Which of the following physiological mechanisms will the researchers most likely observed among the surviving organisms in the lake?

2.2.3 Acidophiles

Acidophiles are organisms that grow at an optimum pH below 3–4. These are a diverse group of organisms included in archaea, bacteria, fungi, algae, and protozoa growing in acidic conditions, reported from natural environments like solfataric fields, sulfuric pools, and geysers, and artificial environments like areas associated with human activities like mining of coal and metal ores (Sharma et al., 2012). While, most of the extremely acidophiles belong to the archaeal group that includes Acidianus, Desulfurococcus, Metallosphaera, Stygiolobus, Sulfolobus, Sulfurisphaera, Pyrococcus, Thermoplasma, and Picrophilus (Bertoldo et al., 2004). Enzymes from microorganisms that can survive under extreme pH could be particularly useful for application under highly acidic conditions. However, one of the striking properties of acidophilic microorganisms is their ability to maintain a neutral pH internally and so the intracellular enzymes from these microorganisms do not need to be adapted to extreme growth conditions as shown in Fig. 2.3A and B (Kumar et al., 2011). However, this does not account for extracellular proteins that have to function in low pH environments in the case of acidophiles. One of the most striking properties of acidophilic microorganisms is their use of proton pumps to maintain a neutral pH internally and so the intracellular enzymes from these microorganisms do not need to be adapted to extreme growth conditions (Kumar et al., 2011). However, the extracellular enzyme proteins of acidophiles have to function at low pH. How these extracellular proteins operate at low pH values is yet poorly understood. For the cells to survive in the aggressive condition of pH, acidophiles utilize several strategies. To withstand low pH, acidophiles employ a range of mechanisms such as a positively charged membrane surface a high internal buffer capacity, over expression of H+ exporting enzymes, and unique transport systems (Wiegel and Kevbrin, 2004). Besides, it has been predicted that the organisms develop specific metabolic properties, genetic features, structural and functional characteristics of their macromolecules that help maintain the pH and distinguish them from their neutrophilic counterparts (Baker-Austin and Dopson, 2007). Other stability features of acidophilic organisms such as DNA-repair proteins, ATP synthase, chaperones, membrane highly impermeable to protons, and potassium antiporter releases protons towards the extracellular medium (Orellana et al., 2018). Many acidophilic proteins have a surface that is negatively charged and low pH as shown in Fig. 2.3A and B (Fushinobu et al., 1998). In this figure: (1) direction of the transmembrane electrochemical gradient (pH) and blocking of H+ by the cell membrane; (2) reversed membrane potential through potassium transport, a modification towards maintaining a stable Donnan potential; (3) secondary transporter protein; the H+ and Na+ gradient is harnessed to drive the transport of nutrients and solutes; (4) proton pump actively removes H+, balancing the energy gained from the H+ entry to the cytoplasm; (5) vesicles containing protons avoid acidification of the cytoplasm, but still generate ATP from the electrochemical gradient (in Acidithiobacillus ferrooxidans); (6) uncouplers (uncharged compounds), such as organic acids, permeate the membrane and release their H+, leading to acidification of the cytoplasm; (7) to avoid this, heterotrophic acidophiles may degrade the uncouplers; and (8) alternatively, cytoplasmic enzymes or chemicals may bind or sequester the protons. Because of their activity and stability at extremely low pH values, the ligninolytic enzymes may be suitable for biotechnological applications.

