Chapter 7 Extreme Environments

Raina M. Maier

7.1 Low Temperature Environments 7.3.1 The Atacama Desert, Chile 7.5.1 Acid Mine Drainage 7.1.1 McMurdo Dry Valleys, 7.4 Environments Based on Questions and Problems Antarctica Chemoautotrophy References 7.2 High Temperature Environments 7.4.1 Deep-Sea Hydrothermal 7.2.1 Geothermal Hot Springs Vents 7.3 Desiccation and UV Stress 7.5 Acidic Environments

Extreme environments are important to environmental microbiologists because there is much speculation that such Information Box 7.1 Ecosystem Services environments harbor unique with activi- ties that are not only of scientific interest but also that have Natural ecosystems provide resources, or ecosystem serv- commercial potential. The interest in extreme environments ices, that benefit society including provision of clean drink- has engendered support for large research efforts focused on ing water and healthy soils for growing crops, regulation of climate, biogeochemical cycling activities, and diver- such sites. Scientific agencies such as the European Science sity. It has been estimated that ecosystems, underpinned Foundation (ESF) and the U.S. National Science Foundation by microbial activity, provide at least $33 trillion per year (NSF) are supporting long-term projects that study specific in global services (e.g., climate regulation, nutrient cycling, ecological systems that focus on extreme environments. waste treatment, water supply and regulation) (Costanza The purpose of these projects is to help provide knowledge et al., 1997). The World Bank 2006 estimate of the gross world that can be used to conserve, protect, and manage unique product (the value of all final goods and services produced global ecosystems and the biodiversity they sustain. The globally) was $48 trillion, a very similar figure. As a global ecosystem services (Information Box 7.1) they provide and society we have thus far considered ecosystem services as the discovery of natural products and processes that can be free. However, as Earth’s human population continues to harnessed for societal benefit in the areas of , grow and place increasing stress on the environment, it is medicine, and remediation are of immense value. The con- increasingly recognized that we must place a value on eco- cept of ecosystem services is becoming more important as system services that is factored into the gross world product. the world population grows and we place increasing stress on the fragile environments that cycle critical nutrients and sustain our Earth and aquatic environments. change. We have chosen five different extreme environ- Microbial communities in extreme environments have ments to describe here including low and high temperature adapted to amazing levels of stress. These adaptations are environments, an arid and high UV stress environment, of interest for development of remediation approaches for an environment based on chemoautotrophy, and a low pH some contaminated sites including acid mine drainage sites environment. This is by no means an exhaustive list of and radioactive waste sites. They also are of interest for extreme environments but will give an idea of how unique applications of novel adapted to temperature or microbial communities develop in such environments and pH extremes. Finally, they are of interest for understanding how novel discoveries are made through the study of such evolutionary history and possible impacts of future climate environments.

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7.1 LOW TEMPERATURE ENVIRONMENTS in the region. Geochemical analysis of the water column shows the presence of oxygen to a depth of 10 m. There is 7.1.1 McMurdo Dry Valleys, Antarctica a sulfide gradient ranging from 0 at the ice–water interface to Ͼ 1 m M sulfide (S 2 Ϫ ) at the sediment surface ( Fig. 7.2 ). The McMurdo Dry Valleys in Antarctica represent one A complementary sulfate gradient occurs with low con- of the driest and coldest ecosystems known. The average centrations at the sediment surface, building to concentra- mean annual surface air temperature is Ϫ 27.6°C and the tions Ͼ 1.5 m M just below the chemocline (Information average surface soil temperature is Ϫ26.1°C. This ecosys- Box 7.2). These gradients suggested the existence of sul- tem has the only permanently ice-covered lakes on Earth, fur cycling (see Section 14.4). In fact, researchers studying varying in ice-cover thickness from 3 to 5 m ( Fig. 7.1 ). The the site found a diverse community of phototrophic purple permanent ice cover greatly impacts several aspects of nor- ( Karr et al. , 2003 ), sulfur chemoautotrophs, and mal lake characteristics (see Chapter 6): heterotrophic sulfate-reducers. For example, cell numbers ● Reduced wind-driven mixing resulting in vertical of sulfur-oxidizing bacteria were found to peak at 200 cells transport that is reduced to the level of molecular diffusion per ml at a depth of 9.5 m (Sattley and Madigan, 2006). As ● Reduced direct gas exchange between liquid water shown in Figure 7.2, this is precisely where both dissolved and the atmosphere oxygen and sulfide coexist in the water column. Three sul- ● Reduced light penetration fur-oxidizers were cultured from lake water samples, all ● Reduced sediment deposition into the water column most closely related to Thiobacillus thioparus, a known sulfur-oxidizer, but the Lake Fryxell strains were classified The long mixing times mean that some chemical gradi- as psychrotolerant. The isolates were able to grow in tem- ents can exist in the water column for at least 20,000 years peratures as low as Ϫ 2°C with a temperature optimum of before they are dissipated by diffusion. Ecosystem proper- 18°C and an upper limit of 31°C. Sulfate-reducing bacte- ties in the water columns of the lakes are also controlled by ria were also found throughout the water column as deter- the seasonal uncoupling of photoautotrophic and hetero- mined by analysis for the dissimilatory sulfite reductase trophic processes resulting from the unusual solar cycle: gene. Interestingly, several clone groups were highly local- 4 months of darkness followed by 4 months of continuous ized with respect to lake depth, suggesting that there are light with twilight in between (MDV, 2007). depth-specific population niches ( Karr et al. , 2005 ). The McMurdo Dry Valleys are part of the NSF Long In addition to the sulfur gradient, a methane gradient Term Ecological Research network and also serve as an begins at a depth of 12 m and increases to nearly 1 m M at NSF Microbial Observatory. One site that has been studied the sediment surface. This gradient implies the presence of extensively from a microbial perspective is Lake Fryxell, a methane cycle and the presence of both methanogenic a freshwater lake that is one of the most productive lakes and methanotrophic microorganisms ( Karr et al. , 2006 ).

