8133–44. proteomics, and transcriptomics of the new species Leptospirillum Group IV UBA BS. Applied Dopson, M, Ossandon, FJ, Lovgren, L & Holmes, DS (2014) Metal resistance or tolerance? Acidophiles and Environmental Microbiology 79: 5384–5393. confront high metal loads via both abiotic and biotic mechanisms. Frontiers in Microbiology 5: Golyshina, OV, Yakimov, MM, Lunsdorf, H, Ferrer, M, Nimtz, M, Timmis, KN, Wray, V, Tindall, BJ 157. & Golyshin, PN (2009) Acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family Dopson, M & Johnson, DB (2012) Biodiversity, metabolism and applications of acidophilic sulfur- Ferroplasmaceae isolated from a hydrothermal pool, and transfer of Ferroplasma cupricumulans metabolizing microorganisms. Environmental Microbiology 14: 2620–2631. to Acidiplasma cupricumulans comb. nov. International Journal of Systematic and Evolutionary Drobner, E, Huber, H & Stetter, KO (1990) Thiobacillus ferrooxidans, a facultative hydrogen oxidizer. Microbiology 59: 2815–2823. Applied and Environmental Microbiology 56: 2922–3. Golyshina, O, Ferrer, M & Golyshin, PN (2016). Diversity and physiologies of acidophilic archaea. In: Druschel, GK, Baker, BJ, Gihring, TM & Banfield, JF (2004) Acid mine drainage biogeochemistry at Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & DB Johnson. Caister Iron Mountain, California. Geochemical Transactions 5: 13–32. Academic Press. Dunfield, PF, Yuryev, A, Senin, P, Smirnova, AV,Stott, MB, Hou, S, Ly, B, Saw, JH, Zhou, Z, Ren, Y, et al. Gonzalez-Toril, E, Llobet-Brossa, E, Casamayor, EO, Amann, R & Amils, R (2003) Microbial ecology (2007) Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. of an extreme acidic environment, the Tinto river. Applied and Environmental Microbiology 69: Nature 450: 879–82. 4853–65. Durvasula, R & Rao, D (2018) Extremophiles: From biology to biotechnology. CRC Press. 399 pp. Goto, K, Mochida, K, Kato, Y, Asahara, M, Fujita, R, An, SY, Kasai, H & Yokota, A (2007) Proposal of Dutrizac J.E.; MacDonald, R (1974) Ferric ion as a leaching medium. Minerals Science and Engineering six species of moderately thermophilic, acidophilic, endospore-forming bacteria: Alicyclobacillus 6: 59–95. contaminans sp. nov., Alicyclobacillus fastidiosus sp. nov., Alicyclobacillus kakegawensis sp. nov., Edgar, RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Alicyclobacillus macrosporangiidus sp. nov., Alicyclobacillus sacchari sp. nov. and Alicyclobacil- Methods 10: 996–8. lus shizuokensis sp. nov. International Journal of Systematic and Evolutionary Microbiology 57: Ehrlich, HL (2001) Past, present and future of biohydrometallurgy. Hydrometallurgy 59: 127–134. 1276–1285.

82 83 Govender, E, Kotsiopoulos, A, Bryan, C & Harrison, S (2014) Modelling microbial transport in simulated Hong, J, Silva, RA, Park, J, Lee, E, Park, J & Kim, H (2016) Adaptation of a mixed culture of acidophiles low-grade heap bioleaching systems: the biomass transport model. Hydrometallurgy 150: 299–307. for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic. Grogan, DW, Carver, GT & Drake, JW (2001) Genetic fidelity under harsh conditions: analysis of Journal of Bioscience and Bioengineering 121: 536–542. spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proceedings Huang, XY, Deng, F, Yamaji, N, Pinson, SR, Fujii-Kashino, M, Danku, J, Douglas, A, Guerinot, ML, of the National Academy of Sciences 98: 7928–7933. Salt, DE & Ma, JF (2016) A heavy metal P-type ATPase OsHMA4 prevents copper accumulation Guay, R & Silver, M (1975) Thiobacillus acidophilus sp. nov. - isolation and some physiological in rice grain. Nature Communications 7: 12138. characteristics. Canadian Journal of Microbiology 21: 281–288. Huber, G, Spinnler, C, Gambacorta, A & Stetter, KO (1989) Metallosphaera sedula gen. nov and sp. nov Gumulya, Y, Boxall, NJ, Khaleque, HN, Santala, V, Carlson, RP & Kaksonen, AH (2018) In a quest for represents a new genus of arobic, metal-mobilizing, thermoacidophilic archaebacteria. Systematic engineering acidophiles for biomining applications: challenges and opportunities. Genes (Basel) 9: and Applied Microbiology 12: 38–47. 