Distribution of Acidophilic Microorganisms in Natural and Man- Made Acidic Environments Sabrina Hedrich and Axel Schippers*

Distribution of Acidophilic Microorganisms in Natural and Man- made Acidic Environments Sabrina Hedrich and Axel Schippers*

Federal Institute for Geosciences and Natural Resources (BGR), Resource Geochemistry Hannover, Germany *Correspondence: [email protected]

https://doi.org/10.21775/cimb.040.025

Abstract areas, or environments where acidity has arisen Acidophilic microorganisms can thrive in both due to human activities, such as mining of metals natural and man-made environments. Natural and coal. In such environments elemental sulfur acidic environments comprise hydrothermal sites and other reduced inorganic sulfur compounds on land or in the deep sea, cave systems, acid sulfate (RISCs) are formed from geothermal activities or soils and acidic fens, as well as naturally exposed the dissolution of minerals. Weathering of metal ore deposits (gossans). Man-made acidic environ- sulfdes due to their exposure to air and water leads ments are mostly mine sites including mine waste to their degradation to protons (acid), RISCs and dumps and tailings, acid mine drainage and biomin- metal ions such as ferrous and ferric iron, copper, ing operations. Te biogeochemical cycles of sulfur zinc etc. and iron, rather than those of carbon and nitrogen, RISCs and metal ions are abundant at acidic assume centre stage in these environments. Ferrous sites where most of the acidophilic microorganisms iron and reduced sulfur compounds originating thrive using iron and/or sulfur redox reactions. In from geothermal activity or mineral weathering contrast to biomining operations such as heaps provide energy sources for acidophilic, chemo- and bioleaching tanks, which are ofen aerated to lithotrophic iron- and sulfur-oxidizing bacteria and enhance the activities of mineral-oxidizing prokary- archaea (including species that are autotrophic, otes, geothermal and other natural environments, heterotrophic or mixotrophic) and, in contrast to can harbour a more diverse range of acidophiles most other types of environments, these are ofen including obligate anaerobes. numerically dominant in acidic sites. Anaerobic growth of acidophiles can occur via the reduction of ferric iron, elemental sulfur or sulfate. While the Populations in natural acidic activities of acidophiles can be harmful to the envi- environments ronment, as in the case of acid mine drainage, they Environments where acid is formed naturally with- can also be used for the extraction and recovery of out the infuence of mining include geothermal metals, as in the case of biomining. Considering terrestrial sites (solfatara) that occur at active volca- the important roles of acidophiles in biogeochemi- noes, deep-sea hydrothermal systems, and naturally cal cycles, pollution and biotechnology, there is a exposed sulfde ore deposits. Tese sites are of great strong need to understanding of their physiology, interest when searching for organisms to be used, biochemistry and ecology. for example in biomining operations, as it is ofen the case that these environments have existed for many years and therefore indigenous microorgan- Introduction isms are potentially adapted to high metal and salt Environments inhabited by acidophiles can be of concentrations, extreme temperature and low pH natural origin where acidic conditions have existed (Table 10.1). for many years, such as volcanic or geothermal

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Table 10.1 Physico-chemical parameters and prokaryotic diversity of selected natural environments where acidophiles have been detected Temperature Site pH (°C) Bacteria Archaea Reference

Geothermal sites Yellowstone 2.0– 30–90 Fx. thermotolerans, Ad. brierleyi, Sulfolobales, Johnson et al. National Park, 4.4 Sulfobacillus-like, Thermoproteales, (2003), Inskeep et Wyoming, Am. ferrooxidans-like, Desulforococales, al. (2004, 2010), USA Methylobacterium- ‘Geoarchaeota’, Kozubal et al. like, Acidisphaera sp., Thaumarchaeota, (2013), Beam et Alicyclobacillus-like, H. Metallosphaera-like, al. (2014) acidophilus, Thiomonas sp. Marinithermus sp. Montserrat, 1.6– 24–99 At. caldus, At. ferrooxidans. Sulfolobus spp., Acidianus Atkinson et al. Lesser Antilles 7.4 L. ferrooxidans, Am. spp., Ferroplasma-like (2000), Burton ferrooxidans, Sulfobacillus and Norris (2000) spp., Acidiphilium-like, Alicyclobacillus spp., Desulfosporosinus sp. St. Lucia, 2.0 42.6 At. caldus, Sulfobacillus Acidianus spp., unknown Stout et al. (2009) Lesser Antilles spp. Eukaryota Palaeochori 2.0 50–60 At. caldus, Acidianus spp., Norris (2011) Bay, Milos ‘Acidithiomicrobium’, Ferroplasma-like Acidithiobacillus spp., Sulfobacillus spp. Vulcano, Italy 2.0– 35–75 Acidithiobacillus spp., T. Tp. volcanium, Ad. brierleyi, Simmons and 3.5 prosperus Ad. inferno Norris (2002) Mutnovsky 3.5– 70 At. caldus, Thermotogae Thaumarchaeota, Wemheuer et al. volcano, 4.0 Acidilobus sp., Vulcanisaeta (2013) Kamchatka sp., Thermoplasma sp. Hveragerji, 2.5– 81–90 NR Sulfolobales, Kvist et al. (2007) Iceland 3.5 8–20 Sulfobacillus spp., Thermoproteales, Davis-Belmar Salar de 1.1– Alicyclobacillus crenarchaeotal group I.1b et al. (2013), Gorbea 4.8 spp., Acidisoma sp., Thermoplastales, Escudero et al. Alkalibacter sp., Proteo- Thermococcales, (2013) and Actinobacteria, Halobacteriales, Cyanobacteria Methanosarcinales and Methanobacteriales, Thermoproteales Copahue <1.0– 8–85 At. ferrooxidans, At. Ferroplasma spp., Ad. Urbieta et al. volcanic area 4.0 thiooxidans, Leptospirillum copahuensis, Sulfolobus (2012, 2014a) including spp., At. caldus-like, spp. Rio Agrio, Sulfobacillus spp. Argentina

Deep sea hydrothermal vents deep sea vent, 3–6 50–80 NR ‘Aciduliprofundum boonei’ Reysenbach et al. Eastern Lau (2006) Spreading Centre Caves Frasassi cave, 0–1.5 13 At. thiooxidans, Ferroplasma spp., Macalady (2007), Italy Sulfobacillus spp., Am. ‘G-plasma’ Jones (2012) ferrooxidans Cueva de Villa 1.4 28 Acidithiobacillus spp., Am. NR Hose et al. (2000) Luz, Mexico ferrooxidans

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Table 10.1 Continued Temperature Site pH (°C) Bacteria Archaea Reference

Gossan Citronen Fjord, 1.9– 2–5 Acidithiobacillus-like, NR Langdahl and Greenland 2.5 heterotrophs Ingvorsen (1997) Antarctica 3.2– 0–4 T. plumbophilus, At. NR Dold et al. (2013) 4.5 ferrivorans

Abbreviations: Acidianus (Ad.), Acidilobus (Al.), Acidimicrobium (Am.), Acidiphilium (A.), Acidisphaera (As.), Acidithiobacillus (At.), ‘Acidithiomicrobium’ (Atm.), Alicyclobacillus (Alb.), Ferrithrix (Fx.), Ferroplasma (Fp.), Hydrogenobaculum (H.), Leptospirillum (L.), Metallosphaera (M.), Picrophilus (P.), Sulfsphaera (Ss.), Sulfobacillus (Sb.), Sulfolobus (S.), Thermoplasma (Tp.), Thiobacillus (T.). NR, not reported.