(A) (B)

FIGURE 7.1 (A) Permanently ice-covered Lake Vanda in the McMurdo Dry Valleys, Anarctica. (B) A sediment core taken by the McMurdo Dry Valley Microbial Observatory researchers from Lake Vanda. Photos courtesy (A), Vladamir Samarkin (B) A. Chiuchiolo.

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In fact, of 13 archaeal clones found in the site, the majority stopped quite easily by simply raising the temperature. Low represent and anoxic methanotrophs. temperature enzymes that have been examined or used in What is interesting about cold-adapted microorganisms? industry include -amylase (breadmaking, textiles, brewing, Their ability to survive and grow in the cold requires spe- and detergents), cellulase (textiles and the pulp and paper cialized adaptations that have the potential to be harnessed industries), -galactosidase (removes lactose from milk), for the benefit of society. For example, these microorgan- lipase (detergents and flavorings), (detergents and isms synthesize cold-adapted enzymes which have had to meat tenderizers), and xylanase (breadmaking) (Cavicchioli evolve specific structural features that make them highly et al., 2002). flexible in comparison to their warm temperature equiva- lents (Siddiqui and Cavicchioli, 2006 ). This flexibility, particularly around the active site of the (the site where the enzyme interacts with its substrate) means that the enzyme can operate efficiently at low temperatures. Information Box 7.2 Chemocline in Lake Fryxell This also means that at high temperatures the enzyme becomes unstable. In fact, it is these two properties of cold- The chemocline is defined as the interface between two dif- active enzymes that makes them suitable for biotechnologi- ferent chemistries in a body of water. In Lake Fryxell, the chemocline is the interface between oxygen-rich and anoxic cal application: their high activity at low temperature and water ( Fig. 7.2 ). In general, both nutrients and bacteria tend their low stability at elevated temperatures. The ability to to accumulate at a chemocline or a thermocline (see Section carry out a reaction at low temperature can have several 6.3.1.3 and Fig. 6.10). Indeed, in Lake Fryxell, the chemocline advantages. Sometimes, low temperature is more suitable harbors the largest numbers of sulfur-oxidizing bacteria and for labile reactants; however, reactions carried out at low also harbors photosynthetic purple sulfur bacteria that con- temperature also require less energy input, which is a direct sume the hydrogen sulfide, completing a sulfur minicycle in cost savings. The fact that these enzymes have low stabil- the chemocline. ity at elevated temperatures means that the reaction can be

FIGURE 7.2 Diagram showing relevant 102 cells/ml physicochemical parameters related to the 0 0.5 1.0 1.5 2.0 sulfur cycle in Lake Fryxell including cell 0 numbers of sulfur-oxidizing bacteria based on most probable number (see Section 10.1.3) analyses. The expanded scale (inset) 2 shows concentrations of sulfide and dissolved oxygen at the chemocline. Diagram from 4 Sattley and Madigan, 2006. Reproduced with permission from the American Society for Microbiology Journals Department. 6

8

Depth (m) 10

12

14

16

18 0 0.25 0.5 0.75 1.0 1.25 1.5 1.75

O2, H2S (mM) 0 12 Temp (°C)