116. Huber, G & Stetter, KO (1991) Sulfolobus metallicus, new species, a novel strictly chemolithoautotrophic Guo, X, You, XY, Liu, LJ, Zhang, JY, Liu, SJ & Jiang, CY (2009) Alicyclobacillus aeris sp. nov., a thermophilic archaeal species of metal-mobilizers. Systematic and Applied Microbiology 14: 372– novel ferrous- and sulfur-oxidizing bacterium isolated from a copper mine. International Journal 378. of Systematic and Evolutionary Microbiology 59: 2415–20. Huber, G, Drobner, E, Huber, H & Stetter, KO (1992) Growth by aerobic oxidation of molecular Halinen, AK, Beecroft, NJ, Maatta, K, Nurmi, P, Laukkanen, K, Kaksonen, AH, Riekkola-Vanhanen, hydrogen in archaea - a metabolic property so far unknown for this domain. Systematic and Applied M & Puhakka, JA (2012) Microbial community dynamics during a demonstration-scale bioheap Microbiology 15: 502–504. leaching operation. Hydrometallurgy 125: 34–41. Huber, H & Stetter, KO (1989) Thiobacillus prosperus sp. nov., represents a new group of halotolerant Hallberg, KB, Gonzalez-Toril, E & Johnson, DB (2010) Acidithiobacillus ferrivorans, sp. nov.; faculta- metal-mobilizing bacteria isolated from a marine geothermal field. Archives of Microbiology 151: tively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine- 479–485. impacted environments. Extremophiles 14: 9–19. Hug, LA, Baker, BJ, Anantharaman, K, Brown, CT, Probst, AJ, Castelle, CJ, Butterfield, CN, Hernsdorf, Hamamura, N, Olson, SH, Ward, DM & Inskeep, WP (2005) Diversity and functional analysis of AW, Amano, Y, Ise, K, et al. (2016) A new view of the tree of life. Nature Microbiology 1: eprint. bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Ingledew, WJ (1982) Thiobacillus ferrooxidans. the bioenergetics of an acidophilic chemolithotroph. Springs, Yellowstone National Park. Applied and Environmental Microbiology 71: 5943–5950. Biochimica et Biophysica Acta 683: 89–117. Harrison, AP (1984) The acidophilic thiobacilli and other acidophilic bacteria that share their habitat. Jacobs, JA & Testa, SM (2014). The Iron Mountain Mine in Shasta County, California. In: Acid mine Annual Review of Microbiology 38: 265–292. drainage, rock drainage, and acid sulfate soils. John Wiley & Sons, Inc., pp. 355–360. Harrison, JJ, Ceri, H & Turner, RJ (2007) Multimetal resistance and tolerance in microbial biofilms. Jiang, CY, Liu, Y, Liu, YY, You, XY, Guo, X & Liu, SJ (2008) Alicyclobacillus ferrooxydans sp. Nature Reviews Microbiology 5: 928–38. nov., a ferrous-oxidizing bacterium from solfataric soil. International Journal of Systematic and Hedrich, S & Johnson, DB (2013) Aerobic and anaerobic oxidation of hydrogen by acidophilic bacteria. Evolutionary Microbiology 58: 2898–903. FEMS Microbiology Letters 349: 40–5. Johnson, DB, Bacelar-Nicolau, P, Okibe, N, Thomas, A & Hallberg, KB (2009) Ferrimicrobium Hedrich, S, Joulian, C, Graupner, T, Schippers, A & Guézennec, AG (2018) Enhanced chalcopyrite acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic, dissolution in stirred tank reactors by temperature increase during bioleaching. Hydrometallurgy iron-oxidizing, extremely acidophilic actinobacteria. International Journal of Systematic and 179: 125–131. Evolutionary Microbiology 59: 1082–9. Hedrich, S & Schippers, A (2016). Distribution of acidophilic microorganisms in natural and man-made Johnson, DB & Hallberg, KB (2009) Carbon, iron and sulfur metabolism in acidophilic micro-organisms. acidic environments. In: Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & Advances in Microbial Physiology, Vol 54 54: 201–255. DB Johnson. Norfolk, UK: Caister Academic Press. Johnson, DB, Hallberg, KB & Hedrich, S (2014) Uncovering a microbial enigma: isolation and character- Hinwood, AL, Horwitz, P, Appleyard, S, Barton, C & Wajrak, M (2006) Acid sulphate soil disturbance ization of the streamer-generating, iron-oxidizing, acidophilic bacterium ’Ferrovum myxofaciens’. and metals in groundwater: implications for human exposure through home grown produce. Applied and Environmental Microbiology 80: 672–80. Environmental Pollution 143: 100–105. Johnson, DB, Okibe, N & Roberto, FF (2003) Novel thermo-acidophilic bacteria isolated from geothermal Hippe, H (2000) Leptospirillium gen. nov. (ex Markoysan 1972), nom. rev., including Leptospirillium sites in Yellowstone National Park: physiological and phylogenetic characteristics. Archives of ferrooxidans sp. nov. (ex Markoysan 1972), nom. rev. and Leptospirillium thermoferrooxidans sp. Microbiology 180: 60–8. nov. (Golovacheva et al. 1992). International Journal of Systematic and Evolutionary Microbiology Johnson, DB, Stallwood, B, Kimura, S & Hallberg, KB (2006) Isolation and characterization of 50: 501–503. Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo- Hiroyoshi, N, Tsunekawa, M, Okamoto, H, Nakayama, R & Kuroiwa, S (2013) Improved chalcopyrite acidophilic heterotrophic Proteobacterium. Archives of Microbiology 185: 212–21. leaching through optimization of redox potential. Canadian Metallurgical Quarterly 47: 253–258. Johnson, DB (2007). Physiology and ecology of acidophilic microorganisms. In: Physiology and Hoffert, JR (1947) Acid mine drainage. Industrial & Engineering Chemistry 39: 642–646. biochemistry of extremophiles. American Society of Microbiology, pp. 257–270. Hogfors-Ronnholm, E, Christel, S, Dalhem, K, Lillhonga, T, Engblom, S, Osterholm, P & Dopson, M Jones, DS, Albrecht, HL, Dawson, KS, Schaperdoth, I, Freeman, KH, Pi, Y, Pearson, A & Macalady, JL (2017) Chemical and microbiological evaluation of novel chemical treatment methods for acid (2012a) Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm. ISME sulfate soils. Science of the Total Environment 625: 39–49. Journal 6: 158–70. Holmes, DS (2017) Did life emerge in thermo-acidic conditions? American Geophysical Union Fall Jones, GC, Hille, RP van & Harrison, STL (2012b) Reactive oxygen species generated in the presence of Meeting 2017 Abstracts. fine pyrite particles and its implication in thermophilic mineral bioleaching. Applied Microbiology Holmes, DS & Bonnefoy, V (2007). Genetic and bioinformatic insights into iron and sulfur oxidation and Biotechnology 97: 2735–2742. mechanisms of bioleaching organisms. In: Biomining. Ed. by DE Rawlings & DB Johnson. Berlin Jones, RM, Hedrich, S & Johnson, DB (2013) Acidocella aromatica sp. nov.: an acidophilic heterotrophic Heidelberg New York: Springer-Verlag, pp. 281–307. Alphaproteobacterium with unusual phenotypic traits. Extremophiles 17: 841–50.

84 85 Govender, E, Kotsiopoulos, A, Bryan, C & Harrison, S (2014) Modelling microbial transport in simulated Hong, J, Silva, RA, Park, J, Lee, E, Park, J & Kim, H (2016) Adaptation of a mixed culture of acidophiles low-grade heap bioleaching systems: the biomass transport model. Hydrometallurgy 150: 299–307. for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic. Grogan, DW, Carver, GT & Drake, JW (2001) Genetic fidelity under harsh conditions: analysis of Journal of Bioscience and Bioengineering 121: 536–542. spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proceedings Huang, XY, Deng, F, Yamaji, N, Pinson, SR, Fujii-Kashino, M, Danku, J, Douglas, A, Guerinot, ML, of the National Academy of Sciences 98: 7928–7933. Salt, DE & Ma, JF (2016) A heavy metal P-type ATPase OsHMA4 prevents copper accumulation Guay, R & Silver, M (1975) Thiobacillus acidophilus sp. nov. - isolation and some physiological in rice grain. Nature Communications 7: 12138. characteristics. Canadian Journal of Microbiology 21: 281–288. Huber, G, Spinnler, C, Gambacorta, A & Stetter, KO (1989) Metallosphaera sedula gen. nov and sp. nov Gumulya, Y, Boxall, NJ, Khaleque, HN, Santala, V, Carlson, RP & Kaksonen, AH (2018) In a quest for represents a new genus of arobic, metal-mobilizing, thermoacidophilic archaebacteria. Systematic engineering acidophiles for biomining applications: challenges and opportunities. Genes (Basel) 9: and Applied Microbiology 12: 38–47. 