1. Acidophile Microbiology in Space and Time   Free download

D. Barrie Johnson and Raquel Quatrini

The study of extreme acidophiles, broadly defined as microorganisms that grow optimally at pH values below 3, was initiated by the discovery by Waksman and Joffe in the early 1900's of a bacterium that was able to live in the dilute sulfuric acid it generated by oxidising elemental sulfur. The number of known acidophiles remained relatively small until the second half of the 20th century, but since then has greatly expanded to include representative of living organisms from within all three Domains of life on Earth, and notably within many of the major divisions and phyla of Bacteria and Archaea. Environments that are naturally acidic are found throughout the world, and others that are man-made (principally from mining metals and coal) are also widely distributed. These continue to be sites for isolating new species, (and sometimes new genera) which thrive in acidic liquor solutions that contain concentrations of metals and metalloids that are lethal to most life-forms. The development and application of molecular techniques and, more recently, next generation sequencing technologies has, as with other areas of biology, revolutionised the study of acidophile microbiology. Not only have these studies provided greater understanding of the diversity of organisms present in extreme acidic environments and aided in the discovery of largely overlooked taxa (such as the ultra-small uncultivated archaea), but have also helped uncover some of the unique adaptations of life-forms that live in extremely acidic environments. Thanks to the relatively low biological complexity of these ecosystems, systems-level spatio-temporal studies of model communities have been achieved, laying the foundations for "multi-omic" exploration of other ecosystems.

This Chapter introduces the subject of acidophile microbiology, tracing its origins to the current status quo, and provides the reader of this textbook with general information which provides a backdrop to the more specific topics described in subsequent chapters.

2. Energy Acquisition in Low pH Environments

Wolfgang Nitschke and Violaine Bonnefoy

Extreme acidophiles thriving in acidic habitats are characterised by low organic substrate inputs and high concentrations in inorganic substances, in particular sulfur compounds and iron. Their strategies for harvesting energy for growth in these harsh environments are described in this chapter. An overview of the general concepts on bioenergetics is given to highlight the particular challenges faced by extreme acidophiles. Pathways involved in oxidation and reduction of iron and sulfur compounds are presented and discussed. From the available data, it is obvious that, for the same electron donor and acceptor, the metabolic pathways differ sufficiently between different prokaryotes to suggest independent origins followed by diversification and horizontal gene transfer.

3. Adaptation to Extreme Acidity and Osmotic Stress

Carla M. Zammit and Elizabeth L.J. Watkin

Environments that are either acidic or have high osmotic potentials are found across the globe in a range of natural and anthropogenic systems. The organisms capable of inhabiting these systems are diverse, including archaea, bacteria and eukaryotes. However, environments where extreme acidity is combined with osmotic stress deriving from elevated concentrations of sodium chloride are seemingly rare. Subsequently, there is a relatively small number of species which have been identified and shown to tolerate both of these stresses simultaneously, and as a result the mechanisms that permit life in these harsh conditions has not been extensively studied. Recent genomic and proteomic studies indicate that several strategies may be employed by acidophilic microorganisms to combat the combined effects of low pH and high osmotic stress, most notably the production of osmo-protectants and the maintenance of membrane integrity. This chapter focuses on iron- and sulfur-oxidising microorganisms, which are able to tolerate acidic conditions, the effect of osmotic stress induced by salinity on their survival, and mechanisms used to survive these stresses both independently and in combination.

4. Oxidative Stress and Metal Tolerance in Extreme Acidophiles

Alonso Ferrer, Omar Orellana and Gloria Levicán

In the extremely acidic conditions found in natural acid rock environments and bioleaching operations, microorganisms have to deal with an abundant supply of (heavy) metals. Iron is required as micronutrient by all acidophiles, and as a primary energy source or alternative electron acceptor by some of them. However iron and other metals can also induce oxidative damage to biomolecules by generating reactive oxygen species (ROS). Acidophilic microorganisms, therefore, are often exposed to highly oxidizing conditions and face the problem of maintaining intracellular metal and redox homeostasis. Although acidophiles fight oxidative stress via a number of ROS scavenging enzymes, they seem to depend mainly on biomolecule repair using a plethora of protein and lipid repair systems. Evidence collected to date indicates that the molecular determinants of this response also serve to protect the cells from other extreme environmental factors such as temperature, pH and metal(loid)s. Acidophiles are well equipped with mechanisms to export, sequester and/or store metals. Reducing the bioavailability of metals also seems to play an important role in their tolerance, via complex formation (with sulfate anions) and binding to extracellular polymeric substances. This chapter summarizes the mechanisms of protection to oxidative stress and metal toxicity in acidophiles, how these are configured to build the robust protection system required to thrive under extreme stress conditions, and how the ability of acidophiles to respond to those conditions may be critical in determining their growth and activity.