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7.2 HIGH TEMPERATURE ENVIRONMENTS large amounts of sulfur that are fed into hot springs by geo- thermal fluids. One study site is called Dragon Spring. The 7.2.1 Geothermal Hot Springs source waters for Dragon Spring are acidic (pH 3.1 at the source), range in temperature from 66 to 73°C, and contain Yellowstone National Park, United States, has over 10,000 up to 80 μ M of dissolved organic carbon. As these fluids unique geothermal features, which contain a wide and var- reach the surface of the springs, the elemental sulfur in the ied range of temperature, pH, and geochemical profiles incoming geothermal fluids forms flocs that are a distinc- ( Fig. 7.3 ). This site has been a focus of research interest tive feature of the entire outflow channel of the spring ( Fig. since it was realized that the thermostable DNA 7.3 ). This flocculent is the basis for a sulfur-cycling micro- enzyme from aquaticus , which was isolated from bial community. Although the flocculent is formed abioti- Yellowstone, could be used for the polymerase chain reaction cally, it is quickly colonized by two groups of organisms. (PCR) (Saiki et al., 1988) (Section 13.4). Thermus aquaticus The first group is composed of two sulfur-respir- is an extreme able to grow between 40 and 79°C ing archaeal populations, Caldisphaera draconis and with an optimum temperature of 70°C ( Brock and Freeze, Acidilobus sulfurireducens. Quantitative PCR analysis 1969). Hot springs, such as those found in Yellowstone, have (Section 13.4.8) shows that these two populations rep- temperatures of up to 100°C. Similarly, deep-sea hydrother- resent a major portion, 17 to 37%, of the floc-associated mal vents can harbor (in fact, one of the DNA (Boyd et al., 2007) (Information Box 7.3). These record holders for high temperature tolerance is a deep-sea isolates face an intriguing problem as sulfur-reducers. It vent organism, , an that can tol- is speculated that these microorganisms do not reduce ele- erate up to 113°C). Genera commonly found in these envi- mental sulfur (S0 ) as it exists in the flocculent but rather ronments include Thermus, Methanobacterium, Sulfolobus, reduce sulfur from polysulfide ( Ϫ S-S-S-S-S-S-S-S Ϫ ). Pyrodictium, and Pyrococcus . Interestingly, polysulfide is not stable at the acidic pH con- The study of the thermophilic microorganisms in ditions found in Dragon Spring, and it disproportionates Yellowstone hot springs has been the subject of a NSF almost completely into S2 Ϫ and S 0 (Rickard and Morse, Microbial Observatory. In this case, researchers have 2007). So these microbes have some, as yet undescribed, examined the microbial community associated with the mechanism to obtain polysulfide.

FIGURE 7.3 The source of Dragon Spring in the Hundred Springs Plain of Norris Basin, Yellowstone National Park. The whitish-yellow material is flocculent elemental sulfur that becomes quickly colonized by sulfur-respiring Crenarchaea in the domain of Archaea and hydrogen sulfide-oxidizing bacteria in the genus Hydrogenobaculum . Photo courtesy Gill G. Geesey, Montana State University.