116. Huber, G & Stetter, KO (1991) Sulfolobus metallicus, new species, a novel strictly chemolithoautotrophic Guo, X, You, XY, Liu, LJ, Zhang, JY, Liu, SJ & Jiang, CY (2009) Alicyclobacillus aeris sp. nov., a thermophilic archaeal species of metal-mobilizers. Systematic and Applied Microbiology 14: 372– novel ferrous- and sulfur-oxidizing bacterium isolated from a copper mine. International Journal 378. of Systematic and Evolutionary Microbiology 59: 2415–20. Huber, G, Drobner, E, Huber, H & Stetter, KO (1992) Growth by aerobic oxidation of molecular Halinen, AK, Beecroft, NJ, Maatta, K, Nurmi, P, Laukkanen, K, Kaksonen, AH, Riekkola-Vanhanen, hydrogen in archaea - a metabolic property so far unknown for this domain. Systematic and Applied M & Puhakka, JA (2012) Microbial community dynamics during a demonstration-scale bioheap Microbiology 15: 502–504. leaching operation. Hydrometallurgy 125: 34–41. Huber, H & Stetter, KO (1989) Thiobacillus prosperus sp. nov., represents a new group of halotolerant Hallberg, KB, Gonzalez-Toril, E & Johnson, DB (2010) Acidithiobacillus ferrivorans, sp. nov.; faculta- metal-mobilizing bacteria isolated from a marine geothermal field. Archives of Microbiology 151: tively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine- 479–485. impacted environments. Extremophiles 14: 9–19. Hug, LA, Baker, BJ, Anantharaman, K, Brown, CT, Probst, AJ, Castelle, CJ, Butterfield, CN, Hernsdorf, Hamamura, N, Olson, SH, Ward, DM & Inskeep, WP (2005) Diversity and functional analysis of AW, Amano, Y, Ise, K, et al. (2016) A new view of the tree of life. Nature Microbiology 1: eprint. bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Ingledew, WJ (1982) Thiobacillus ferrooxidans. the bioenergetics of an acidophilic chemolithotroph. Springs, Yellowstone National Park. Applied and Environmental Microbiology 71: 5943–5950. Biochimica et Biophysica Acta 683: 89–117. Harrison, AP (1984) The acidophilic thiobacilli and other acidophilic bacteria that share their habitat. Jacobs, JA & Testa, SM (2014). The Iron Mountain Mine in Shasta County, California. In: Acid mine Annual Review of Microbiology 38: 265–292. drainage, rock drainage, and acid sulfate soils. John Wiley & Sons, Inc., pp. 355–360. Harrison, JJ, Ceri, H & Turner, RJ (2007) Multimetal resistance and tolerance in microbial biofilms. Jiang, CY, Liu, Y, Liu, YY, You, XY, Guo, X & Liu, SJ (2008) Alicyclobacillus ferrooxydans sp. Nature Reviews Microbiology 5: 928–38. nov., a ferrous-oxidizing bacterium from solfataric soil. International Journal of Systematic and Hedrich, S & Johnson, DB (2013) Aerobic and anaerobic oxidation of hydrogen by acidophilic bacteria. Evolutionary Microbiology 58: 2898–903. FEMS Microbiology Letters 349: 40–5. Johnson, DB, Bacelar-Nicolau, P, Okibe, N, Thomas, A & Hallberg, KB (2009) Ferrimicrobium Hedrich, S, Joulian, C, Graupner, T, Schippers, A & Guézennec, AG (2018) Enhanced chalcopyrite acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen. nov., sp. nov.: heterotrophic, dissolution in stirred tank reactors by temperature increase during bioleaching. Hydrometallurgy iron-oxidizing, extremely acidophilic actinobacteria. International Journal of Systematic and 179: 125–131. Evolutionary Microbiology 59: 1082–9. Hedrich, S & Schippers, A (2016). Distribution of acidophilic microorganisms in natural and man-made Johnson, DB & Hallberg, KB (2009) Carbon, iron and sulfur metabolism in acidophilic micro-organisms. acidic environments. In: Acidophiles - life in extremely acidic environments. Ed. by R Quatrini & Advances in Microbial Physiology, Vol 54 54: 201–255. DB Johnson. Norfolk, UK: Caister Academic Press. Johnson, DB, Hallberg, KB & Hedrich, S (2014) Uncovering a microbial enigma: isolation and character- Hinwood, AL, Horwitz, P, Appleyard, S, Barton, C & Wajrak, M (2006) Acid sulphate soil disturbance ization of the streamer-generating, iron-oxidizing, acidophilic bacterium ’Ferrovum myxofaciens’. and metals in groundwater: implications for human exposure through home grown produce. Applied and Environmental Microbiology 80: 672–80. Environmental Pollution 143: 100–105. Johnson, DB, Okibe, N & Roberto, FF (2003) Novel thermo-acidophilic bacteria isolated from geothermal Hippe, H (2000) Leptospirillium gen. nov. (ex Markoysan 1972), nom. rev., including Leptospirillium sites in Yellowstone National Park: physiological and phylogenetic characteristics. Archives of ferrooxidans sp. nov. (ex Markoysan 1972), nom. rev. and Leptospirillium thermoferrooxidans sp. Microbiology 180: 60–8. nov. (Golovacheva et al. 1992). International Journal of Systematic and Evolutionary Microbiology Johnson, DB, Stallwood, B, Kimura, S & Hallberg, KB (2006) Isolation and characterization of 50: 501–503. Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo- Hiroyoshi, N, Tsunekawa, M, Okamoto, H, Nakayama, R & Kuroiwa, S (2013) Improved chalcopyrite acidophilic heterotrophic Proteobacterium. Archives of Microbiology 185: 212–21. leaching through optimization of redox potential. Canadian Metallurgical Quarterly 47: 253–258. Johnson, DB (2007). Physiology and ecology of acidophilic microorganisms. In: Physiology and Hoffert, JR (1947) Acid mine drainage. Industrial & Engineering Chemistry 39: 642–646. biochemistry of extremophiles. American Society of Microbiology, pp. 257–270. Hogfors-Ronnholm, E, Christel, S, Dalhem, K, Lillhonga, T, Engblom, S, Osterholm, P & Dopson, M Jones, DS, Albrecht, HL, Dawson, KS, Schaperdoth, I, Freeman, KH, Pi, Y, Pearson, A & Macalady, JL (2017) Chemical and microbiological evaluation of novel chemical treatment methods for acid (2012a) Community genomic analysis of an extremely acidophilic sulfur-oxidizing biofilm. ISME sulfate soils. Science of the Total Environment 625: 39–49. Journal 6: 158–70. Holmes, DS (2017) Did life emerge in thermo-acidic conditions? American Geophysical Union Fall Jones, GC, Hille, RP van & Harrison, STL (2012b) Reactive oxygen species generated in the presence of Meeting 2017 Abstracts. fine pyrite particles and its implication in thermophilic mineral bioleaching. Applied Microbiology Holmes, DS & Bonnefoy, V (2007). Genetic and bioinformatic insights into iron and sulfur oxidation and Biotechnology 97: 2735–2742. mechanisms of bioleaching organisms. In: Biomining. Ed. by DE Rawlings & DB Johnson. Berlin Jones, RM, Hedrich, S & Johnson, DB (2013) Acidocella aromatica sp. nov.: an acidophilic heterotrophic Heidelberg New York: Springer-Verlag, pp. 281–307. Alphaproteobacterium with unusual phenotypic traits. Extremophiles 17: 841–50.

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Sanyal, A, Antony, R, Samui, G & Thamban, M (2018) Microbial communities and their potential Ragon, M, Restoux, G, Moreira, D, Moller, AP & Lopez-Garcia, P (2011) Sunlight-exposed biofilm for degradation of dissolved organic carbon in cryoconite hole environments of Himalaya and microbial communities are naturally resistant to chernobyl ionizing-radiation levels. PLoS One 6: Antarctica. Microbiological Research 208: 32–42. e21764. Sapp, A, Huguet-Tapia, JC, Sanchez-Lamas, M, Antelo, GT, Primo, ED, Rinaldi, J, Klinke, S, Goldbaum, Ram, RJ, Verberkmoes, NC, Thelen, MP, Tyson, GW, Baker, BJ, Blake R. C., 2, Shah, M, Hettich, RL & FA, Bonomi, HR, Christner, BC, et al. (2018) Draft genome sequence of Methylobacterium Banfield, JF (2005) Community proteomics of a natural microbial biofilm. Science 308: 1915–20. sp. strain V23, isolated from accretion ice of the antarctic subglacial Lake Vostok. Genome Rampelotto, P (2013) Extremophiles and extreme environments. Life 3: 482–485. Announcements 6: eprint. Rawlings, DE (2002) Heavy metal mining using microbes. Annual Review of Microbiology 56: 65–91. Schelert, J, Dixit, V, Hoang, V, Simbahan, J, Drozda, M & Blum, P (2004) Occurrence and characteri- Rawlings, DE (2005) Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used zation of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of for the recovery of metals from minerals and their concentrates. Microbial Cell Factories 4: 13. gene disruption. Journal of Bacteriology 186: 427–437. Rawlings, DE & Johnson, DB (2007) The microbiology of biomining: development and optimization of Schippers, A & Sand, W (1999) Bacterial leaching of metal sulfides proceeds by two indirect mechanisms mineral-oxidizing microbial consortia. Microbiology 153: 315–24. via thiosulfate or via polysulfides and sulfur. Applied and Environmental Microbiology 65: 319– Rawlings, DE, Tributsch, H & Hansford, GS (1999) Reasons why ’Leptospirillum’-like species rather 21. than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial Schleper, C, Puehler, G, Holz, I, Gambacorta, A, Janekovic, D, Santarius, U, Klenk, HP & Zillig, W processes for the biooxidation of pyrite and related ores. Microbiology 145 ( Pt 1): 5–13. (1995) Picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus Reddy, KJ, Wang, L & Gloss, SP (1995) Solubility and mobility of copper, zinc and lead in acidic and family comprising archaea capable of growth around pH 0. Journal of Bacteriology 177: 7050– environments. Plant and Soil 171: 53–58. 