5. Physiological and Phylogenetic Diversity of Acidophilic Bacteria

Mark Dopson

Acidophilic bacteria can be found in natural and anthropogenic acidic environments such as acid sulfate soils and biomining operations. These environments range in temperatures from below zero where low temperature adapted, acidophilic bacteria accelerate metal and acid release from sulfide minerals, through mesophilic environments, to hot solfataric fields containing Hydrogenobaculum acidophilum with a temperature optimum of 65°C. Acidophilic bacteria have been isolated from the Actinobacteria, Aquificae, Firmicutes, Nitropsora, Proteobacteria, and Verrucomicrobia phyla, and are capable of oxidizing both inorganic and organic electron donors coupled to the reduction of oxygen or ferric iron, though no extremely acidophilic bacteria are known to ferment organic substrates. Acidophilic bacteria also exhibit a range of carbon metabolisms, from obligate autotrophs such as Leptospirillum spp., facultative autotrophs such as Sulfobacillus spp. that can both fix carbon dioxide (CO2) or assimilate organic carbon, to obligate heterotrophs such as Alicyclobacillus tolerans. This chapter summarizes present knowledge of the physiological and phylogenetic diversity of acidophilic bacteria and highlights differences in growth characteristics between the various species.

6. Diversity and Physiologies of Acidophilic Archaea

Olga V. Golyshina, Manuel Ferrer and Peter N. Golyshin

The majority of cultured acidophilic archaea are represented by two phyla, the Crenarchaeota and the Euryarchaeota, that inhabit sulfur-rich areas in volcanically-active regions worldwide and sulfidic deposits rich in metals, acid mine drainage systems, macroscopic growths in forms of streamers, slimes, mats and microbial stalactites. A metabolic network formed by these archaea is determined by niche adaptation and is characterized by sulfur-, hydrogen- and iron-dependency, and by the organotrophy of scavenger species and strains. Acidophily is a major trait in the organisms from the above environments and most characterised species are moderate or extreme thermophiles, though there are some exceptions. Isolation sites of acidophilic archaea are almost exclusively limited to terrestrial habitats, with marine environments being poorly explored as a reservoir for these organisms, with only one such acidophilic euryarchaeote isolated to date. The nature and mechanisms of acidophily are believed to be determined by a number of factors with special archaeal membranes and lipids being of the greatest importance. Genome analysis of a number of species of acidophilic archaea has identified certain metabolic traits that make them distinct from bacterial counterparts and suggests some unusual variants of classical pathways.

7. Physiological and Phylogenetic Diversity of Acidophilic Eukaryotes

Angeles Aguilera, Sanna Olsson and Fernando Puente-Sánchez

The study of extremely acidic environments has grown significantly over the past 50 years. Highly acidic environments are generally unfavorable for the growth of photosynthetic organisms. However, these ecosystems are usually inhabited by acidophilic and acid-tolerant eukaryotic microorganisms such as algae, amoeba, ciliates, heliozoa and rotifers, as well as fungi and yeasts. This chapter provides an overview of our knowledge of the diversity and ecophysiology of eukaryotic acidophilic microorganisms, as well as summarizing recent data from one of the most extensive extreme acidic environments, the Río Tinto, Spain.

8. Microbial Communities and Interactions in Low pH Environments

D. Barrie Johnson

Most of the types of interactions that occur between microorganisms in environments considered to be non-extreme have also been observed in those which are extremely acidic. These include interactions that are both positive (mutualism, commensalism, synergy and syntrophy) and negative (competition, amensalism, predation and pathogenicity) from the perspective of one or more of the co-habiting microorganisms. Carbon flow between autotrophic (chemolithotrophs and phototrophs) and heterotrophic acidophiles is now known to operate in both directions and can help sustain stable consortia of these prokaryotes. By catalysing redox transformations of iron and sulfur, bacteria and archaea can generate electron donors and acceptors used by other species of acidophiles and thereby induce rapid biogeochemical cycling of these elements, a key characteristic of many low pH environments. Examples of relatively simple and more complex communities that have been studied in acidic streams, pit lakes and anthropogenic (biomining) environments are used to illustrate the importance of microbial interactions at low pH.

9. Biofilm Formation by Acidophile Bacteria and Archaea

Alvaro Orell and Nicolas Guiliani

The ability of microbes to grow as surface-associated communities, or biofilms, is seen as a life-style widely spread in all biomes on planet Earth, including extremely acidic ecosystems. Within these harsh and constantly changing habitats, the biofilm mode of growth is thought to offer the microbial community a regular condition so as to cope with the adverse scenery. Though acidophilic biofilm communities are mainly dominated by bacterial species, archaeal and eukaryotic populations appear to play crucial roles, broadening metabolic diversity and maintaining the community structure.