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ether linkage OϪ Information Box 7.3 Isolation of Novel Microorganisms branched isoprene chains Ϫ H2C OPO O Analysis of DNA sequences from the environment can reveal C O CH the existence of novel and potentially intriguing microorgan- OC CH2 isms. But to actually study the physiology of these microbes, L-glycerol they must be cultured. Culturing an unknown microorgan- ism is a major challenge and has been approached in two O D-glycerol ways. First, by sequencing the 16S rRNA gene of a novel C O CH2 microbe, one can phylogenetically analyze the organism in C OCH OϪ relation to its nearest neighbors. This can provide clues to O OPOϪ possible metabolic preferences, which can be tested by con- unbranched fatty acid H2C structing specialized culture media. The second approach ester linkage OϪ was used by researchers at Dragon Spring in Yellowstone FIGURE 7.4 A comparison of membrane phospholipids from archaea National Park (Boyd et al., 2007). In this case the 16S rRNA (top) and bacteria (bottom). Note that in the archaea the lipids are linked genes of the two novel sulfur-respiring microbes recovered to glycerol through an ether linkage whereas for bacteria an ester link- from Dragon Spring did not closely match any known sulfur- age is used. Further, archaeal lipids are based on a repeating 5-carbon iso- reducers. Therefore, the scientists analyzed the geochemis- prene unit, usually 20 carbons in length. Bacterial lipids are straight chain try of Dragon Spring and mimicked these conditions in the fatty acids of 16 to 18 carbons in length. laboratory. Using this approach they successful cultured the archaeal sulfur-reducers Caldisphaera draconis and Acidilobus sulfurireducens . Figure 7.4. The archaeal membrane is more thermostable than bacterial membranes (although it should be noted that the membrane structure is the same whether or not an The second group that colonizes the flocculent is com- archaea is thermophilic). Finally, in terms of nucleic acids, posed largely of chemoautotrophic sulfide-oxidizing bac- all hyperthermophiles produce a unique enzyme called teria from the genus Hydrogenobaculum . This thermophile DNA gyrase. This gyrase acts to induce positive supercoils oxidizes the sulfide produced by the sulfur-respiring archaea in DNA, theoretically providing considerable heat stability back to elemental sulfur (S 0). Thus, a very dynamic and ( Kikuchi and Asai, 1984 ; Bouthier de la Tour et al. , 1990). tightly coupled sulfur cycle occurs at the very source of What has been learned about microbial adaptation Dragon Spring. Interestingly, as the water flows away from to life as an extreme thermophile? It was reported that the spring over a distance of a few meters, there are dras- HB27, which was isolated from a tic changes in the biogeochemistry of the system as pH hot spring in Japan, has a DNA translocator system that increases, temperature decreases, oxygen is introduced, and allows it to take up DNA very broadly from various mem- as iron and arsenic become important in the energy flow in bers of all three domains of life, Bacteria, Archaea , and the system ( Inskeep and McDermott, 2005 ). Eucarya ( Schwarzenlander and Averhoff, 2006 ). Further, Many mechanisms allow microorganisms to survive this translocator system exhibits extremely high rates of at temperatures that would normally denature proteins, DNA uptake. In fact, this organism has a DNA uptake cell membranes, and even genetic material (Bouzas et al. , velocity of 40 kilobase pairs per second (i.e., 40,000 base 2006 ). The key to enzyme function, whether in cold or hot pairs). This can be compared to some known mesophilic environments, is the maintenance of an appropriate balance organisms, which have uptake velocities that are 2.5 between molecular stability and structural flexibility. One to 10 times lower, including (4 kbp/s), general adaptive mechanism exhibited by thermophilic Haemophilus influenzae (16 kbp/s), and Streptococcus microorganisms is the production of chaperonins, which pneumoniae (4 kbp/s). The authors suggested that the DNA are specialized thermostable proteins that help refold and translocator system has been of benefit to this organism by restore other proteins to their functional form follow- allowing it to adapt quickly to life in an extreme environ- ing thermal denaturation. In addition there are microbe- ment. Moreover, this DNA translocator might be one of specific adaptations to increase protein stability at high the most powerful tools for interdomain transfer of ther- temperature including these: mophilic and physiological traits between microorganisms thriving in extreme environments. ● An increased number of disulfide bridges What do have to offer biotechnology? ● Increased interactions among aromatic peptides There are numerous biotechnological applications for ● Increased hydrogen bonding among peptides enzymes isolated from thermophilic microorganisms and In terms of cell membranes, most hyperthermophiles the number of applications is growing rapidly, especially in belong to the Archaea . The archaeal cell membrane differs commercial industry. The prime example is the thermosta- in structure from the bacterial cell membrane as shown in ble DNA polymerase used in PCR. Other examples include

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proteases, lipases, amylases, and xylanases that are used in organic carbon in temperate soils ranges from 1 to 5% (see the agricultural, paper, pharmaceutical, water purification, Section 4.2.2.6). bioremediation, mining, and petroleum recovery industries. A commonly studied feature in arid environments is the lithic microbial communities, those that inhabit rock surfaces and subsurface rock pores. These communities are dominated by photoautotrophic nitrogen-fixing cya- 7.3 DESICCATION AND UV STRESS nobacteria and are capable of colonizing a diverse group 7.3.1 The Atacama Desert, Chile of minerals including dolomite, granite, gypsum, halite, limestone, quartz, and sandstone. These communities have The deserts of the world represent both hot and cold semi- been found in a range of hot and cold deserts including the and hyperarid environments where extreme conditions Atacama, the Mohave, the al-Jafr Basin (Jordan), the des- severely limit primary productivity and thus the diversity erts of northwestern China, and the McMurdo Dry Valleys of life. Factors limiting microbial life in the arid deserts of Antarctica ( Fig. 7.6 ). These hypolithic (inhabit rock sur- include water availability, temperature, and the intensity faces) and endolithic (inhabit pore spaces within the rocks) of UV radiation. Arid deserts are characterized by mean communities are believed to exploit the protection offered annual rainfall levels of 25–200 mm while hyperarid des- by rock surfaces that scatter UV radiation and presum- erts have mean annual rainfall of Ͻ 25 mm. Water avail- ably trap limited water supplies (Dong et al. , 2007 ). The ability in a desert is determined not only by mean annual dominant photoautotroph found in the majority of these rainfall, but also by the combined effects of precipitation communities is the desiccation and radiation tolerant cya- (P) and potential evapotranspiration (PET). Hyperarid nobacterium . areas are defined as those with a P/PET ratio less than 0.05 The desiccation and UV tolerance of has ( Houston and Hartley, 2003 ). been examined (Potts, 1999). For example, Nostoc sp. can One of the driest deserts on Earth is the Atacama Desert, remain in a state of desiccation for months or years at a Chile, where the hyperarid core of the desert experiences time. This has been measured at a level of 2–5% cell water intervals of years to decades with no rain (Betancourt content, which is one order of magnitude lower than that et al., 2000). Due to the lack of available moisture, plants of eubacterial spores (Gao and Ye, 2007)! Adaptation to are sparse or completely absent, creating soil conditions with desiccation is unique among the extremes experienced by extremely low soil organic carbon and nitrogen levels, further bacteria (i.e., temperature, pH, and pressure) because the limiting the potential diversity of microbial life (Fig. 7.5). cells do not grow while desiccated and the greater por- Examples of total organic carbon levels in a range of des- tion of their viable lifetime may be spent in the dehydrated ert soil samples include 0.7% in the Mojave (Eureka Valley, state. Thus, cycles of desiccation appear to induce survival California), 0.17% in the Sahara (near Abu Simbel, Egypt) strategies for the cells rather than the ability to function ( Lester et al. , 2007 ), and 0.02–0.09% in samples taken along under extreme conditions. The survival strategies identified an elevational transect through the hyperarid core region of include the following: the Atacama Desert (Drees et al. , 2006 ). For comparison, ● The ability to protect and repair DNA exposed to UV radiation ● Maintenance of protein stability in the dehydrated state ● Maintenance of membrane integrity The primary adaptative mechanism of the cyanobacteria is the production of an extracellular polysaccharide (EPS) sheath. This sheath regulates the uptake and loss of water, serves as a matrix for immobilization of cellular components produced by the cell in response to desiccation, and may pro- tect cell walls during shrinking and swelling ( Potts, 1999 ). Several molecules are produced by the cell in response to desiccation and UV exposure. These are often found in the EPS sheath and include UV absorbing compounds such as mycosporine-like amino acids and scytonemin, carotenoids and detoxifying enzymes or radical quenchers that provide protection from harmful radicals and oxygen species, and water stress proteins ( Gao and Ye, 2007 ). Water stress pro- FIGURE 7.5 The hyperarid core region of the Atacama Desert, Chile. teins are extremely stable and have been found to comprise The photo was taken at 974 m elevation (24.073°S, 70.204°W) southeast up to 70% of the soluble proteins in environmental samples of Antofagasta. Courtesy of Julia W. Neilson. of Nostoc commune. In addition, N. commune cells contain