9. Remonsellez, F, Orell, A & Jerez, CA (2006) Copper tolerance of the thermoacidophilic archaeon Schleper, C, Puhler, G, Klenk, HP & Zillig, W (1996) Picrophilus oshimae and Picrophilus torridus Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 152: 59–66. fam. nov., gen. nov., sp. nov., two species of hyperacidophilic, thermophilic, heterotrophic, aerobic Ren-Long, J, Wu, J, Chaw, SM, Tsai, CW & Tsen, SD (1999) A novel species of thermoacidophilic archaea. International Journal of Systematic Bacteriology 46: 814–816. archaeon, Sulfolobus yangmingensis sp. nov. International Journal of Systematic Bacteriology 49: Schoonen, MAA, Cohn, CA, Roemer, E, Laffers, R, Simon, SR & O’Riordan, T (2006) Mineral-induced 1809–1816. formation of reactive oxygen species. Medical Mineraology and Geochemistry 64: 179–221. Rensing, C, Fan, B, Sharma, R, Mitra, B & Rosen, BP (2000) Copa: an Escherichia coli Cu(I)- Schrenk, MO, Edwards, KJ, Goodman, RM, Hamers, RJ & Banfield, JF (1998) Distribution of Thiobacil- translocating P-type ATPase. Proceedings of the National academy of Sciences of the United States lus ferrooxidans and Leptospirillium ferrooxidans: implications for generation of acid mine of America 97: 652–6. drainage. Science 279: 1519–1522. Reysenbach, AL, Liu, Y, Banta, AB, Beveridge, TJ, Kirshtein, JD, Schouten, S, Tivey, MK, Von Damm, Schwartz, E, Fritsch, J & Friedrich, B (2013). H2-metabolizing prokaryotes. In: The prokaryotes. Springer KL & Voytek, MA (2006) A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal Berlin Heidelberg, pp. 119–199. vents. Nature 442: 444–7. Seemann, T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30: 2068–9. Riekkola-Vanhanen, M (2010) Talvivaara Sotkamo Mine - bioleaching of polymetallic nickel ore in Segerer, AH, Trincone, A, Gahrtz, M & Stetter, KO (1991) Stygiolobus azoricus gen. nov., sp. nov. subarctic climate. Nova Biotechnologica 10: 7–14. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Riekkola-Vanhanen, M (2013) Talvivaara mining company - from a project to a mine. Minerals Sulfolobales. International Journal of Systematic Bacteriology 41: 495–501. Engineering 48: 2–9. Segerer, A, Langworthy, TA & Stetter, KO (1988) Thermoplasma acidophilum and Thermoplasma Ruepp, A, Graml, W, Santos-Martinez, ML, Koretke, KK, Volker, C, Mewes, HW, Frishman, D, Stocker, volcanium sp. nov from solfatara fields. Systematic and Applied Microbiology 10: 161–171. S, Lupas, AN & Baumeister, W (2000) The genome sequence of the thermoacidophilic scavenger Segerer, A, Neuner, A, Kristjansson, JK & Stetter, KO (1986) Acidianus infernus gen. nov., spec. nov., and Thermoplasma acidophilum. Nature 407: 508–13. Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur- Sabath, N, Ferrada, E, Barve, A & Wagner, A (2013) Growth temperature and genome size in bacteria metabolizing archaebacteria. International Journal of Systematic Bacteriology 36: 559–564. are negatively correlated, suggesting genomic streamlining during thermal adaptation. Genome Shah, AR, Shah, R & Madamwar, D (2006) Improvement of the quality of whole wheat bread by Biology and Evolution 5: 966–977. supplementation of Xylanase from Aspergillus foetidus. Bioresource Technology 97: 2047–2053. Saha, D, Panda, A, Podder, S & Ghosh, TC (2014) Overlapping genes: a new strategy of thermophilic Sharma, HP, Patel, H & Sugandha (2016) Enzymatic added extraction and clarification of fruit juices - a stress tolerance in prokaryotes. Extremophiles 19: 345–353. review. Critical Reviews in Food Science and Nutrition 57: 1215–1227. Saitoh, N, Nomura, T & Konishi, Y (2017) Bioleaching of low-grade chalcopyrite ore by the thermophilic She, Q, Singh, RK, Confalonieri, F, Zivanovic, Y, Allard, G, Awayez, MJ, Chan-Weiher, CC, Clausen, archaean Acidianus brierleyi. Solid State Phenomena 262: 237–241. IG, Curtis, BA, De Moors, A, et al. (2001) The complete genome of the crenarchaeon Sulfolobus