2.4.4 Acidophiles

Acidophiles thrive under highly acidic conditions such as marine volcanic vents, and acidic sulfur springs, acid rock drainage (ARD) and acid mine drainage. These microorganisms have adapted themselves by maintaining their cellular pH neutral and also acquire resistance towards metals [24,63,64]. Members of the genus Acidobacterium, Leptospirillum, Picrophilus and Ferroplasma are found in acidic sites. These microorganisms generally require intracellular neutral pH but the mechanism of homeostasis in acidophiles is not well understood. The major factor that contributes to intracellular pH is membrane impermeability which controls the proton influx inside the cell. Archaea have been adapted to withstand low pH because tetraether lipids in their cell membrane have low permeability for proton influx. Similarly, Leptospirillum ferriphilum genome possesses genes for the biosynthesis of cell membrane and cell wall structural components for tolerance towards acidic pH [65]. Moreover, adaptations like reduction in the size of membrane pores, proton efflux systems such as antiporters, symporters and H+ ATPases and accumulation of buffering components such as arginine, histidine and lysine help in the proton sequestration. Besides this, other adaptation strategies include degradation of organic acids for proton dissociation, synthesis of chaperones that protect proteins and DNA at low pH from damage.

Specialized and Generalist Acidophilic Prokaryotes

Acidophiles as a group are highly versatile, and are able to utilize a wide variety of energy sources (solar, and inorganic and organic chemicals), grow in the presence or complete absence of oxygen, and at temperatures of between 4°C and 96°C. However, individual species display very different degrees of metabolic versatility. Among the most specialized acidophiles are Leptospirillum spp. and Ferrovum myxofaciens, all of which are known only to use a single electron donor (ferrous iron) to support their growth and, because of the high redox potential of the ferrous/ferric couple these bacteria have, by necessity, to use molecular oxygen as electron acceptor, restricting them to being metabolically active only in aerobic environments. Leptospirillum spp. and Ferrovum myxofaciens are obligate autotrophs, and can also fix dinitrogen. Their metabolic limitations appear, however, to be compensated for by other physiological advantages that they have over other iron-oxidizing acidophiles. In the case of Leptospirillum spp., their greater affinity for ferrous iron and tolerance of extremely high concentrations of ferric iron and for growth in temperatures >40°C than, for example, the iron-oxidizing acidithiobacilli, enables them to out-compete the latter bacteria in many natural and anthropogenic environments, such as stirred tank bioreactors used to bioleach or bio-oxidize sulfide ores.

Species of the genus Acidithiobacillus are, in contrast, more generalist bacteria. Some can use a variety of inorganic electron donors (iron, sulfur and hydrogen) while others use a more limited range. Even within species, there may be differences in this regard. For example, only one strain of At. ferrivorans and one of At. thiooxidans have been found to use hydrogen as electron donor. While all iron-oxidizing acidithiobacilli have been confirmed to be facultative anaerobes, species, such as At. thiooxidans, that cannot oxidize iron do not grow under anoxic conditions.

The most generalist of all acidophiles are spore-forming Firmicutes of the genus Sulfobacillus, and to a lesser extent Alicyclobacillus and Acidibacillus spp.. Sulfobacillus are Gram-positive bacteria that can grow in aerobic or anaerobic environments, and are facultative autotrophs. They use a wide range of electron donors, including ferrous iron, hydrogen, zero-valent sulfur and reduced sulfur oxy-anions, and a variety of organic compounds (such as glucose and glycerol) as carbon sources and electron donors, though their capacities for heterotrophic growth are more limited than the more heterotrophically-inclined Alicyclobacillus spp..

Glossary

Acidophiles

From the Latin acidus (sour) and the Greek philos (loving). Includes organisms that grow optimally at low pH.

One of three domains of life. From the Greek archaios (ancient, primitive). Prokaryotic cells; formerly called Archaeobacteria.

One of three domains of life. From the Greek bacterion (staff, rod). Prokaryotic cells; formerly called Eubacteria.

One of two kingdoms of organisms of the domain Archaea. From the Greek crene (spring, fountain), for the resemblance of these organisms to the ancestor of the Archaea, and archaios (ancient). Include sulfur-metabolizing, extreme thermophiles.

One of three domains of life. From the Greek eu (good, true) and karion (nut; refers to the nucleus). Eukaryotic cells; formerly called Eucaryotes.