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FIGURE 7.6 Samples of soil gyp- sum with cyanobacteria: (A) Soil gypsic crust sample AT326b from the Atacama Desert; (B) soil gypsic crust sample DG from the Mojave Desert; (C) fibrous gypsum sample JB1 from a secondary vein exposed by erosion at the surface from al-Jafr Basin, Jordan; (D) light micrograph of cyanobacte- rium Chroococcidiopsis in an enrich- ment culture from sample AT326b from the Atacama Desert. From Dong et al. , 2007. Reproduced with permis- sion of AUG.

trehalose and sucrose, which have the ability to stabilize pro- hypolithic cyanobacteria declined from 28 to Ͻ 0.1%, from teins and protect the integrity of the membrane during desic- the less arid to the most hyperarid region, reinforcing the cation. Electron microscopy indicates that the nucleoplasm conclusion that the absence of available water is the limit- of N. commune cells requires five days of rehydration before ing factor to microbial life in the extreme hyperarid deserts. appearing similar to fully hydrated cells. In addition, a very What knowledge can be gained from the study of organ- ordered recovery of gene expression has been documented isms capable of survival in extreme hyperarid regions? during rehydration beginning with respiration, followed by First, an understanding of where life can occur in the , and finally nitrogen fixation. Although des- hyperarid regions on Earth helps to narrow and focus the iccation and UV tolerance is widespread among cyanobac- search for life beyond our planet. For example, it has been teria, the interactions are complex and considerable work is suggested that life would have been forced into endolithic needed to fully understand the mechanisms involved. habitats on Mars as liquid water slowly disappeared from Due to the presence of these photoautotrophic nitro- the planet. Second, research suggests that expanded knowl- gen-fixing primary producers, the lithic communities in edge of microbial diversity in hyperarid regions could be arid environments are not dependent on exogenous carbon used to evaluate precipitation history. For example, a study or nitrogen supplies. This has allowed the investigation of of soil bacterial diversity in the Atacama Desert was per- other physical factors that may limit microbial life in hyper- formed along a west/east elevational transect (400–4500 m) arid environments. Two studies have examined such lithic through the driest region of the core absolute desert ( Drees systems to determine the physical factors most limiting to et al., 2006). Mean annual temperature and precipita- life in hyperarid regions. In the first study, the microbial tion along the transect ranges from 17 to 7°C and 0.6 to diversity was characterized in hypolithic crusts on quartz 35.7 mm, respectively. In 2002, soil samples were col- substrates from three hyperarid desert locations in north- lected at a depth of 25 cm to obtain bacteria unaffected western China (Turpan Depression, Taklimakan Desert, by surface radiation or eolian (windborne) dispersion. and Qaidam Basin). Regression analysis revealed a posi- Community DNA was extracted from the samples, ampli- tive correlation between the availability of liquid water and fied by PCR using universal bacterial 16S rDNA prim- two diversity indices: species richness (R 2 ϭ 0.738) and ers, and the diversity analyzed by denaturing gradient gel the Shannon diversity index ( R 2 ϭ 0.650). The availability electrophoresis (DGGE) (see Sections 13.4 and 13.7). The of liquid water was calculated from the interaction of tem- results revealed the presence of two distinct microbial com- perature and rainfall (Pointing et al., 2007). A similar study munity types, one found in samples taken from the central was conducted by Warren-Rhodes et al . (2006) in which hyperarid core and the other occurring in samples taken translucent quartz and quartzite stones were collected from at elevations above and below the core region (Fig. 7.7). four locations along an aridity gradient in the Atacama The two microbial community groups did not correlate Desert where rainfall declined from 21 to Յ 2 mm per year. exactly with any obvious factors such as temperature, The percentage of rocks collected that were colonized by precipitation, total organic carbon, or percent plant cover