90 91 Quatrini, R, Appia-Ayme, C, Denis, Y, Jedlicki, E, Holmes, DS & Bonnefoy, V (2009) Extending the Sanchez-Andrea, I, Rodriguez, N, Amils, R & Sanz, JL (2011) Microbial diversity in anaerobic sediments models for iron and sulfur oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. BMC at Rio Tinto, a naturally acidic environment with a high heavy metal content. Applied and Genomics 10: 394. Environmental Microbiology 77: 6085–93. Quatrini, R & Johnson, DB (2016) Acidophiles: Life in Extremely Acidic Environments. Caister Academic Sánchez-Andrea, I, Sanz, JL, Bijmans, MF & Stams, AJ (2014a) Sulfate reduction at low pH to remediate Press. acid mine drainage. Journal of Hazardous materials 269: 98–109. Quatrini, R & Johnson, DB (2018) Microbiomes in extremely acidic environments: functionalities Sánchez-Andrea, I, Stams, AJM, Hedrich, S, Ňancucheo, I & Johnson, DB (2014b) Desulfosporosinus and interactions that allow survival and growth of prokaryotes at low pH. Current Opinion in acididurans sp. nov.: an acidophilic sulfate-reducing bacterium isolated from acidic sediments. Microbiology 43: 139–147. Extremophiles 19: 39–47. Quehenberger, J, Shen, L, Albers, SV, Siebers, B & Spadiut, O (2017) Sulfolobus - a potential key Sandstrom, A, Shchukarev, A & Paul, J (2005) XPS characterisation of chalcopyrite chemically and bio- organism in future biotechnology. Frontiers in Microbiology 8: 2474. leached at high and low redox potential. Minerals Engineering 18: 505–515. Raddadi, N, Cherif, A, Daffonchio, D, Neifar, M & Fava, F (2015) Biotechnological applications of Santos, ECD, Mendonça Silva, JC de & Duarte, HA (2016) Pyrite oxidation mechanism by oxygen in extremophiles, extremozymes and extremolytes. Applied Microbiology and Biotechnology 99: aqueous medium. The Journal of Physical Chemistry C 120: 2760–2768. 7907–7913. Sanyal, A, Antony, R, Samui, G & Thamban, M (2018) Microbial communities and their potential Ragon, M, Restoux, G, Moreira, D, Moller, AP & Lopez-Garcia, P (2011) Sunlight-exposed biofilm for degradation of dissolved organic carbon in cryoconite hole environments of Himalaya and microbial communities are naturally resistant to chernobyl ionizing-radiation levels. PLoS One 6: Antarctica. Microbiological Research 208: 32–42. e21764. Sapp, A, Huguet-Tapia, JC, Sanchez-Lamas, M, Antelo, GT, Primo, ED, Rinaldi, J, Klinke, S, Goldbaum, Ram, RJ, Verberkmoes, NC, Thelen, MP, Tyson, GW, Baker, BJ, Blake R. C., 2, Shah, M, Hettich, RL & FA, Bonomi, HR, Christner, BC, et al. (2018) Draft genome sequence of Methylobacterium Banfield, JF (2005) Community proteomics of a natural microbial biofilm. Science 308: 1915–20. sp. strain V23, isolated from accretion ice of the antarctic subglacial Lake Vostok. Genome Rampelotto, P (2013) Extremophiles and extreme environments. Life 3: 482–485. Announcements 6: eprint. Rawlings, DE (2002) Heavy metal mining using microbes. Annual Review of Microbiology 56: 65–91. Schelert, J, Dixit, V, Hoang, V, Simbahan, J, Drozda, M & Blum, P (2004) Occurrence and characteri- Rawlings, DE (2005) Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used zation of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of for the recovery of metals from minerals and their concentrates. Microbial Cell Factories 4: 13. gene disruption. Journal of Bacteriology 186: 427–437. Rawlings, DE & Johnson, DB (2007) The microbiology of biomining: development and optimization of Schippers, A & Sand, W (1999) Bacterial leaching of metal sulfides proceeds by two indirect mechanisms mineral-oxidizing microbial consortia. Microbiology 153: 315–24. via thiosulfate or via polysulfides and sulfur. Applied and Environmental Microbiology 65: 319– Rawlings, DE, Tributsch, H & Hansford, GS (1999) Reasons why ’Leptospirillum’-like species rather 21. than Thiobacillus ferrooxidans are the dominant iron-oxidizing bacteria in many commercial Schleper, C, Puehler, G, Holz, I, Gambacorta, A, Janekovic, D, Santarius, U, Klenk, HP & Zillig, W processes for the biooxidation of pyrite and related ores. Microbiology 145 ( Pt 1): 5–13. (1995) Picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus Reddy, KJ, Wang, L & Gloss, SP (1995) Solubility and mobility of copper, zinc and lead in acidic and family comprising archaea capable of growth around pH 0. Journal of Bacteriology 177: 7050– environments. Plant and Soil 171: 53–58. 