Environmental signals and molecular mechanisms that underlay the biofilm life-style have been largely characterized for bacteria that colonize human cavities. In contrast, the genetic basis that enable environmental acidophiles to form and develop biofilms are far from comprehended. Nonetheless, the molecular mechanisms that govern the biofilm developmental processes in a few model acidophiles have begun to be elucidated. This chapter describes what is currently known on the cellular and molecular mechanisms that promote biofilm formation of bacteria and archaea inhabiting extremely acidic ecosystems.

10. Distribution of Acidophilic Microorganisms in Natural and Man-made Acidic Environments   Free download

Sabrina Hedrich and Axel Schippers

Acidophilic microorganisms can thrive in both natural and man-made environments. Natural acidic environments comprise hydrothermal sites on land or in the deep sea, cave systems, acid sulfate soils and acidic fens, as well as naturally exposed ore deposits (gossans). Man-made acidic environments are mostly mine sites including mine waste dumps and tailings, acid mine drainage and biomining operations. The biogeochemical cycles of sulfur and iron, rather than than those of carbon and nitrogen, assume centre stage in these environments. Ferrous iron and reduced sulfur compounds originating from geothermal activity or mineral weathering provide energy sources for acidophilic, chemolithotrophic iron- and sulfur-oxidizing bacteria and archaea (including species that are autotrophic, heterotrophic or mixotrophic) and, in contrast to most other types of environments, these are often numerically dominant in acidic sites. Anaerobic growth of acidophiles can occur via the reduction of ferric iron, elemental sulfur or sulfate. While the activities of acidophiles can be harmful to the environment, as in the case of acid mine drainage, they can also be used for the extraction and recovery of metals, as in the case of biomining. Considering the important roles of acidophiles in biogeochemical cycles, pollution and biotechnology, there is a strong need to understanding of their physiology, biochemistry and ecology.

11. Progress in Acidophile Genomics

Juan Pablo Cárdenas, Raquel Quatrini and David S. Holmes

By March 2015, 161 genomes of acidophilic microorganisms had been published, including 126 permanent draft and closed genomes and 35 partial genomes reconstructed from metagenomic data, distributed between Archaea (71), Bacteria (86) and Eukarya (4). This provides a rich source of latent data that can be exploited for understanding the biology of this group of organisms and for advancing biotechnological applications. The genomic data are already yielding valuable insights into the genome architecture and cellular metabolism of acidophiles, allowing the construction of useful models to probe their evolution and ecophysiology both in naturally occurring acidic environments and in industrial operations such as in the biorecovery of metals.

12. The Flexible Genome of Acidophilic Prokaryotes   Free download

Raquel Quatrini, Francisco J. Ossandon and Douglas Rawlings

Over the last couple of decades there has been considerable progress in the identification and understanding of the mobile genetic elements that are exchanged between microbes in extremely acidic environments, and of the genes piggybacking on them. Numerous plasmid families, unique viruses of bizarre morphologies and lyfe cycles, as well as plasmid-virus chimeras, have been isolated from acidophiles and characterized to varying degrees. Growing evidence provided by omic-studies have shown that the mobile elements repertoire is not restricted to plasmids and viruses, but that a plethora of integrative elements ranging from miniature inverted repeat transposable elements to large integrative conjugative elements populate the genomes of acidophilic bacteria and archaea. This chapter reviews the diversity of elements that have been found to constitute the flexible genome of acidophiles. Special emphasis is put on the knowledge generated for Sulfolobus (archaea) and species of the bacterial genera Acidithiobacillus and Leptospirillum. Also, recent knowledge on the strategies used by acidophiles to contain deletereous exchanges while allowing innovation, and the emerging details of the molecular biology of these systems, are discussed. Major lacunae in our understanding of the mobilome of acidophilic prokaryotes and topics for further investigations are identified.