Te frst sulfur-metabolizing archaeon, subse- Hydrothermal environments quently named as Acidianus brierleyi, was isolated Solfatara are characterized by high temperatures by James Brierley in 1965 from geothermal sites in (up to 100°C) and elevated concentrations of sulfur Yellowstone (Brierley, 1973). Further cultivation- (and RISCs), hydrogen and ofen also soluble based studies on acidophiles from YNP resulted in metals and arsenic. When elemental sulfur, formed the enrichment of novel thermophilic iron- and/

by the condensation of volcanic gases such as H2S or sulfur-metabolizing and heterotrophic bacteria and SO2, is oxidized by acidophilic prokaryotes, (Johnson et al., 2003), including the isolation of the sulfuric acid is formed which results in low-pH type strain of the acidophilic, moderately thermo- environments. Owing to the low pH and high philic species Ferrithrix thermotolerans (Johnson et temperatures in volcanic areas, acid-labile minerals al., 2009) and Acidicaldus organivorans (Johnson et are dissolved and release elevated concentration al., 2006). of transition metals and metalloids. Te nature of While a number of described acidophilic hydrothermal sites is dictated by the subterranean archaeal species have been detected in samples geology and water fow. In particular, the degree of YNP, several studies have shown that the hot of mixing of deep hydrothermally heated water springs of YNP are a source of novel and deeply

(bufered in the neutral–alkaline region by CO2/ branching archaea (e.g. Beam et al., 2014; Inskeep – HCO3 ) with cold shallow groundwater (strongly et al., 2010, 2004; Table 10.1). Archaea in an acidic acidifed by microbial and chemical oxidation iron oxide geothermal spring were further analysed of sulfur compounds) determines pH and tem- by Kozubal et al. (2013) and proposed as a new can- perature of the environment (Atkinson, 2000). Te didate phylum within the domain Archaea referred microbiology of acidic geothermal areas has been to as ‘Geoarchaeota’ or ‘novel archaeal group 1 studied for more than four decades (e.g. Johnson (NAG1)’. Tese organisms were found to contain et al., 2003; Brock, 2001; 1978) and microbial pathways necessary for the catabolism of peptides communities in these environments host a variety and complex carbohydrates and genes involved in of deeply rooted known and unknown Archaea, the metabolism of oxygen. Furthermore Beam et al. Bacteria and Eukarya. (2014) applied metagenome sequence analysis to One of the most intensively studied natural study thermophilic populations (65–72°C) at acidic acidic hydrothermal environment is Yellowstone iron oxide- and sulfur-rich sediment environments National Park (YNP; Wyoming, USA) which of YNP. Tese deeply rooted Taumarchaeota were houses the most numerous and diverse geothermal proposed to be chemo-organotrophic and couple terrestrial systems on Earth with widely varying growth to the reduction of oxygen or nitrate in iron geochemical properties, such as temperature, pH, oxide habitats, or sulfur in hypoxic (low-oxygen) and concentrations of dissolved ions and oxygen. sulfur sediments. Possible carbon sources for these

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archaea include aromatic compounds, complex Leptospirillum spp. at moderate temperatures, while carbohydrates, oligopeptides and amino acids or the later was dominant in samples of 50–58°C. carbon dioxide. No evidence for the oxidation of Tese low to moderate temperature site popula- ammonia was obtained from the de novo sequence tions were also accompanied by heterotrophic assemblies. Acidiphilium-like spp., Sulfobacillus spp., Acidimi- Some of the geothermal springs within YNP crobium ferrooxidans, Alicyclobacillus species, and ofen contain elevated concentrations of arsenic an unknown actinobacterium (RIV 14). 16S rRNA (1–150 mg/l) which occurs predominantly as gene sequences of two potentially moderate sulfur- arsenious acid [As(III)] when the water is dis- reducing genera (one of the Desulfurella group) charged, but is rapidly oxidized downstream from were detected in the same pools, while sulfate- the spring source. Te oxidation of As(III) to reducing bacteria could be isolated from moderate arsenate (As(V)) can occur through abiotic and pools (30°C, pH 3.2) one of which (strain M1) was biotic processes and, although biological oxida- later assigned as the type strain of the novel species tion is typically much faster (Cullen and Reimer, Desulfosporosinus acididurans (Sánchez-Andrea et 1989), only a few studies have examined the al., 2015). Higher temperature pools of Montserrat organisms that carry out this process. Studies on contained clone sequences related to Ferroplasma- the microbial communities of acid-sulfate-chloride like organisms, while the dominant group clustered springs within YNP (pH ~3.1, T = 53–74°C) in the Crenarchaeota, distantly related to classifed indicated the dominance of hydrogen-oxidizing Sulfolobus spp. and Acidianus spp. Members of the Hydrogenobaculum acidophilum, Desulphurella and thermoacidophilic crenarchaeotal genus Sulfolobus smaller numbers of Acidimicrobium and Tiomonas were isolated from sites of 65–98°C including one (Inskeep et al., 2004). Archaeal sequences of char- isolate representing a new species which has also acterized members in these springs were related to been isolated from the Azores (P.R. Norris, unpub- thermophilic, metal-mobilizing Metallosphaera and lished data). Marinithermus, but the majority were unknown Two more sites with similar microbiological crenarchaeotes and euryarchaeotes, most closely composition such as Montserrat have been stud- related to sequences from marine hydrothermal ied. One is located on the island St. Lucia, where vents and which are proposed to be responsible for volcanic, acidic sulfur-springs occur (pH of 2.0 and the oxidation of As(III) in the spring (Jackson et al., 42.6°C) and the other one is a geothermal site at 2001). D’Imperio et al. (2007) described the isola- Palaeochori Bay of the island of Milos in the Aegean tion of a novel arsenite-oxidizing Acidicaldus species Sea (T = 55–60°C). Both microbial communi- and documented arsenite oxidation inhibition by ties were dominated by At. caldus and Acidianus hydrogen sulfde and its infuence on the distribu- spp. and samples from St. Lucia also contained tion of arsenite-oxidizing chemolithotrophs. low numbers of Sulfobacillus spp. and unknown Another interesting and intensively studied Euryarchaeota (Stout et al., 2009; Table 10.1). Te hydrothermal site is located on the Caribbean Milos samples additionally harboured unknown island of Montserrat (Atkinson et al., 2000; Burton Ferroplasma/Acidiplasma-like clone sequences as and Norris, 2000; Table 10.1), the topography of well as a novel sulfur-oxidizing actinobacterium which is dominated by the active Chances Peak ‘Acidithiomicrobium’ (also isolated from Milos) and volcano (914 m) and the eroded peaks of three a novel Acidithiobacillus sp. V1 (Norris et al., 2011). extinct volcanoes, Centre Hills (676 m), Silver Hill Copahue-Caviahue, a geothermal area in Argen- (403 m) and South Soufrière Hills (756 m). tina, is dominated by the Copahue Volcano which Microbial studies on Montserrat samples is responsible for the thermal activity in this area. identifed the thermophilic sulfur-oxidizer Acid- Several acid ponds, pools and hot springs occur ithiobacillus caldus, which is known to grow over a in this region, some of which are the source of the wide temperature range, as the dominant organism acidic river Rio Agrio that discharges in the acidic at moderate to higher temperature (Burton and Caviahue lake. Te various habitats harbour a broad Norris, 2000). Cultivation studies furthermore range of acidophilic, moderate to thermophilic indicated that iron-/sulfur-oxidizing Acidithioba- microorganisms as they vary in their physico-chem- cillus ferrooxidans dominated over iron-oxidizing ical characteristics (mainly pH and temperature).

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Molecular analysis of the microbial river commu- and is characterized by active volcanoes, fssure nity (Urbieta et al., 2012) revealed the presence of swarms, numerous normal faults, and high-temper- moderate, sulfur-oxidizing bacteria (Acidithiobacil- ature geothermal felds. In the famous Hveragerði lus thiooxidans and Acidithiobacillus albertensis) and high-temperature geothermal feld, located about moderately thermophilic iron- and sulfur-oxidizing 50 km southwest of Reykjavik, geothermal manifes- bacteria (Alicyclobacillus spp. and Sulfobacillus spp.) tations consist of fumaroles and hot springs, which ubiquitous along the river. Iron-oxidizing bacteria have diferent values of temperatures and pH. (Leptospirillum spp. and Ferrimicrobium spp.) and Studies on the prokaryotic diversity of the sedi- archaea (Ferroplasma spp.) were present at the ment samples near the Mutnovsky volcano (pH source of the river where iron concentrations were 3.5–4.0, T = 70°C) revealed the dominance of much lower than further downstream. Te biodi- sulfur-oxidizing At. caldus and Termotogae (accom- versity of various ponds studied in the Copahue panied by small numbers of Desulfurella, Kosmotoga area (Urbieta et al., 2014a; Table 10.1) is deter- and Hydrogenobaculum spp.) and sequences cluster- mined by the temperature, resulting in archaea ing with the thaumarchaeal ‘terrestrial hot spring (novel Sulfolobales spp. and Termoplasmatales spp.) group’ as well as minor numbers of Acidilobus spp., colonizing higher-temperature ponds, whereas Vulcanisaeta spp. and uncultivated Termoplasma ponds of moderate temperature are colonized spp. (Wemheuer et al., 2013; Table 10.1). Te type by sulfur-oxidizing bacteria, e.g. Tiomonas spp., strain of the hyperthermophile Acidilobus aceticus Acidithiobacillus spp., Hydrogenobaculum spp. and has previously been isolated from the same area and Acidiphilium spp. Also a novel Acidianus species, described as a strictly anaerobic acidophile with a Acidianus copahuensis, whose genome has recently heterotrophic lifestyle (Prokofeva et al., 2000). been sequenced, has been isolated from hot springs Te archaeal composition of samples from Hver- of Copahue (Urbieta et al., 2014b). Te presence of agerði (Iceland) hot springs (pH 2.5, T = 81–90°C) sulfate-reducing bacteria in anaerobic sediments of was more diverse, comprising members of the acido- an acidic hot spring of Copahue was confrmed by philic, sulfur-oxidizing Sulfolobales, Termoproteales the isolation of several strains related to the sulfate- and uncultured members of the crenarchaeal group reducer ‘Desulfobacillus acidavidus’ strain CL4 from I.1b (Kvist et al., 2007). Further studies on archaeal these sediments (Willis et al., 2013). Several eukar- communities of water and mud samples from yotes, such as yeasts and flamentous fungi, have hot springs in Kamchatka and Iceland detected also been detected in the geothermal area (Chiac- archaeal ammonia monooxygenase (amoA) genes chiarini et al., 2009). In microbial terms, Copahue in agreement with considerable in situ nitrifcation is a very diverse environment and analyses have rates (Reigstadt, 2010). Te wide temperature and shown that the distribution of microorganisms in pH range (T = 38–97°C and pH 2.5–7) at which this system is determined by pH, temperature and ammonium-oxidizing archaea (AOA) have been conductivity, rather than mineral chemistry as it is detected, together with the geographically disparate the case in Yellowstone National Park (Urbieta et sampling locations, provide evidence for an active al., 2014a). role of hyperthermophilic AOA in nitrogen cycling Numerous hydrothermal vents are also located in hot spring microbial communities throughout on the Kamchatka Peninsula in the northeast of the world. Russia. Tese vents constantly release geothermal Natural acidic, saline lakes are relatively rare