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gradients. Further analysis of the sample sites in the core events at higher elevations. In contrast, the sample sites in the absolute desert group shows no evidence of either historic Andean group either are presently sparsely vegetated or have precipitation events or runoff from occasional precipitation evidence of historic vegetation or runoff events. A second sampling two years later, in 2004, indicated that the observed 0.15 2510 microbial communities were stable over time. These results 0.10 Core Absolute Desert Group 400 suggest that microbial diversity patterns in hyperarid envi- Andean Vegetation Group 2792 ronments can reveal information concerning the frequency of 0.05 3107 4270 precipitation events or even historical climatic variations. 4500 0.00 3593 3900 Ϫ0.05 7.4 ENVIRONMENTS BASED ON CHEMOAUTOTROPHY Ϫ0.10 987 1315 Ϫ0.15 1931 7.4.1 Deep-Sea Hydrothermal Vents 703 Dimension 2 (1.04% of variance) Ϫ0.20 In 1977 geologists first described deep-sea hydrothermal Ϫ1.5 Ϫ1.0 0.5 0.0 0.5 1.0 vents ( Fig. 7.8 ). These are areas on the ocean floor where, Dimension 1 (98.82% of variance) driven by magma-derived hydrothermal convection, hot FIGURE 7.7 Kruskal’s multidimensional scaling analysis of the DGGE water laced with minerals flows up through cracks and fis- profiles from the Atacama Desert elevational transect analyzed in three sures. The cracks, which are known as hydrothermal vents, dimensions with 99.5% of the variance explained. Data labels represent often have a buildup of chemical precipitates that resemble sample elevations (meters) along the transect. The microbial communities chimneys surrounding them ( Fig. 7.9 ). Water, reaching in samples along the transect clustered into one of two groups: the core absolute desert group and the Andean group. From Drees et al. , 2006 . temperatures of up to 400°C, is emitted from these vents at Reproduced with permission from the American Society for Microbiology rates of 1 to 5 m/s. In addition, most vent fluids are anoxic, Journals Department. highly reduced, acidic (pH 2–4), and enriched in CO 2 ,

Water temperature (2°C)

2ϩ FeS, Mn + O2 FeO(OH), MnO2

Water temperature (3.5°C) Basalt Basalt

Precipitation chimney (350°C)

Reaction zone (400oC)

Ϫ HCO3 CO2,CH4 2ϩ Fe 2ϩ Ca2ϩ Mn Zn2ϩ ° H2 ϩ H2S Magma (1200 C) Cu2

FIGURE 7.8 Schematic representation of a depicting a black smoker rising from the ocean floor creating a plume of chemical-rich superheated water around it.