13. Metagenomics of Acid Mine Drainage at Iron Mountain California, Expanding Our View from Individual Genes and Cultures to Entire Communities

Brett J. Baker and Jillian F. Banfield

The microbial world is vast and complex, which makes addressing fundamental questions related to ecology and evolution challenging. Until recently, microbial ecology relied on cultivation-based methods, which can only target a tiny faction of the diversity of microorganisms present in the environment. Therefore, microbiologists have turned to new tools to interrogate natural communities. This revolution has been fuelled by new DNA sequencing technologies that probe community composition and metabolic potential (metagenomics), and linked "omics" approaches, such as such as metaproteomics and metatranscriptomics that provide metrics for activity. Metagenomic and metaproteomics methods were first applied to study natural microbial communities at an acid mine drainage site (Richmond Mine at Iron Mountain, California). Research conducted there demonstrated that it is possible to obtain complete genomes representing natural populations and to track their metabolic activity in situ. As a result dozens of novel genomes (down to the strain level) of uncultured acidophiles are now available. These genomes have provided a robust framework upon which it has been possible to address fundamental questions related to ecological processes such as colonization and to document the modes and rate of genome evolution.

14. Proteomics of Acidophilic Prokaryotes

Francisco Remonsellez, Fernando Pagliai, Claudio Navarro, Rodrigo Almárcegui and Carlos A. Jerez

Proteomics, metaproteomics, genomics and metagenomics, together with metabolomics, have been widely used in recent years to study the global regulatory responses of all kinds of cells. By this approach it may be possible to explore the new properties of microorganisms that arise from the interplay of genes, proteins, other macromolecules, small molecules, and the environment. Proteomics offers direct information of the dynamic protein expression in tissue or whole cells, giving us a global analysis of the cell proteins and their cellular behaviour. In this chapter, recent advances in standard and quantitative proteomics applied to individual acidophilic bacteria and archaea are reviewed. Specifically, adaptation responses of acidophiles to extremely high metal concentrations, different minerals or solid substrates and possible applications of some of these findings are illustrated.

15. Metabolomic Approaches to the Study of Acidophiles

Patricio Martínez and Pilar Parada

After the rapid development of genome sequencing tools in recent decades, the holistic study of biological systems has been gaining strength. Additionally, new technologies of high-throughput analysis such as transcriptomics, proteomics and metabolomics, have helped to integrate different types of data favouring a more comprehensive understanding of cells physiology and ecology. Metabolomics, in particular, has allowed the study of the complexities of cellular metabolism, facilitating the overall analysis of metabolic pathways and the understanding of the biochemistry associated. Metabolic biomarkers derived from metabolomic studies, that allow monitoring cell behaviour, have become important tools in cellular physiology studies and in biotechnological applications. In acidophiles, advanced scientific studies have provided a better understanding of their physiology and utilization in the biotechnological industry. However, much remains to be investigated, and many of these microorganisms are still unknown or have not been successfully isolated. The use of high-throughput "omics" tools, such as metabolomics, has contributed to integrated data analysis of their behaviour and complexity, giving a new view of the acidophiles system biology. In this chapter, we review the use of metabolomics as an advanced platform for the study of acidophile biology and its contribution and prospects for biotechnological applications.

16. Biotechnologies that Utilise Acidophiles

Susan T.L. Harrison

The bioleaching of mineral sulfides provides the most prevalent anthropogenic application of chemolithotrophic acidophiles. The same reactions, occurring unconstrained in the environment, are also the cause of acid mine drainage (AMD), a major issue with respect to water pollution. In this chapter, the role of these microorganisms in processes for metal recovery through tank, heap, dump and in situ leaching systems is described. Their potential in gold and copper recovery is demonstrated through discussion of existing processes and operations. The potential to expand the relevance of these technologies to other metals, low grade and complex mineral ores and concentrates is explored. Further, the importance of developing processes for the environmentally responsible processing of wastes from both mining operations and end-of-life products is raised as are the challenges for ensuring process efficiency while minimising the environmental footprint.

17. Acidophiles and Astrobiology

Ricardo Amils and David Fernández-Remolar

The NASA Astrobiology roadmap underlines the importance of extreme environments and the microorganisms that live in them to evaluate the possible existence of life outside of planet Earth. Acidophiles are of special astrobiological interest because the environments in which they thrive are generated by the metabolism of chemolithotrophic microorganisms that obtain energy from inorganic mineral substrates, a property that places them among the best candidates for a successful primitive energy conservation system. A thorough geomicrobiological characterization of an extreme acidic environment, the Río Tinto basin, has revealed the importance of the iron cycle in the water column of the river and detected a higher level of metabolic diversity in the sediments and subsurface of the Iberian Pyrite Belt (IPB), due to the presence of micro-niches with environmental conditions different from the acidic waters with high redox potentials existing in the river. The identification of iron oxides and iron sulfates on the surface of Mars, similar to those produced in the Tinto basin by the metabolic activity of chemolithotrophic microorganisms, has given Río Tinto the status of a geochemical and mineralogical terrestrial analogue of Mars. The argument that Mars' environmental conditions are not suitable for methanogenesis could be challenged by the methane production observed in the sediments and the subsurface of the IPB.