9. Remonsellez, F, Orell, A & Jerez, CA (2006) Copper tolerance of the thermoacidophilic archaeon Schleper, C, Puhler, G, Klenk, HP & Zillig, W (1996) Picrophilus oshimae and Picrophilus torridus Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 152: 59–66. fam. nov., gen. nov., sp. nov., two species of hyperacidophilic, thermophilic, heterotrophic, aerobic Ren-Long, J, Wu, J, Chaw, SM, Tsai, CW & Tsen, SD (1999) A novel species of thermoacidophilic archaea. International Journal of Systematic Bacteriology 46: 814–816. archaeon, Sulfolobus yangmingensis sp. nov. International Journal of Systematic Bacteriology 49: Schoonen, MAA, Cohn, CA, Roemer, E, Laffers, R, Simon, SR & O’Riordan, T (2006) Mineral-induced 1809–1816. formation of reactive oxygen species. Medical Mineraology and Geochemistry 64: 179–221. Rensing, C, Fan, B, Sharma, R, Mitra, B & Rosen, BP (2000) Copa: an Escherichia coli Cu(I)- Schrenk, MO, Edwards, KJ, Goodman, RM, Hamers, RJ & Banfield, JF (1998) Distribution of Thiobacil- translocating P-type ATPase. Proceedings of the National academy of Sciences of the United States lus ferrooxidans and Leptospirillium ferrooxidans: implications for generation of acid mine of America 97: 652–6. drainage. Science 279: 1519–1522. Reysenbach, AL, Liu, Y, Banta, AB, Beveridge, TJ, Kirshtein, JD, Schouten, S, Tivey, MK, Von Damm, Schwartz, E, Fritsch, J & Friedrich, B (2013). H2-metabolizing prokaryotes. In: The prokaryotes. Springer KL & Voytek, MA (2006) A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal Berlin Heidelberg, pp. 119–199. vents. Nature 442: 444–7. Seemann, T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30: 2068–9. Riekkola-Vanhanen, M (2010) Talvivaara Sotkamo Mine - bioleaching of polymetallic nickel ore in Segerer, AH, Trincone, A, Gahrtz, M & Stetter, KO (1991) Stygiolobus azoricus gen. nov., sp. nov. subarctic climate. Nova Biotechnologica 10: 7–14. represents a novel genus of anaerobic, extremely thermoacidophilic archaebacteria of the order Riekkola-Vanhanen, M (2013) Talvivaara mining company - from a project to a mine. Minerals Sulfolobales. International Journal of Systematic Bacteriology 41: 495–501. Engineering 48: 2–9. Segerer, A, Langworthy, TA & Stetter, KO (1988) Thermoplasma acidophilum and Thermoplasma Ruepp, A, Graml, W, Santos-Martinez, ML, Koretke, KK, Volker, C, Mewes, HW, Frishman, D, Stocker, volcanium sp. nov from solfatara fields. Systematic and Applied Microbiology 10: 161–171. S, Lupas, AN & Baumeister, W (2000) The genome sequence of the thermoacidophilic scavenger Segerer, A, Neuner, A, Kristjansson, JK & Stetter, KO (1986) Acidianus infernus gen. nov., spec. nov., and Thermoplasma acidophilum. Nature 407: 508–13. Acidianus brierleyi comb. nov.: facultatively aerobic, extremely acidophilic thermophilic sulfur- Sabath, N, Ferrada, E, Barve, A & Wagner, A (2013) Growth temperature and genome size in bacteria metabolizing archaebacteria. International Journal of Systematic Bacteriology 36: 559–564. are negatively correlated, suggesting genomic streamlining during thermal adaptation. Genome Shah, AR, Shah, R & Madamwar, D (2006) Improvement of the quality of whole wheat bread by Biology and Evolution 5: 966–977. supplementation of Xylanase from Aspergillus foetidus. Bioresource Technology 97: 2047–2053. Saha, D, Panda, A, Podder, S & Ghosh, TC (2014) Overlapping genes: a new strategy of thermophilic Sharma, HP, Patel, H & Sugandha (2016) Enzymatic added extraction and clarification of fruit juices - a stress tolerance in prokaryotes. Extremophiles 19: 345–353. review. Critical Reviews in Food Science and Nutrition 57: 1215–1227. Saitoh, N, Nomura, T & Konishi, Y (2017) Bioleaching of low-grade chalcopyrite ore by the thermophilic She, Q, Singh, RK, Confalonieri, F, Zivanovic, Y, Allard, G, Awayez, MJ, Chan-Weiher, CC, Clausen, archaean Acidianus brierleyi. Solid State Phenomena 262: 237–241. IG, Curtis, BA, De Moors, A, et al. (2001) The complete genome of the crenarchaeon Sulfolobus

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