One of two kingdoms within the domain Archaea. From the Greek eurys (broad, wide), for the relatively broad patterns of metabolism of these organisms, and archaios (ancient). Includes halophiles, methanogens, and some anaerobic, sulfur-metabolizing, extreme thermophiles.

From the Greek halos (salt) and philos (loving). Includes organisms that grow optimally at high salt concentrations.

From the Greek hyper (over), therme (heat), and philos (loving). Includes organisms that grow optimally at temperatures higher than 80 °C.

Proposed third kingdom within the domain Archaea. From the Greek koros (young man), for the early divergence of this group during the evolution of the Archaea, and archaios (ancient). Includes a small group of ribosomal RNA sequences retrieved from geothermally heated sediments.

From the Greek mesos (middle) and philos (loving). Includes organisms that grow optimally at temperatures between 20 and 50 °C.

Strictly anaerobic Archaea that produce (Greek gen: to produce) methane.

The study of the evolutionary relationships among organisms or genes.

Universally distributed molecule among cellular life forms. Widely used to infer the evolutionary relationships among organisms.

From the Greek therme (heat) and philos (loving). Includes organisms that grow optimally at temperatures between 50 and 80 °C.

Physiological Versatility in Acidophilic Prokaryotes: Specialized and Generalist Microorganisms

Acidophiles as a group are highly versatile and are able to utilize a wide variety of energy sources (solar and inorganic and organic chemicals), grow in the presence or complete absence of oxygen, and at temperatures of between 4 and 96 °C. However, individual species display very different degrees of metabolic versatility. On the one end of this spectrum are members of the genus Leptospirillum. Three species are known: Leptospirillum ferrooxidans, Leptospirillum ferriphilum, and Leptospirillum ferrodiazotrophum. All grow as highly motile curved rods and spirilli, and species and strains vary in temperature and pH characteristics. All three species, however, appear to use only one energy source – ferrous iron. Because of the high redox potential of the ferrous/ferric couple (see ‘Aerobic and anaerobic acidophiles’), these Bacteria, by necessity, have to use molecular oxygen as an electron acceptor, restricting them to being active only in aerobic environments. All three species fix carbon dioxide (but not organic carbon) and two of the three (L. ferrooxidans and L. ferrodiazotrophum) are also able to fix molecular nitrogen. Leptospirillum spp. are, therefore, highly specialized acidophiles. Their metabolic limitations appear, however, to be compensated by their abilities to outcompete other iron-oxidizing bacteria in many natural and anthropogenic environments, such as stirred tank bioreactors used to bioleach or biooxidize sulfide ores. This is achieved, at least in part, by their greater affinities for ferrous iron and greater tolerance of ferric iron than most other iron oxidizers.

At. ferrooxidans is, in contrast, a more generalist bacterium. Initially it was described as an obligate aerobe that obtains energy by oxidizing ferrous iron, elemental sulfur, sulfide, and RISCs, and fixes CO2 as its sole source of carbon. The first hint of a more extensive metabolic potential was in a report by Thomas Brock and John Gustafson in 1976 who showed that the bacterium could couple the oxidation of elemental sulfur to the reduction of ferric iron, though it was not confirmed at the time whether this could support growth of the acidophile in the absence of oxygen, though the free energy of the reaction (ΔG = –314 kJ mol−1; eqn [6]) suggested that this might be the case.

(6)S+6Fe3++4H2O→HSO4− +6Fe2++7H+

Later, Jack Pronk and colleagues at Delft University showed conclusively that At. ferrooxidans is, indeed, a facultative anaerobe and can grow anaerobically by ferric iron respiration using not only sulfur as electron donor, but also formic acid (which can also be used as sole energy source under aerobic conditions). The finding that this acidophile can use formic acid, although somewhat unexpected, does not imply that it is capable of heterotrophic as well as autotrophic growth, as C1 compounds, such as formate and methanol, are also used by other autotrophic prokaryotes. About the same time, it was discovered that some strains of At. ferrooxidans (including the type strain) can use hydrogen as an energy source, but that bacteria cultivated on hydrogen are less acidophilic than when grown on sulfide ores. It was shown later that hydrogen oxidation could also be coupled to ferric iron reduction by some At. ferrooxidans isolates.