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2 ϩ 2 ϩ 2 ϩ 2 ϩ H2 S, CH4 , H2 , Fe , Zn , Cu , and other transition (acceptors); Fe (donor) with O2 (acceptor). In addition, metals. As the hot, mineral-rich, hydrothermal water the presence of CH 4 in vent fluids can support heterotro- emerges from the vent, it quickly mixes with cold seawater phic growth of methanotrophic microorganisms in the pres- 2 Ϫ and forms a dark cloud of mineral precipitates. The appear- ence of either O 2 or SO4 as terminal electron acceptors. ance of this dark cloud has given the name “ black smok- The vent communities support macrofauna that rely on ers ” to these vent chimneys. It was surprising to find whole the chemoautotrophic bacterial populations as a source of self-contained ecosystems consisting of microscopic and organic carbon. There are at least three major mechanisms macroscopic life in this environment, which has no light for transfer of this bacterial carbon and energy to the next and extremely high temperature and pressure. trophic level. The first is an endosymbiotic relationship It is the confluence of the superheated hydrothermal between vent bacteria and an invertebrate, Riftia pachyp- vent water, which contains reduced minerals that can act as tila, which has been dubbed “ tube worm” (Markert et al. , electron donors, and the oxidized seawater, which contains 2007 ). are large tube-shaped creatures a variety of electron acceptors, that serves as the basis for a that grow from the seafloor (Fig. 7.10). These worms have chemoautotrophic community of microorganisms that sus- no mouth, gut, or any other digestive system and depend tains the entire heterotrophic component of the vent com- completely on bacteria for their nutrition. Instead of con- munity ranging from microorganisms to animals (Fisher suming the bacteria, the worms have interior surfaces that et al. , 2007 ). Thus, the entire food web in a hydrothermal are colonized by massive quantities (3ϫ 1 0 11 bacteria per vent community is based on chemoautotrophy, not photo- ounce of tissue) of sulfur-oxidizing chemoautotrophs autotrophy as in surface environments (see Section 14.2.2). (Karl, 1995) (see Section 14.4.3.1). Chemoautotrophy is Examples of electron donor/acceptor pairs supporting sustained by the presence of H2 S originating from vent flu- autotrophy that have been identified in hydrothermal vents ids and oxygen in the seawater. The worm’s body is filled 3 ϩ Ϫ 2 Ϫ 0 include: H2 (donor) with O2 , Fe , NO3 , CO2 , SO4 , S with blood containing large amounts of hemoglobin that 0 2 Ϫ Ϫ (acceptors); H2 S, S , S2 O3 (donors) with O 2 , NO3 binds H2 S. The blood transports the H 2S to the bacteria, which oxidize it and fix CO2 into organic compounds that nourish the worm. The symbionts benefit from high nutri- ent concentrations within the worm’s body, which results in high microbial metabolic activity. In turn, the microbi- ally fixed organic carbon is transferred to the host, making R. pachyptila one of the fastest growing marine inverte- brates that has been studied ( Markert et al. , 2007 ). The second mechanism by which microbially produced carbon and energy is transferred to the next trophic level method is termed microbial gardening. In this case, bac- terial cultures are maintained by mussels and other inver- tebrates on specialized appendages such as tentacles and gills. These invertebrates periodically harvest and consume

FIGURE 7.9 Photograph of a black smoker vent, which was first pub- lished on the cover of Science magazine. The vent water is exploding out FIGURE 7.10 An adult Riftia pachyptila tube worm community in situ . of the vent at 1–5 m/s and is 380°C. From Spiess et al., 1980. Reprinted Note the clams surrounding one of the worms in the community. Photo with permission from AAAS. courtesy Andrea D. Nussbaumer, Charles R. Fisher, and Monika Bright.