gases and fuids dominated by N2, CO2 and H2; compared to alkaline salt lakes, but can be found CH4 and H2S also frequently occur. Te hot springs in Australia and the Atacama region of northern found in this area have multiple origins, including Chile. Te Salar de Gorbea and Salar Ignorado meteoric and magmatic water. Active hydrothermal are basins in Chile where the oxidation of native springs with a large variety in chemical composi- volcanic sulfur occurrences leads to the release of tion, temperature, and pH are located in the area of sulfuric acid and strong hydrothermal alteration the Uzon Caldera, Geyser Valley near the volcanoes of the country rocks lowers the bufering capacity Karymskii and Mutnovsky. Similar sites can also and thereby cause these habitats to be acidic. Te be found in Iceland, which is located on the Mid- water bodies of Salar de Gorbea are character- Atlantic Ridge and harbours a zone of active rifing ized by high salinity (100–300% total dissolved

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solids) and acidic pH (1.1–4.8) with temperatures 50–80°C (Le Bris et al., 2005). Acidity is also pro- between 8 and 19.5°C. Te dominant bacteria in duced locally in the chimneys by the precipitation these samples were members of the Proteo-and of minerals such as pyrite and chalcopyrite. Te Actinobacteria, Cyanobacteria as well as Firmicutes majority of thermophiles isolated from deep-sea (e.g. Sulfobacillus, Alicyclobacillus) (Davis-Belmar et vents are, however, neutrophiles or acid-tolerant al., 2013; Escudero et al., 2013). Cultivation based organisms. A notable exception is the thermoacido- studies revealed the presence of novel strains of the philic sulfur- and ferric iron-reducing heterotroph genus Acidisoma (Davis-Belmar et al., 2013). Te ‘Aciduliprofundum boonei’, a member of the ‘deep- archaeal community of Salar de Gorbea was domi- sea hydrothermal vent Euryarchaeota 2’ (DHVE2) nated by various members of the Euryarchaeota group, which grows between pH 3.3 and 5.8 (Termoplastales, Termococcales, Halobacteriales, (Reysenbach et al., 2006; Table 10.1). Further ther- Methanosarcinales and Methanobacteriales) and moacidophilic strains related to ‘Acp. boonei’ could minor numbers of Crenarchaeota (Termoproteales) be isolated from vents along the East Pacifc Rise (Escudero et al., 2013). and Mid-Atlantic Ridge, showing that the DHVE 2 Salt-tolerant acidophiles appear to be relatively are widespread in various hydrothermal vents and uncommon. One such is the acidophilic iron- constitute a major part of the archaeal population oxidizer ‘Tiobacillus prosperus’ (Huber and Steter, (Flores et al., 2012). 1989), which was isolated from the Mediterranean island of Volcano (located to the north of Sicily; Cave systems Table 10.1) and which can grow in media contain- Another example of natural-acidic environments ing up to 6% NaCl. 16S rRNA gene clone libraries are cave systems, such as the Lechugilla Cave (New of samples from Volcano sites were dominated by a Mexico, USA; Hill, 1995), Frasassi cave (Italy; novel Acidithiobacillus sp. at 35–45°C, while novel Macalady et al., 2007) and Cueva de la Villa Luz iron-oxidizing bacteria related to ‘T. prosperus’ were (Mexico; Hose et al., 2000) (Table 10.1). Tese isolated but not detected in gene banks (Simmons develop in subterranean environments containing and Norris, 2002). Acidophilic archaea at Volcano sulfde-rich ground-waters where, under aerobic included Acidianus spp. and Termoplasma spp., all conditions, microbial oxidation of sulfde to sulfu- of which displayed a broad tolerance (up to 4%) to ric acid leads to extensive dissolution of carbonate sodium chloride (Simmons and Norris, 2002). rock strata. One of the most comprehensively stud- While acidophilic prokaryotic communities in ied sites of this type, in terms of microbiology and hydrothermal areas have been studied quite inten- chemical parameters, is the sulfdic Frasassi cave in sively, eukaryotes in geothermal sites have received Italy where bioflms (‘snotites’) of pH 0–1 have far less atention. In one study, eukaryotes observed been described (Macalady et al., 2007). Commu- in Nymph Creek, YNP (pH 2.5, T = 40°C), com- nity analysis of theses snotites revealed a limited prised Cyanidium-like algae, the acidophilic diatom biodiversity with sulfur-oxidizing At. thiooxidans Pinnularia, the amoeba Tetramitus thermoacidophi- as the most abundant bacterium, and smaller lus and Naegleria spp., fagellates and novel members numbers of Sulfobacillus spp., Acidimicrobium fer- of the amoeba Vampyrellidae (Amaral-Zetler et al., rooxidans and archaea related to the uncultivated 2013). ‘G-plasma’ clade of the Termoplasmatales (Jones et al., 2012). Also some protists and fungal flaments Deep-sea hydrothermal vents were observed via microscopy in the Frasassi cave Te walls of deep-sea hydrothermal vent deposits samples (Macalady et al., 2007). (chimneys) with their steep chemical and thermal Te hypogenic cave Cueva de Villa Luz in gradients provide a wide range of microhabitats for southern Mexico also comprises extremely acidic microorganisms. In situ measurement of pH at the microenvironments (pH 0.1–3.0) and sulfur-rich exterior of such a deposit wall on the East Pacifc springs and shows elevated hydrogen sulfde emis- Rise reported pH values of 4.1 at 120°C (von sion. Te microbial composition of water drips from Damm et al., 1995). Calculated pH values based snotites is similar to the Frasassi community with on various fow rates and exchange of fuids within sulfur-oxidizing Acidithiobacillus spp. as key players the pores can range from 3 to 6 at temperatures of and smaller numbers of moderately thermophilic