The most generalist of all acidophiles are, however, Sulfobacillus spp. These Gram-positive bacteria can grow as chemolithotrophs, heterotrophs, or mixotrophs in aerobic or anaerobic environments. Although there are no reports of Sulfobacillus spp. using hydrogen, they can (unlike At. ferrooxidans) use a variety of organic compounds (such as glucose and glycerol) as carbon and energy sources, though their capacities for heterotrophic growth are more limited than Alicyclobacillus spp. (related acidophilic Firmicutes, some of which can also oxidize ferrous iron and sulfur).

Thermoplasma acidophilum

The genome of an acidophile that is also a slightly thermophilic member of the Euryachaeota, T. acidophilum DSM 1728 (growing best at 59 °C and pH 2), was sequenced and yielded a 1.56 Mbp circular chromosome. The lack of a rigid cell wall and the presence of eucarya-type proteases and chaperones have been of interest in this archaeon. Analysis of the 1509 predicted genes showed typical archaeal characteristics as well as a relatively large fraction of bacterial genes (about 10%) likely acquired through lateral gene transfers. A substantial number of genes similar to the phylogenetically distant Crencharchaeote, S. solfataricus, which coinhabits the same environments, were identified, also indicating candidates for laterally transferred genes.

4.3 Lipothrixviridae family

Members of the Lipothrixviridae family infect hyperthermophilic acidophiles of the order Sulfolobales and have filamentous virions that are decorated at each end with diverse terminal structures that can resemble claws (Acidianus filamentous virus 1 [AFV1]), brushes (AFV2) or mops (Sulfolobus islandicus filamentous virus [SIFV]) and are likely to be involved in the virus attachment to the host cell (Arnold et al., 2000b; Bettstetter et al., 2003; Häring et al., 2005a) (Fig. 6D). High-resolution structures are available for three members of the Lipothrixviridae, namely, AFV1 (Kasson et al., 2017), Sulfolobus filamentous virus 1 (SFV1) (Liu et al., 2018) and SIFV (Wang et al., 2020a). Lipothrixviruses share extensive gene content with rudiviruses and, accordingly, the two families were grouped into the order Ligamenvirales (Prangishvili and Krupovic, 2012). However, unlike rudiviruses, lipothrixviruses are enveloped with a lipid membrane, and the nucleocapsid is built from heterodimers of two homologous MCPs (Fig. 6E and F) that closely resemble the symmetrical homodimer of rudiviruses (Fig. 6C). Importantly, as in rudiviruses, the lipothrixvirus genomes are also stored in the virions as A-form DNA. The mechanism of lipothrixvirus nucleocapsid assembly is thus likely to be very similar to the assembly of rudivirus capsids (Fig. 6M), with multiple copies of the MCP heterodimer binding to the linear dsDNA and transforming it into A-form (Fig. 6H). Notably, lipothrixviruses display considerable variation in the properties of the superhelical nucleocapsid, including the diameter, flexibility and surface electrostatic potential. Furthermore, one of the two MCPs in SFV1 and SIFV, but not in AFV1, contains C-terminal hook-like extension which makes contacts across the helical groove of the nucleocapsid, stapling the nucleocapsid and thereby probably stabilizing it (Liu et al., 2018; Wang et al., 2020a).