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the bacteria, retaining small inocula to initiate the next community (Handelsman et al., 1998). At the time, the crop. The third mechanism for carbon transfer to higher focus of metagenomic analysis was on the discovery of trophic levels is direct consumption of free-living bacterial new natural products and biochemical pathways. However, cells, filaments, or mats. Crabs, amphipods, predatory fish, the metagenome can also be explored to better understand and even other microorganisms, including bacteria, have ecological and evolutionary processes that drive com- been observed to feed directly on the chemoautotrophic or munity development (Allen and Banfield, 2005). This chemoheterotrophic primary producers ( Karl, 1995 ). approach was used to study a subsurface AMD community What new insights are hydrothermal vent communi- from the Richmond Mine Site, Iron Mountain, California ties offering to environmental microbiologists? It has ( Tyson et al., 2004). Like the hydrothermal vent commu- been hypothesized, through comparison of genomic DNA nity discussed in Section 7.4, the AMD community taken of vent bacteria and closely related pathogenic bacteria, beneath the surface of this site is self-sustaining and based that there are evolutionary links between the chemolitho- on chemoautotrophy rather than photoautotrophy. The pH autotrophic vent bacteria (which operate as symbionts to and temperature of the biofilm sample taken from the site vent animals) and important closely related human patho- were 0.83 and 42°C, respectively. Solution concentrations gens ( Nakagawa et al. , 2007 ). In this study the genomes of metals in the AMD were 317 m M Fe, 14 m M Zn, 4 m M of two deep-sea vent  -Proteobacteria strains, Sulfurovum Cu, and 2 m M As. sp. NBC37-1 and Nitratiruptor sp. SB155-2, were com- In the metagenomic analysis, a total of 76.2 million pared to their pathogenic relatives, Helicobacter and base pairs of usable DNA sequence were obtained. These Campylobacter (Chapter 22). Although they are not patho- sequences were reconstructed to give nearly complete genic, the two deep-sea vent bacteria share many virulence genomes for two iron-oxidizing bacteria, Leptospirillum genes with the pathogens. These traits provide an ecologi- group II and Ferroplasma type II, as well as partial recov- cal advantage for the hydrothermal vent bacteria which ery of three other genomes. Fluorescent in situ hybrid- need to form symbiotic relationships with vent animals. In ization (Section 9.4.3) analysis of an AMD sample their pathogenic relatives, however, these traits are used for confirmed the presence of these organisms and suggests efficient colonization and persistent infection of the host. that the Leptospirillum group II is dominant (75%), with the Ferroplasma type II representing approximately 10% of the community ( Figs. 7.11 and 7.12 ). What did the researchers learn in terms of evolution and 7.5 ACIDIC ENVIRONMENTS ecology at this site? In terms of evolution, analysis of the Leptospirillum 7.5.1 Acid Mine Drainage group II genome indicated very few nucleo- tide polymorphisms (changes in DNA sequence), implying Although there are naturally occurring acidic environments that only a single strain of this isolate dominates the com- such as Dragon Spring discussed in Section 7.2, acid mine munity (Tyson et al., 2004). In contrast, for Ferroplasma drainage is an excellent example of an anthropogenically type II, the researchers observed between one and three caused extreme environment. Mineral mining usually distinct patterns of nucleotide polymorphisms in the assem- focuses on pyrite (FeS2 ) deposits that contain metals such bled genome. These data suggest that Ferroplasma type II as silver (Au), gold (Ag), copper (Cu), zinc (Zn), and lead has undergone evolutionarily recent homologous recombi- (Pb), usually as impurities in the pyrite ore. Alternatively, nation resulting in three distinct strains of the organism. these metals are part of the sulfide minerals such as chal- In terms of ecology, analysis of the genomes of the copyrite (CuFeS2 ), chalcocite (CuS), sphalerite (ZnS), and two microbes allowed these scientists intriguing insights galena (PbS). In either case, mining exposes these sulfide into how each bacterium processes carbon, fixes nitrogen, minerals to both air and water, resulting in biologically and generates energy. For example, several CO2 fixation mediated acid generation (Section 14.4.3). In sites where pathways were identified for Leptospirillum group II, indi- excess water is generated, acidic leachates form (pH Ͻ 2 ) cating that it is definitely chemoautotrophic. In contrast, which are sometimes also high in toxic metals (Fig. 15.5). although the Ferroplasma type II has a mechanism to fix These leachates are called acid mine drainage (AMD). CO2 , it also contains a large number of sugar and amino Despite extremes of acidity, heat, and high concentrations acid transporters, suggesting that it may prefer to metabo- of sulfate and toxic metals, a range of specialized microor- lize heterotrophically. Both genomes were also examined ganisms populate AMD environments. for nitrogen fixation genes, which were found to be absent. Given the difficulty of culturing the majority of envi- However, such genes were found in one of the partially ronmental microorganisms (and especially those found in sequenced genomes for Leptospirillum group III, indicat- extreme environments), there has been great interest in the ing an important role for this organism in the AMD biofilm study of the community based on its DNA (Section 13.5.2). system although it represents only about 10% of the com- The term metagenome was introduced in 1998 to describe munity as analyzed by fluorescence in situ hybridization the entire DNA that is represented by the microbial ( Fig. 7.12 ). The researchers note that the metagenomic

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FIGURE 7.11 Fluorescent in situ hybridization (FISH) image of the AMD biofilm taken from a subsurface site at the Richmond Mine at Iron Mountain, California. Three different FISH probes were used. The first was specific for bacteria and used a fluorescein isothiocyanate probe, which fluoresces green. The second probe was specific for archaea and used the Cy5 fluor, which appears blue. This probe detected the Ferroplasma . The third probe was one that targeted the Leptospirillum genus and used the Cy3 fluor, appearing red. Note that overlap of red and green appears yellow and indicates Leptospirillum cells. The dominance of yellow in this image shows the dominance of Leptospirillum . From Tyson et al. , 2004 .

Eukaryotes 4% Sulfobacillus spp. 1% QUESTIONS AND PROBLEMS

Archaea 10% 1. Define the term ecosystem services. Leptospirillum 2. Give two examples of extreme environments not gp lll 10% discussed in this chapter. 3. Which of the extreme environments discussed in this chapter likely has the slowest growth rates? 4. Of the five extreme environments discussed Leptospirillum in this chapter, which two are based on gp ll 75% chemoautotrophy? FIGURE 7.12 Relative microbial abundances in AMD determined 5. If you had an unlimited research budget, what using quantitative FISH counts. From Tyson et al., 2004 . type of extreme environment would you choose to study? analysis of the AMD site was successful in part because 6. Discuss adapations for enzymes at low and high the diversity in the biofilm was low and the frequency of temperatures. genomic rearrangements and gene insertions/deletions was 7. Compare and contrast a thermocline (Chapter 6) and a relatively low (allowing reconstruction of each genome). chemocline.

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