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Am. ferrooxidans mediating redox reactions within heterotrophic acidophiles from this environment. the cave (Hose et al., 2000). Te bacteria assimilated 14C-labelled bicarbonate and glucose below 0°C, but were found to be only Acid sulfate soils and acidic fens cold-tolerant rather than psychrophilic. Acid sulfate soils (ASS) form predominantly when Acid rock drainage (ARD), resulting from the anoxic marine sediments or salt-marshes rich in microbially catalysed oxidation of sulfde minerals, pyrite and other sulfde minerals are aerated, e.g. becomes increasingly signifcant in polar regions when drained for agricultural or industrial purposes, as global warming causes increased glacier melting and oxidation reactions generate proton acidity and and thereby the exposing of more sulfde-rich rock mobilize metals. ASS are widespread in coastal strata to air. Schwertmannite present in glacier ice regions and cause a serious environmental risk due and icebergs of the Antarctica and Artic was the to severe soil acidity and acid metal-rich run-of frst indication of pyrite oxidation in this area. Dold efecting adjacent fora and fauna. As described et al. (2013) investigated the microbial community earlier, microorganisms catalyse the oxidation of causing ARD on the Antarctic landmass, which sulfdic minerals and acid production and are there- was exclusively dominated by bacteria especially fore the key drivers in the formation of acid sulfate related to Tiobacillus plumbophilus and At. fer- soils. Initial studies on the formation of ASS by rivorans, both of which are psychrotolerant. Other Arkesteyn (1980) resulted in the isolation of iron-/ bacteria in these Antarctic samples were species sulfur-oxidizing At. ferrooxidans and sulfur-oxidiz- typically detected in acidic environments including ing At. thiooxidans from the acidifying soil material Frateuria-like species, Acidisphaera, Actinobacteria but were not atributed to contribute to the initial and Acidobacteria. pH decrease to pH 4. Sulfur-oxidizing acidophiles were also detected in a buried potential acid sulfate soil layer of a Japanese paddy feld which were pro- Populations in mine-impacted posed to contribute to ASS formation by oxidizing environments sulfur compounds to sulfate and thereby producing Te best-studied low-pH environments are man- acidity (Ohba and Owa, 2005). Wu et al. (2013) made and mostly associated with the mining of studied the zones of the Risöfadan experimental metals and coals. Pyrite becomes exposed to air feld, Finland, which had been drained for more (oxygen) and water during mining and oxidizes to than 40 years. Bacteria present in the plough and sulfuric acid and ferric iron. However, the prime oxidized layers were related to known acidophilic oxidant of pyrite is ferric iron, and sulfate is only heterotrophs and iron- and sulfur-oxidizers previ- the fnal product with the highest oxidation state, ously found in acidic, metal- and sulfur-containing meaning that sulfur compound intermediates such environments. Enrichment cultures of partial oxi- as thiosulfate occur in the oxidation pathway (Vera dized soil at pH 3.0 included iron-/sulfur-oxidizing et al., 2013). At. ferrivorans, At. ferrooxidans, Sulfobacillus spp. Organisms in such habitats mainly catalyse and Tiomonas spp.. carbon, iron and sulfur transformations and are widespread among the domains Bacteria and Naturally exposed ore deposits Archaea, and some Eukarya. In general, members Acidophilic microorganisms also occur naturally of the Proteobacteria phylum are by far the most in the uppermost weathered zones of sulfde ore common representatives of Bacteria in mine- deposits (gossans; Table 10.1). Such deposits have impacted environments followed by Nitrospirae, been mainly exploited by mining and remain only Actinobacteria, Firmicutes and Acidobacteria, while in remote areas e.g. in high altitudes. Langdahl members of the Bacteriodetes, and Candidate divi- and Ingvorsen (1997) studied the microbiology sion TM7 occur only in low numbers. of gossan material at the Citronen Fjord in North In contrast to geothermal sites, which mainly Greenland, which was characterized by subzero comprise sulfur-oxidizing microorganisms that do temperatures and therefore a potential habitat not oxidize ferrous iron, few of these organisms for psychrophilic microorganisms. Tey success- are detected in AMD and one explanation might fully enriched Acidithiobacillus-like organisms and be that reduced sulfur compounds resulting from

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pyrite oxidation can also be oxidized to sulfate by Microbial populations at various ferric iron (Druschel et al., 2003) and are thus not mine sites available as substrate for acidophiles. Te numeri- cally dominant microorganisms in AMD sites Richmond Mine, Iron Mountain are ofen iron-oxidizing microorganisms (many One of the most intensively studied and most of which also oxidize sulfur), as iron is ofen the extreme acidic site is the Richmond Mine at Iron dominant metal in these environments. A well- Mountain in California, comprising natural under- known iron-oxidizer frequently found in acidic ground water pools of negative pH which are the mine waters is At. ferrooxidans (Colmer et al., lowest pH environments discovered yet (Nord- 1950), which is also capable of oxidizing reduced strom et al., 2000). Te Richmond Mine harbours a sulfur compounds and hydrogen, and of ferric iron broad range of biodiversity and has been the source respiration under anaerobic conditions. At. ferroox- of several new species of iron-oxidizing prokaryotes idans (and related iron-oxidizing acidithiobacilli) (e.g. Tyson et al., 2004; Edwards et al., 1999; Table is mostly found in AMD-environments of low 10.2). Oxidative dissolution of pyrite and other pH and moderate temperature. Another common sulfde minerals occurs at greatly accelerated rates iron-oxidizing bacterium in acidic environments within Iron Mountain causing increased tempera- is L. ferrooxidans, which has a higher afnity for tures within the mine (Nordstrom et al., 2000). ferrous iron and a greater tolerance of ferric iron Te higher temperature sites of the mine have than At. ferrooxidans, and therefore correlates with been found to be dominated by sulfur-oxidizing changing ferrous iron concentrations in the mine Sulfobacillus spp., while low-pH sites of the same waters. Heterotrophic acidophilic bacteria, mostly temperature range were colonized by the iron-oxi- belonging to the Alphaproteobacteria class (e.g. dizing archaeon ‘Ferroplasma acidarmanus’ (Bond et Acidiphilium, Acidocella, Acidisphaera), also play an al., 2000). Higher pH and cooler temperature areas important role in the carbon cycle of mine waters. are mainly dominated by At. ferrooxidans, while Archaea have also been reported to be less abun- Leptospirillum spp. are widely distributed through- dant than bacteria in mine-impacted environments out the mine. All three groups of leptospirilli, L. as most of them are (moderate) thermophiles and ferrooxidans (‘Group I’), L. ferriphilum and ‘L. are more frequently found in geothermal areas. rubarum’ (‘Group II’), ‘Leptospirillum ferrodiazotro- One archaeon, however, which is ofen found in phum’ (‘Group III’), are present in the Richmond association with AMD is the mesophilic, iron- Mine and recently a fourth group ‘Leptospirillum oxidizer Ferroplasma acidiphilum (Golyshina et group IV UBA BS’ has been detected (Aliaga Golts- al., 2000). Eukaryotes ofen only account for mann et al., 2013). Underground slime bioflms a minor portion of the microbial community in and acid streamer growths within the Richmond some AMD environments, but have been studied Mine were dominated by Leptospirillum spp. and to a great extent e.g. in the Rio Tinto, Spain (e.g. ‘Ferroplasma acidarmanus’, accompanied by the González-Toril et al., 2003) and to some extent facultative heterotroph Ferrimicrobium acidiphilum at the Richmond Mine, California, USA (e.g. and Deltaproteobacteria (Edwards et al., 1999). Edwards et al., 1999). Genomic (Tyson et al., 2004) and proteomic Microorganisms in these environments ofen (Ram et al., 2005) studies of bioflm communi- occur as bioflms on mineral surfaces, microbial ties within Iron Mountain carried out by the stalactites, snotites, macroscopic growths in Banfeld group have provided further insights streams as well as in the planktonic phase of acidic into the nature of the microbial communities and water bodies, streams and pit lakes. Examples motivated revision of the Leptospirillum and Fer- of the most intensively studied AMD environ- roplasma taxonomy (e.g. Leptospirillum group IV, ments are listed in Table 10.2 and some relevant Ferroplasma group II). One particularly fnding and microbial diverse sites are further described revealed by community genomic analysis was the below. presence of novel archaeal lineages designated as ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms; Baker et al., 2006), which form a deep branch within the Euryarchaeota. Te cells of

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Table 10.2 Characteristics of selected mine-impacted sites, and dominant microorganisms detected Dyfryn Adda Richmond Mine, Parys Mountain Drei Kronen und (draining Mynydd Iron Mountain (Mynydd Parys) Cae Coch Ehrt Parys) Cantereras Rio Tinto Location California (USA) North Wales North Wales Germany North Wales Spain Spain Type of mine Copper Copper Pyrite Pyrite Copper Copper Pyrite Microbial growth Mine water Underground lake Water pools Stalactites Mine water Mine water Mine water Bioflms Drapes Water droplets Streamer growth Streamer growth Bioflms Streamer growth Streamer growth Streamer growth Stalactites Sediments 33 pH <0–2.0 2.3 1.8–2.3 2.6 2.5 2.5–2.8 2.2–4.7 Temperature (°C) 30–50 8–9 8–9 15 11 – 17.9 Dominant bacteria Leptospirillum spp. Iron-oxidizing At. ferrivorans Leptospirillum sp. At. ferrivorans At. ferrivorans L. ferrooxidans Sulfobacillus spp. acidithiobacilli ‘Fv. myxofaciens’ ‘Ferrovum’ sp. ‘Fv. myxofaciens’ Acidobacteriaceae At. ferrooxidans At. ferrooxidans Acidobacteriaceae Gallionella sp. ‘Atx. ferrooxidans’ Acidiphilium spp. Acidiphilium spp. Dominant archaea ‘Fp. acidarmanus’ Euryarchaeota Euryarchaeota ARMAN-group Euryarchaeota Euryarchaeota Ferroplasma spp. ARMAN group Thermoplasmatales Thermoplasma spp. Eukarya Algae – – – Euglena mutabilis Chlamydomonas Algae Fungi acidophila Protists