A remarkable feature of lipothrixvirus virions is their membranes, which are half as thin as the cytoplasmic membrane of the host (Kasson et al., 2017; Liu et al., 2018; Wang et al., 2020a). Lipid analysis has shown that AFV1 specifically selects for a flexible glycerol dibiphytanyl glycerol tetraether lipid lacking cyclopentane rings (GDGT-0), which can be bent into a U-shaped “horseshoe” conformation. Molecular dynamics simulation has shown that such thin lipid envelope containing horseshoe GDGT-0 lipids would be stable and have the necessary curvature to surround the nucleocapsid (Kasson et al., 2017). The horseshoe lipid conformation has been observed in vitro at the air-water interface, but never before in a biological system (Kohler et al., 2006). By contrast, the thin SFV1 membrane is strongly enriched in archaeol (~ 80% of all lipids), a short lipid molecule that is half as long as GDGT tetraethers and present at ~ 1% in the host membrane. Thus, the thickness of the SFV1 membrane is consistent with the monolayer composed of archaeol (Liu et al., 2018). Consequently, lipothrixviruses have evolved different routes to converge on thin lipid membranes. However, the function of the viral envelope as well as the actual mechanism of envelopment and lipid selection remain to be studied in detail.

1.18.5.1 Background

Microorganisms living in permanently acidic environments are known as ‘acidophiles’. Some are extreme acidophiles typically growing at pH <3, and others are moderate acidophiles with an optimal growth in the range of pH 3–5. Numerous processes generate acidic environments such as fermentations and nitrification or the oxidation of sulfur, sulfides, and sulfur-based ores, ultimately leading to the production of sulfuric acid in amounts capable to compensate for the pH-buffering capacity of aquatic or terrestrial habitats. This process is responsible for the development of the most acidic niches on the Earth. They are spontaneously formed in volcanic regions in which hydrogen sulfide can react with sulfur dioxide to produce elemental sulfur, according to the equation: H2S + SO2 → 3S + 2H2O. The resulting sulfur can, therefore, be oxidized by bacteria to form sulfuric acid: 2S + 2H2O + 3O2 → 2H2SO4. The high level of acidity can then lead to the dissolution of surrounding rocks to give rise to acidic muds, known as solfatara fields, found, for example, near Naples (Italy) or in Yellowstone National Park (USA). Acidic environments can also result from anthropogenic activities such as excavation in search of mineral ores, and that has inadvertently led to the exposure of sulfide mineral-rich rocks, and in the generation of extremely acid environments throughout the world. Pyrite (FeS2) is one of these minerals; it is extremely abundant and although it is stable in conditions where oxygen or water is absent, when exposed to moist air, it can be oxidized by oxygen or any other oxidant, in particular Fe3+, to also lead to the production of sulfuric acid via thiosulfate ions. The effect of acidic conditions on the environment can be extremely harmful due to the dissolution of various minerals leading to high concentrations of metal in dissolved forms, highly toxic to most living organisms. Acidophiles are widely distributed in the archaeal and eubacterial domains; they often also display a thermophilic character due to the origin of the acidity. These organisms can be of importance in the context of evolution as it has been proposed that metabolic processes typical of living organisms have originated on the surface of the sulfide mineral and that the ability of acidophiles to thrive in acidic and metal-rich conditions might be similar to the conditions that prevailed in the primordial volcanic aqueous environments, which were probably abundant shortly after the evolution of our planet. Acidophiles could therefore represent some of the most ancient forms of life on the Earth. Although they live in acidic environments, they maintain an intracellular pH close to neutrality, and the protons involved in energy transduction have to be excreted against an unfavorable pH gradient. They solve the problem by developing membranes highly impermeable to H3O+, by inversing the membrane potential, the inside being positive when compared to the external fluid, contrary to neutrophiles, and by actively pumping protons out of the cell next to an increased capacity in cytoplasmic pH buffering [13]. The positive membrane potential derives from the import of K+ ions; it culminates as the proton motive force declines, and has a protective role when the metabolism of the microorganism is low, counteracting, in this way, proton influx. One consequence is that their tolerance to metal cations is high, but, by contrast, they are much more sensitive to anions than neutrophiles. Up to now, no acidophilic cyanobacteria or anaerobic photosynthetic bacteria have been discovered. Inorganic electron donors are significant energy sources due to the fact that substances such as ferrous ions and reduced forms of sulfur are particularly abundant in the environments colonized by acidophiles. They often exceed that of organic compounds. Some mesophilic acidophiles, however, can use, as electron donors, some low-molecular-weight monomeric compounds such as sugar, alcohols, and a few amino acids, but not apparently polymeric substances. The electron acceptor is mostly oxygen but, in acidic environments, the ferric ion can also be an alternative due to the high redox potential of the Fe2+/Fe3+ couple (770 mV at pH 2). A few entire genomes have been sequenced from organisms characterized by an iron/sulfur-derived metabolism: the bacterium Acidithiobacillus ferrooxidans and the Archaea Thermoplasma acidophilum, Picrophilus torridus, S. tokodaii, and F. acidarmanus. Among them, P. torridus and its close cousin, P. oshimae, have pH optima close to zero and the recently discovered group of Ferroplasma species is able to grow at a pH range of 0–2.5; they belong to the order of Thermoplasmatales to which also belongs the family of Picrophilaceae. They were first extracted in 1999 from a microbial community found in a bioleaching plant in Chile, and the strain designated F. acidiphilum was isolated in pure culture in 2000 from a pyrite-leaching bioreactor fed with pyrite ores from Kazakhstan [14]. The microorganism grows at pH 1.3–2.2 at temperatures between 15 and 45 °C, uses ferrous ions as a sole source of energy, and fixes CO2 as the sole carbon source. Ferroplasma species constitute some of the most abundant genera in environments of very low pH and are heavily charged in metals such as iron, copper, arsenic, cadmium, and zinc. They are important contributors to the geochemical cycling of iron and sulfur. One of the most unusual features of these organisms is that they lack a cell wall, contrary to Picrophilus species. The stability of the membrane in high acid conditions is apparently secured, thanks to the presence of a unique tetraether lipid bound to glucose, mannose, and galactose units, and to the presence of glycoproteins, lipoglycans, and liposaccharide-like material. The isoprenyl chains also contain cyclopropane rings. The pH gradient across the cell membrane approaches 5 pH units and the membrane permeability to protons is very low. A detailed investigation of the proteome of F. acidiphilum reveals that out of 189 proteins, unequivocally identified, 163 contained iron; many of them are housekeeping proteins, which, in other organisms, do not contain iron and, in many cases, not even a metal. The iron, bound to these proteins, is apparently essential for both activity and structure. By comparison, the analysis of the metalloproteome of the closely related archaeon P. torridus and unrelated bacterium A. ferroxidans, which occupies niches similar to those of F. acidiphilum, demonstrates that the much lower number of detected Fe-containing proteins corresponds to proteins also found in other organisms. It has been proposed that this unique capacity of F. acidiphilum to use iron is a relic of an ancient property, which could fit particularly well with one theory of the origin of life based on the idea that the formation and transformation of biomolecules took place on iron–sulfur-rich surfaces such as pyrite. It is supposed that Ferroplasma sp. have been always confronted by Fe-rich and acidic habitats, which did not offer to the microorganism the selective advantage to evolve toward alternative, non-iron-based, mechanisms. The annotation of the genome of P. torridus also shows that these extreme acidophiles encode for 12% of transporters, including H+/K+-transporting adenosine triphosphatase (ATPase), predicted to import K+ ions in order to keep the pH of the cytoplasm close to neutrality. The genome also contains a large number of sequences coding for chaperones that probably play a role in the stabilization and folding of proteins in acidic conditions. Surprisingly, it was also shown from a genomic expression library that some intracellular enzymes from F. acidiphilum have acidic pH optima close to 3 and are active and stable only between pH 1.7 and 4.0, about 3 pH units lower than the mean pH of the cytoplasm. It has been suggested that there is some sort of compartmentalization in the cytoplasm, or the existence of unknown selective forces favoring the expression of these enzymes.