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ARMAN-members are of extremely small size and on the occurrence of these proposed acidophilic/ sequences have been detected in various locations acid-tolerant iron-oxidizing Gallionella spp. in sev- of the Richmond Mine and also other habitats eral mine-impacted environments (e.g. Fabisch et (e.g. the Drei Kronen und Ehrt mine; Ziegler et al., 2013; Heinzel et al., 2009). Minor numbers of al., 2013). Te Richmond Mine, similar to the Rio Alphaproteobacteria (Acidiphilium spp. and a novel Tinto in Spain, is also one of the best studied habi- Sphingomonas-isolate), Gammaproteobacteria (At. tats of acidophilic eukaryotes, and harbours species ferrivorans and bacteria related to the unclassifed of, for example, red algae, fungi and protists (Vahl- species ‘WJ2’), Actinobacteria (Ferrimicrobium-like kampfidae, Dothideomycetes and Eurotiomycetes; bacteria), Nitrospirae (L. ferrooxidans) and Firmi- Baker et al., 2009, 2004). cutes were also detected in the streamer and slime growths. Archaeal 16S rRNA genes related to a single, novel euryarchaeotal species were detected in one pool characterized by lower pH and higher Cae Coch pyrite mine dissolved solutes than in the other sampled water In contrast to the relatively high temperature Rich- bodies. With At. ferrivorans, ‘Fv. myxofaciens’, L. mond Mine site, two low-temperature and extreme ferrooxidans and the proposed iron-oxidizing Gal- acidic sites in north Wales, UK, Cae Coch mine lionella spp. identifed as the dominant organisms, and Mynydd Parys mine, have been extensively iron oxidation seems to be the main metabolism in studied by Johnson and colleagues. Te abandoned this pyrite mine (Johnson, 2012). Cae Coch pyrite mine contains several water accu- mulations in form of several small pools and one Mynydd Parys copper mine single drainage stream. Microbial growth occurs as Te fooded underground copper mine, Mynydd bioflms on the walls, stalactites, streamer growths Parys (Parys Mountain), north Wales, represents a within the drainage stream and in water droplets similar extreme acidic subsurface environment to on the ceiling and stalactites. Te microbial com- the Cae Coch mine, though the underground lake munity of the water droplets is relatively simple and is essentially anoxic. When the mine was partially dominated by the iron-oxidizing At. ferrivorans and dewatered, due to concerns that a concrete dam smaller numbers of iron-oxidizing L. ferrooxidans could fail and result in the fooding and severe pol- (Table 10.2). Te streamer and slime communi- lution of a nearby coastal town, an opportunity to ties, in contrast, are more diverse and vary between study the microbial community within the mine the diferent sampling locations, but are mostly arose. Te subterranean waters were dominated by dominated by the streamer-forming iron-oxidizer iron-oxidizing Acidithiobacillus spp., and even so ‘Ferrovum myxofaciens’, which has been detected as the exact species was not determined at this time, a dominant bacterium in a number of acidic envi- the dominance of At. ferrivorans is most likely due ronments (Johnson et al., 2014). ‘Fv. myxofaciens’ to the low temperatures and subsequent studies ofen occurs as macroscopic streamer growths (Coupland and Johnson, 2004). As in the Cae Coch due to its ability to produce copious amounts of mine, L. ferrooxidans, Gallionella-related species, extracellular polymeric substances (EPS). Tis heterotrophic acidophiles (Acidiphilium, Acidobac- bacterium, like L. ferrooxidans, appears to be only terium, Acidisphaera) and Fm. acidophilum were capable of growth by ferrous iron oxidation and present in the samples. Archaeal 16S rRNA gene fxes carbon dioxide and nitrogen (Johnson et sequences detected in the Parys Mountain under- al., 2014). Te dominance of ‘Fv. myxofaciens’ in ground lake were, however, very diferent to the the stream correlates well with the occurrence Cae Coch archaea with clones related to potential of At. ferrivorans in the mine, as both species are methanogens and only minor numbers of Termo- psychrotolerant and capable of growth at low tem- plasma/Ferroplasma-like species (Coupland and peratures as is characteristic of the Cae Coch mine Johnson, 2004; Table 10.2). Macroscopic growths (9 +/– 1°C). One of the stalactites analysed was hanging from pit props within the mine had a dif- predominantly colonized by bacteria closely related ferent microbial composition and were dominated to the neutrophilic iron-oxidizer Gallionella fer- by the heterotrophic iron-bacteria Ferrimicrobium, ruginea. Tere have been a number of other reports Acidimicrobium and the iron-reducing genus

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Actinobacterium as well as a variety of archaea simi- a study of this stream carried out for 9 years follow- lar to those detected in the streamers (Coupland ing its inception (Table 10.2). A few months afer and Johnson, 2008). Owing to the limited oxygen it carried AMD, the stream became heavily colo- ingress reported for Mynydd Parys, activities of nized with macroscopic streamer growths, which iron-oxidizing microorganisms seemed to be low, continued to occupy a large part of the stream but ferric iron reduction catalysed by autotrophic over the entire study period. Te prokaryotic com- and heterotrophic bacteria, as well as due to reac- munity of the streamer growths was dominated tion with pyrite and other residual sulfde minerals, by the autotrophic iron-oxidizers At. ferrivorans led to ferrous iron being the dominant form in the and ‘Fv. myxofaciens’ and a novel heterotrophic subterranean lake (Johnson, 2012). iron-oxidizer (‘Acidithrix ferrooxidans’). As time progressed acidophiles, such as iron-reducing Other mine sites heterotrophs of the genus Acidiphilium, Acidobacte- Similar structures and microbial communities as rium, Acidocella and Metallibacterium, as well as the described for the two former mine sites were also autotrophic iron-oxidizers At. ferrooxidans and L. found in the Drei Kronen und Ehrt pyrite mine ferrooxidans, were detected in the stream. Archaeal in the Harz mountains in Germany. Ziegler et al. species found in the Dyfryn Adda were all distantly (2009) studied small microbial stalactites and related to known Euryarchaeota. Te surface of the identifed the iron-oxidizers L. ferrooxidans and stream becomes occasionally colonized by the algae ‘Fv. myxofaciens’ as dominant bacteria and smaller Euglena mutabilis, which might be one cause of the numbers of Acidiphilium spp.. Tey also detected varying abundance of heterotrophic bacteria within archaea related to the ARMAN-group, previously the stream as the algae provide organic carbon detected in the Richmond Mine, and uncultivated that can sustain their growth (Ňancucheo and members of the Termoplasmatales (Ziegler et al., Johnson, 2012). Te relatively constant physico- 2013; Table 10.2). chemical parameters supported a stable microbial Mine sites with a more moderately acidic to community in the stream over the years; with most circumneutral pH, as a habitat for moderate acido- organisms originating from the underground mine philic and acid-tolerant microorganisms, have also water of Parys Mountain. Te only notable difer- been studied. One of these is the former Wheal Jane ence between the Parys mine underground water tin mine in Cornwall (England) which harbours and the Dyfryn Adda stream is the dominance similar microorganism as the Parys mine but is of ‘Fv. myxofaciens’ in the stream, which was not dominated by moderate acidophilic iron-oxidizing detected within the mine itself, possibly because it species closely related to Halothiobacillus neapolita- is an obligate aerobe. nus (Hallberg and Johnson, 2003). Various other mine sites across the world, which Cantareras will not be discussed in detail, have been analysed Similar physico-chemical parameters to the waters for their microbial composition: e.g. metal mines in draining Mynydd Parys was reported in a stream China (e.g. Hao et al., 2010; He et al., 2007), the draining the small abandoned Cantareras copper Königsstein uranium mine in Germany (Seifert et mine in south-west Spain (Table 10.2). Te micro- al., 2008) and the Los Rueldos mercury mine in bial community inhabiting stream water draining Spain (Mendez-Garcia et al., 2014). the mine and the stream sediment was found to be dominated by the psychrophilic iron-oxidizer Acidic streams draining abandoned At. ferrivorans and heterotrophic bacteria (Rowe et mines al., 2007). Te microbial mat-like structures were populated by acidophilic algae (Chlamydomonas Dyfryn Adda acidophila). Even so the mat was dominated by Closely connected to the Mynydd Parys copper iron-oxidizing bacteria, high concentrations of fer- mine microbiology described above is the micro- rous iron could be detected, which is explained by bial colonization of a stream bed, the Dyfryn Adda, the presence of heterotrophic iron-reducing micro- into which AMD draining the abandoned mine organisms (Acidiphilium spp., Acidobacteriaceae) was diverted in 2003. Kay et al. (2013) described whose growth was postulated to be supported by

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organic carbon released by the algae. Another aci- Acidic pit lakes dophilic alga, Chlorella protothecoides var. acidicola, Pit lakes form as a result of opencast mining of coal was isolated from the mat at Cantareras (Nancucheo or metals when the abandoned voids become flled and Johnson, 2012) and had an optimum growth with groundwater. Oxidation of sulfde minerals pH of 2.5 tolerating elevated concentrations of in adjacent rocks or dumps leads to acidifcation various transition metals. Novel acidophilic sulfate- of these lakes and release of sulfate, iron and other reducing bacteria (designated as ‘Desulfobacillus metals (Geller et al., 1998). Due to the high iron acidavidus’) were detected and isolated from the content of pit lakes, ferric minerals are precipi- anaerobic, sulfate-rich botom layer of the Cantar- tated and form sediments which ofen leads to the eras stream (Rowe et al., 2007). Archaeal sequences adsorption of phosphorus and lead to oligotrophic related to the Euryarchaeota could be detected in the conditions developing in most acid pit lakes (Klee- mine water and the streamer growths of Cantareras. berg and Grüneberg, 2005). Acidic pit lakes can be found in mining areas of e.g. China, Australia, Rio Tinto Poland, Spain and Germany, whereby the two later Te Rio Tinto is a long (ca. 100 km) and extremely areas have been studied most intensively. acidic (pH 2.0–2.5), river, which originates from Many pit lakes in Germany resulting from lig- the Peňa de Hierro in south-west Spain and enters nite coal mining have an acidic pH (2.0–4.0) and the Atlantic Ocean at Huelva (Table 10.2). Te are rich in sulfate and iron with minor amounts of microbial communities of the Tinto river have toxic metals (Geller et al., 1998), while concentra- intensively been studied by Amils and colleagues, tions of nutrients such as nitrate, phosphate, and and are described in more detail elsewhere. Te ammonium, are very low. Te maximum depth biogeochemical transformations of iron dominates of the lakes is 11 m and they receive most of their the microbial ecology of the Tinto river, acting as infow by a ditch connecting the lakes and only sec- both electron donor and electron acceptor for che- ondarily from the aquifer. Most of the pit lakes have moautotrophic (L. ferrooxidans, At. ferrooxidans) an anoxic hypolimnion during summer stratifca- and heterotrophic (Acidiphilium spp., Fm. acidiphi- tion and a complete mixture of the water column lum and a moderately acidophilic iron-oxidizer occurs during winter (Blodau et al., 1998). Te related to strain WJ2) acidophiles (Gonzalez-Toril phytoplankton community commonly observed et al., 2003). Bacteria involved in the sulfur redox in these lakes is dominated by the phytofagellates cycle in Rio Tinto include At. ferrooxidans, At. Ochromonas, Chlamydomonas and Gymnodinium thiooxidans and the sulfate-reducing Desulfospor- (Beulker et al., 2003) which is infuenced by the osinus spp.. Macroscopic growth occurring in some availability of inorganic carbon. Microbial oxida- parts of the river are devoid of iron-metabolizing tion of iron occurring in the oxic zones of these acidophiles, predominantly colonized by Alphapro- lakes results in the sedimentation of ferric iron teobacteria (including Sphingomonas-like bacteria) minerals (e.g. schwertmannite, goethite) serving with smaller numbers of Betaproteobacteria, Actino- as electron acceptor for acidophilic iron reducing bacteria and Firmicutes (Lopez-Archilla et al., 2001). bacteria (Acidiphilium spp., Acidobacterium spp.) Archaea related to the genera Ferroplasma and under oxygen-limited conditions. Te pH of the Termoplasma have also been detected in Rio Tinto, lakes increases with depth causing the microbial although they account for only a small proportion communities (including Fe(III)-reducing micro- of the total prokaryotic population. Te most organisms) to be more heterogeneous than those remarkable trait of the Tinto river is, however, the in the surface waters. Te acidic sediments of ‘lake unexpectedly high diversity of eukaryotes detected 77’ in the Lower Lusatia area (Germany) were (Aguilera, 2013). Te algae are the main primary found to be dominated by moderately acidophilic producers in the river and are mainly represented Acidobacterium spp., which were able to catalyse the by diatoms and Euglena mutabilis (Lopez-Archilla reductive dissolution of ferric iron minerals (Blöthe et al., 2001). Further eukaryotes detected in the et al., 2008). Acidithiobacillus spp. and Acidocella Rio Tinto are other photosynthetic algae, protists, spp. were more dominant in the acidic sediments, yeasts and flamentous fungi (Aguilera, 2013). while Bacillus spp. and Alicyclobacillus spp. were isolated from moderate acidic sediments of the

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lake and Acidiphilium spp. could be found in almost leptospirilli, Actinobacteria, and archaeal members all sediment depths (Lu et al., 2010; Blöthe et al., of the Termoplasmata. Te microbial diversity 2008). Acidic conditions in the upper sediments of in the Concepción pit lake was more diverse, but the lakes are stabilized by the cycling of iron which dissolved iron concentrations were low and restricts fermentative and sulfate-reducing activi- therefore less typical AMD microorganisms ties, but as reactive iron decreases and pH increases could be detected. In the lower layers, however, with sediment depth, sulfate-reducers (e.g. Desul- where dissolved iron concentrations increased, fosporosinus spp.) have been detected (Koschorrek, iron-oxidizing ‘Fv. myxofaciens’ and potential iron- 2008). Enhanced sulfate-reduction occurred in the reducing Acidiphilium spp., Acidimicrobaceae and sediments of the acidic mining lake ‘Gruenewalder Acidobacteria were present. Possibly because of the Lauch’ where the sediment was covered by a thick low concentrations of sulfate and transition metals layer of periphytic algae separating the sediment in this lake, members of AMD atypical genera (pH 6) from the acidic water (pH 3.1) and provided (Erwinia, Legionella and Halomonas) were also large amounts of organic carbon as electron donors detected. (Koschorreck et al., 2007). Sulfate-reduction in lake Falagan et al. (2014) studied two other meromic- sediments below pH 4.5 has also been reported for tic stratifed pit lakes in the IPB, Cueva de la Mora other environments (e.g. Koschorreck et al., 2007; and Guadiana, which were claimed to have the most Gyure et al., 1990; Satake, 1977) and is usually dramatic vertical pH gradients and water chemistry related to increased infux of organic mater and the of pit lakes in the region. Cueva de la Mora has three inhibition of competing iron-reducing bacteria and layers, the oxygenated mixolimnion, the chemo- methanogens (Koschorreck, 2008). cline and the more anoxic monimolimnon. Most Pit lakes located in Spain are the consequence interesting was the observed enhanced accumula- of metal mining rather than coal mining, and are tion of algal biomass well below the lake surface, biochemically more complex as they can contain which was thought likely due to the highly variable various transition metals (e.g. Zn) and metalloids bioavailability of phosphorus in these water bodies. in addition to iron and sulfate. Tere are more than Acidophilic micro-algae were reported to be the pri- 25 pit lakes in the Iberian Pyrite Belt (IPB), which mary producers in the lake, providing oxygen and hosts one of the world’s largest accumulations of organic carbon and thereby supporting growth of mine wastes and AMD, including the Rio Tinto. heterotrophic acidophiles (e.g. Acidobacteriaceae), Te lakes in the IPB are characterized by low pH which were dominant in the mixolimnion and the and high concentration of heavy metals, but each chemocline. Te chemocline was quite diverse and lake presents unique characteristics. Te microbiol- populated by iron-oxidizing L. ferrooxidans, iron-/ ogy of two acidic, sulfate- and metal-rich pit lakes in sulfur-oxidizing and facultative iron-reducing At. the IPB, Nuestra Señora del Carmen and Concep- ferrooxidans and sulfate-reducing Desulfomonile ción, was described by Santofmia et al. (2013). In sp. Based on chemical parameters and the various Nuestra Señora del Carmen, a chemically and ther- microbial metabolisms present, the chemocline mally stratifed pit lake consisting of two diferent seems to be the zone where dynamics of carbon, layers, they detected typical AMD microorganisms iron and sulfur redox transformations were most involved in iron redox-cycling. Te upper layer of dynamic. Archaeal sequences detected in the water the lake was dominated by heterotrophic Acidiphi- column and sediments of this lake belonged to lium spp. and accompanied by iron-oxidizing the Eukaryota and few sequences to the recently Leptospirillum spp. and Planctomycetes. Eukaryotic proposed Taumarchaeota, possibly mediating the sequences detected in the upper layer belonged to transformation of nitrogen. the genus Chlamydomonas and flamentous species and diatoms were observed by microscopy. Te Mine water treatment systems lower layer (15 m) of the lake, which exhibited a Mine waters pose serious threats to the environ- slightly reduced environment and equal concentra- mental, owing to their high metal and sulfate loads tions of ferrous and ferric iron, was more diverse and (ofen) their low pH, and therefore require and mainly represented by Acidithiobacillus spp., treatment prior to release in receiving water courses. Acidimicrobiaceae, both facultative iron-reducers, Te ability of bacteria found in acidic environments