6.5.2 Physiological adaptations to high salt concentration

Halophilicity can go together with other extremophilic characteristics. Halo-acidophiles and halo-alkaliphiles both exist in nature. The latter are more common, and this characteristic is sometimes combined with thermal tolerance [19] to produce halophilic alkalithermophiles. It can also be combined with cold adaptation. The Antarctic haloarchaea Halorubrum lacusprofundi and Halohastalitchfieldiae are dominant in the microbial community in permanently cold and hypersaline Deep Lake, where temperatures can drop to −20 °C, and a proteomic study showed that they do so by modifying their cell envelope, which maintains osmotic balance and translation initiation, and altering their mechanisms for RNA turnover and tRNA modification [143]. The alga Dunaliella also survives in this environment and is the lake’s primary producer [144], providing nutrients for haloarchaea.

There are many adaptations employed by halophilic organisms to adapt to a life in salt (see [145] for a detailed review). These include robust cell walls that can adapt their own hydrophobicity depending on the environmental NaCl, variable amounts of negatively charged polar lipids, as well as the ether linkages found in archaea, which make them less permeable to ions. Halophiles employ two main strategies to resist high salinity and water stress. One is a ‘high salt-in’ strategy. Some halophiles increase the internal osmolarity by accumulating K+ ions in the cytoplasm, requiring the expenditure of two molecules of ATP for each K+. The alternative strategy, commonly seen in Bacteria and Eukarya, is a ‘low-salt-in’ one, in which the cells accumulate compatible solutes. These small molecules, e.g. sugars, alcohols, amino acids, N-acetylated diamino acids, glycine betaine, ectoine and hydroxyectoine can be either taken from the environment or synthesized intracellularly and can act as general stress protectants. They prevent the ‘salting-out’ of proteins in the cell. Halotolerant and moderately halophilic organisms tend to use this mechanism, whereas extreme halophiles also use the ‘high salt-in’ strategy [145].

These adaptations were accompanied by certain features commonly observed in proteins from halophiles (see [146] for review). In general, such proteins had a large number of negative charges, and were more hydrophobic than their mesophilic counterparts. Sequencing of the genome of Halobacterium sp. NRC-1 revealed that the proteome is remarkably high in negative charges [147], with a median pI of 4.9 and few basic proteins, which contrasts with non-halophilic proteomes which have an average pI close to neutral [146]. They tend to have fewer bulky hydrophobic side chains on the surface, compared to small and borderline hydrophobic residues [148]. This property aligns with the higher flexibility and surface hydration observed for halophilic proteins [149].

Most of the information about life in high salt has been gathered from studies of organisms that reside in high NaCl. However, some organisms live in high concentrations of kosmotropic (stabilizing) salts, but others survive in high concentrations of chaotropic (destabilizing) salts, e.g. NaBr, CaCl2 and MgCl2. These include some fungi from various extreme environments, which tolerate up to 2.1 M MgCl2 or 2 M CaCl2 and could therefore be described as chaophilic [150].

3.06.3.3.3 Extremophiles

Extremophilic microbes (extremophiles) abound in extreme habitats. These include acidophiles (acidic sulfurous hot springs), alkalophiles (alkaline lakes), halophiles (salt lakes), piezo (baro-) and (hyper)thermophiles (deep-sea vents),57–61 and psychrophiles (Arctic and Antarctic waters, alpine lakes).62 Thus far, investigations have centered on the isolation of thermophilic and hyperthermophilic enzymes (extremozymes),63–67 but there is little doubt that these extreme environments will also yield novel bioactive chemotypes. Abandoned mine-waste disposal sites have yielded unusual acidophiles, which thrive in the acidic, metal-rich waters, polluted environments that are generally toxic to most prokaryotic and eukaryotic organisms.68 The novel sesquiterpenoid and polyketide-terpenoid metabolites, berkeleydione (12; Figure 6) and berkeleytrione (13; Figure 6) showing activity against metalloproteinase-3 and caspase-1, activities relevant to cancer, Huntington’s disease, and other diseases, have been isolated from Penicillium species found in the surface waters of Berkeley Pit Lake in Montana.68–70