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to mediate iron and sulfur redox reactions can be accompanied by an acidophilic organism related to advantageous in mine water treatment to remove the neutrophilic iron-oxidizer Gallionella ferruginea toxic metals and sulfate and raise pH. (Heinzel et al., 2009). Despite being inoculated A pilot passive treatment plant was constructed with more familiar iron-oxidizing acidophiles (At. at the Wheal Jane tin mine in Cornwall (England) ferrooxidans and L. ferrooxidans) at the beginning of to remediate acidic (pH 3.4), metal-rich water the plant operation, ‘Ferrovum’ and the Gallionella- draining the mine utilizing naturally occurring related bacterium prevailed in the plant. microorganisms (Hallberg and Johnson, 2005). Te treatment plant consisted of three separate Mine heaps and waste rock dumps composite systems, comprising a series of aerobic From an industrial viewpoint, sulfdic mine depos- wetlands for iron oxidation and precipitation, a its arrangements can be divided into commercial compost bioreactor for removing chalcophilic heap and dump bioleaching operations, and waste metals and to generate alkalinity, and rock flter rock dumps and tailings which may generate acid ponds for removing soluble manganese and organic mine drainage (AMD). Heap and dump bioleach- carbon. Moderately acidophilic iron-oxidizing ing is used extensively around the world for copper bacteria (related to Halothiobacillus neapolitanus) extraction with an estimated 15% of the world’s and later heterotrophic acidophiles were the domi- copper produced (Brierley and Brierley 2013; nant cultivatable bacteria in the Wheal Jane AMD. Schippers et al. 2014). Heterotrophic acidophiles (Acidiphilium spp., Waste rock originates from mining and com- Acidobacterium-like) and smaller numbers of mod- prises of rocks of diferent size in which the metal erately and extremely acidophilic iron-oxidizing content is too low for an economically feasible ore bacteria (Acidithiobacillus spp., Leptospirillum processing. Tailings are the fne grained remains of spp.) were isolated from the surface waters and ore processing which are dumped as a slurry (Fig. sediments of the constructed aerobic wetlands. 10.1). Both kinds of mine waste ofen contain high Te dominant microbial isolate in waters draining amounts of iron sulfde minerals (pyrite or pyrrho- the anaerobic compost bioreactors was an iron- tite) and ofen produce AMD due to chemical and and sulfur-oxidizing moderate acidophile closely microbial metal sulfde oxidation. Over a period related to Tiomonas intermedia. Acidophiles enu- of several years or decades, an oxidized zone with merated at the Wheal Jane plant only accounted for depleted sulfde content, low pH, and enrichment of up to 25% of the total microbial population. secondary minerals develops above an unoxidized Te Carbondale constructed wetland in Water- zone with unaltered material in tailings dumps (Fig. loo Township (Ohio, USA) was constructed to 10.2; Korehi et al., 2013; Schippers et al., 2010). treat coal mine drainage of more or less constant Te biogeochemical processes in sulfdic waste pH (2.0–3.9), metal and sulfate concentrations. rock dumps and tailings are similar to those in Each wetland cell was layered with mushroom or commercial heap bioleaching operations. At low manure compost followed by a limestone layer. pH < 4, the biological metal sulfde oxidation by Te microbial community of the oxidized surface acidophilic, chemolithoautotrophic iron- and of the wetland was dominated by iron–sulfur oxi- sulfur-oxidizing bacteria and archaea, dominates dizing Acidithiobacillus spp. and accompanied by over chemical oxidation. Tese prokaryotes can iron-oxidizing Actinobacteria and ‘Fv. myxofaciens’ dissolve pyrite very efciently, and the biological (Nicomrat et al., 2008). pyrite oxidation rate has shown to be two orders Another example is an acid mine water treat- of magnitude or greater than the chemical rate. ment plant in the Lusatia area (Germany) operated At pH below 2.5, ferric iron is soluble and serves at pH 3.0 and continuously fed with mine water as a more efcient oxidant for metal sulfdes than pH 4.5 containing an elevated concentration of molecular oxygen. By reacting with a metal sulfde, iron (and sulfate) which is microbially oxidized to ferric iron is being reduced to ferrous, the substrate form schwertmannite. Te iron-oxidizing commu- for Fe(II)-oxidizing acidophiles. In addition, ferric nity in the plant, which proofed to be very stable iron can act as electron acceptor for acidophiles that (Heinzel et al., 2009), was dominated by the iron- oxidize RISCs (or organic carbon) in the absence oxidizing betaproteobacterium ‘Ferrovum’ and ofen of oxygen. Elemental sulfur and RISCs are formed

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Figure 10.1 Depositing sulfdic mine tailings as slurry on top of the tailings dump (ore processing plant in the background).

Figure 10.2 Acid mine drainage releasing sulfdic mine tailings with developed diferent zones due to microbially driven pyrite oxidation (modifed from Korehi et al., 2013).

as intermediates in the metal sulfde oxidation pro- microorganisms, with the consequent release of cess with sulfate as the most oxidized sulfur form adsorbed metals and metalloids (such as As). In (Schippers et al., 2010, 2014; Vera et al., 2013). contrast, if sulfate reduction is occurring, metals Since concentrations of dissolved organic may precipitate as metal sulfdes within the dump carbon are usually very low in mine heaps and (Schippers et al., 2010). dumps, microbial metal sulfde oxidation is the As the main geochemical processes in sulfdic dominant biogeochemical process at low pH and mine dumps and heaps are catalysed by microor- chemolithotrophic, acidophilic iron- and sulfur- ganisms, analysis of microbial communities is vital oxidizing bacteria are ofen the dominant members to understand the scale and nature of these transfor- of the microbial community. In addition, ferric oxy- mations. Microbial analysis helps understand which hydroxides can be dissolved by ferric iron-reducing organisms are responsible for these processes,

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where in a dump or heap a particular process takes facultatively anaerobic acidophiles under anoxic place, where zones of high or low microbial activity conditions (Schippers et al., 2010). occur, and how the kinetics of particular processes, In addition to exploring the microbial diversity e.g. pyrite oxidation or sulfate reduction, can be of sulfdic mine dumps and heaps, the quantifcation infuenced by stimulating or inhibiting microbial of particular organisms, e.g. acidophilic iron- activity. Monitoring microbiology is also useful for oxidizers, in diferent areas and depth layers of a evaluating the success of AMD countermeasures dump is of interest especially for monitoring pur- (Sand et al., 2007) or heap leaching operations poses. Primarily cultivation techniques, i.e. solid (Remonsellez et al., 2009). media and most-probable-number (MPN), have In the Escondida mine in northern Chile, the been used to enumerate prokaryotes involved in extensive industrial copper heap leaching opera- oxidation and reduction processes in sulfdic mine tion has been monitored by analysing 16S rRNA dumps (Schippers et al., 2010). gene copy numbers of acidophiles in the pregnant Far more studies have been carried out for leach solution over time. At. ferrooxidans was the mine tailings than for waste rock dumps. A study most abundant organism during the frst part of by Schippers et al. (2010) compared the numbers the leaching cycle, while the abundance of L. fer- of relevant groups of microorganisms in selected riphilum and Fp. acidiphilum increased with age of waste rock and tailings dumps with diferent metal the heap. At. thiooxidans remained at similar levels contents, pH values and located in eight countries throughout the leaching cycle, and Firmicutes group in diferent climate zones. Te maximum number showed a low and a patchy distribution in the heap. of total cells ranged between 106 and 108 cells per Acidiphilium-like bacteria reached their highest gram waste rock or tailings material, and acidophilic abundance corresponding to the amount of auto- iron- and sulfur-oxidizing bacteria represented a trophs (Remonsellez et al., 2009). signifcant proportion of these. Large numbers Te microbial diversity in sulfdic mine dumps of these organisms were detected in waste rock and heaps comprises aerobic and anaerobic spe- dumps and tailings all over the world. Neutrophilic

cies which are autotrophic (CO2-fxation) or sulfur-oxidizing bacteria oxidize the sulfur com- heterotrophic (Corg as carbon source) as well as pounds formed by chemical sulfde oxidation at lithotrophic (inorganic compounds as energy circumneutral pH. Tese bacteria were detected in

source) or organotrophic (Corg as energy source). most of the cases in similar numbers to acidophilic A comprehensive list of microorganisms on the representatives. Acidophilic heterotrophic bacteria genus level from the three domains of life, Bacteria, can facilitate the continued growth of autotrophic Archaea and Eukarya, which have been detected in iron- and sulfur-oxidizing bacteria, and have also mine dumps or heaps by cultivation and molecular been detected in some cases. In waste rock dumps, biological techniques, is shown in Tables 10.3 and maximum numbers of heterotrophic acidophiles 10.4. Tese lists are based on the results of pub- were in the range of 103–106 cells/g dry sample, and lished papers. Important physiological properties tailings contained lesser numbers. relevant to biogeochemical oxidation and reduc- A quantifcation of the microbial communi- tion processes are given for each genus. ties in four diferent mine tailings was carried out Most microbial genera detected in dumps and by Kock and Schippers (2008) and Korehi et al. heaps belong to the domain Bacteria and comprise (2013) using both cultivation and molecular tech- acidophilic species. Te Archaea are mainly ther- niques. Depth profles of cell numbers showed that mophiles and all of them are acidophiles. Eukarya the compositions of the microbial communities are detected in these environments include algae, fungi, signifcantly diferent at the four sites, and varied yeasts and protozoa. Te iron- and sulfur-oxidizing greatly between zones of oxidized and non-oxidized bacteria or archaea are responsible for metal sulfde tailings. qPCR data indicated that Bacteria domi- oxidation. Tese microorganisms have been widely nated over Archaea and Eukarya and that the genus detected in mine waste tailings located in difer- Acidithiobacillus represented the most abundant ent climate zones. Ferric iron and oxidized sulfur iron- and sulfur-oxidizers detected. Leptospirillum produced by these Bacteria or Archaea may be used and Sulfobacillus were detected in smaller numbers as terminal electron acceptors by obligately and and not in all tailings dumps. Anaerobes such

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Table 10.3 Bacteria detected in mine dumps or heaps and their physiological properties. Genus names are listed in alphabetical order. Genus names in bold means that the genus comprises acidophilic species Oxidation of Reduction of

Genus Pyrite Other MS Fe(II) Sulfur Corg Fe(III) Sulfate

Bacteria Acidimicrobium + + + Acidiphilium + + + Acidisphaera + Acidithiobacillus + + + + + Acidobacterium + Acidovorax + Acinetobacter + Alicyclobacillus + + + + + + Amycolatopsis + Arthrobacter + Bacillus + + Bradyrhizobium + Brevibacterium + Caulobacter + Cellulomonas + Chryseobacterium + Comamonas + Corynebacterium + Desulfobacter + + + Desulfobacterium + + + Desulfobotulus + + Desulfobulbus + + + Desulfococcus + + Desulfonema + + Desulfosarcina + + Desulfosporosinus + + Desulfotomaculum + + + Desulfovibrio + + + Enhydrobacter + Ferribacterium + + Ferrimicrobium + + + + + Ferritrophicum + Gallionella + Gemmatimonas Geobacter + + Hydrogenophaga Hyphomicrobium + Legionella + Leptospirillum + + + Leptothrix + Meiothermus + Methylobacterium +

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Table 10.3 Continued Oxidation of Reduction of

Genus Pyrite Other MS Fe(II) Sulfur Corg Fe(III) Sulfate Methylococcus + Microbacterium + Microvirgula + Nocardiopsis + Ochrobactrum + Paenibacillus + Peredibacter + Pseudomonas + + + Ralstonia + Rubrobacter + Sedimentibacter + Sphingomonas + Subtercola + Sulfobacillus + + + + + + Thiobacillus + + + Thiomonas + + + Thiovirga + +

MS, metal sulfde other than pyrite; sulfur, inorganic sulfur compounds; +, physiological property shown (modifed from Schippers et al., 2010).

Table 10.4 Archaea and Eukarya detected in mine dumps or heaps and their physiological properties. Genus names are listed in alphabetical order. Genus names in bold means that the genus comprises acidophilic species Oxidation of Reduction of

Genus Pyrite Other MS Fe(II) Sulfur Corg Fe(III) Sulfate Archaea Acidianus + + + + + Ferroplasma + + + + Metallosphaera + + + + + Sulfolobus + + + + + Sulfurisphaera + + Thermogymnomonas + Eukarya Amoeba + Cladosporium + Euglena + Eutrepia + Hormidium Penicillium + Rhodotorula + Trichosporon + Ulothrix

MS, metal sulfde other than pyrite; sulfur, inorganic sulfur compounds; +, physiological property shown (modifed from Schippers et al., 2010).

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as sulfate-reducers or the neutrophilic iron- and characteristic of acidic environments. Te micro- manganese-reducing Geobacteraceae were detected bial composition of these growths is determined by in lesser abundance. the physico-chemical parameters of the site, result- Several tailings studies have reported the ing in warm and extremely acidic streams being predominance of iron- and sulfur-oxidizing aci- dominated by iron-oxidizing bacteria (Leptospiril- dophiles at low pH, but microbial communities at lum spp.) and archaea (‘Fp. acidarmanus’), while in the moderately acidic oxidation stage (between higher pH and more moderate mine waters other the initial circumneutral to alkaline pH and the iron-oxidizing bacteria (Acidithiobacillus spp. and strong acidic fnal stage) have only been studied ‘Fv. myxofaciens’) are more abundant. Numbers for few mine tailings sites (Liu et al., 2014; Chen of heterotrophic species are ofen relatively low in et al., 2013). Microbial communities detected acidic environments, as dissolved organic carbon were found to be very diferent to those in tailings levels are usually extremely low. However, extrane- of < pH 3. For example microbial communities ous inputs of dissolved organic carbon and that in lead/zinc mine tailings samples in China with originating from indigenous acidophilic algae in a pH range of 1.8–7.5 revealed a predominance acidic streams and pit lakes promotes the growth of Proteobacteria, including the hydrogen- and of heterotrophic bacteria, which may then the out- sulfur-oxidizing genera Hydrogenophaga, Tiovirga number chemolithotrophic bacteria. Te microbial and Tiobacillus, respectively, in the circumneutral ecology of extremely acidic environments can samples at the initial weathering stage, while gene therefore be both complex and dynamic, and sequences related to the acidophilic, iron-oxidizing provides an exciting area for microbiological and genera Ferroplasma (Euryarchaeota), Acidithiobacil- molecular-based research. lus (Proteobacteria), Leptospirillum (Nitrospira) and Sulfobacillus (Firmicutes) were mainly detected in Acknowledgement samples of lower pH and more intense weathering S. Hedrich was supported by the German Federal and iron precipitations (Chen et al., 2013). Te Ministry of Education and Research (BMBF, Grant fnding that the composition of microbial commu- ID 033RF001). nities in sulfdic mine tailings is mainly controlled by pH was also shown for copper mine tailings References (Liu et al., 2014) and supported by experimental Aguilera, A. (2013). Eukaryotic organisms in extreme acidic evidence (Chen et al., 2014). environments, the Río Tinto case. Life 3, 363–374. Aliaga Goltsman, D.S., Dasari, M., Tomas, B.C., Shah, M.B., VerBerkmoes, N.C., Hetich, R.L., and Banfeld, J.F. (2013). New group in the leptospirillum clade: Conclusions Cultivation-independent community genomics, Acidophiles can thrive in both natural and man- proteomics, and transcriptomics of the new species “Leptospirillum group IV UBA BS”. Appl. Environ. made environments. Te most ancient sites where Microbiol. 79, 5384–5393. acidophilic life has developed over many decades Amaral-Zetler, L.A. (2013). Eukaryotic diversity at pH represent geothermal areas which display high tem- extremes. Front. Microbiol. 3, 441. peratures and are rich in sulfur compounds. Due Arkesteyn, G.J.M.W. (1980). Pyrite oxidation in acid sulphate soils: Te role of microorganisms. Plant Soil 54, to these physico-chemical parameters, such sites 119–134. are mostly colonized by organisms that metabolize Atkinson, T., Cairns, S., Cowan, D.A., Danson, M.J., Hough, reduced sulfur compounds, and archaea dominate D.W., Johnson, D.B., Norris, P.R., Raven, N., Robinson, over bacteria at high temperatures. Mine-impacted C., Robson, R., et al. (2000). A microbiological survey of Montserrat Island hydrothermal biotopes. environments, in contrast, ofen contain more Extremophiles 4, 305–313. soluble iron than reduced sulfur compounds and Baker, B.J., Tyson, G.W., Webb, R.I., Flanagan, J., are therefore predominantly inhabited by microor- Hugenholtz, P., Allen, E.E., and Banfeld, J.F. (2006). ganisms that can catalyse redox transformations of Lineages of acidophilic archaea revealed by community genomic analysis. Science 314, 1933–1935. iron. Microbial growths, occurring as flamentous Baker, B.J., Tyson, G.W., Goosherst, L., and Banfeld, J.F. ‘acid streamers’ – growths in fowing waters, thick (2009). Insights into the diversity of eukaryotes in acid and ofen dense ‘acid mats’ – and ‘pipes’/’snotites’ mine drainage bioflm communities. Appl. Environ. – pendulous growths atached to mine roofs, are Microbiol. 75, 2192–2199.

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