gn133 Featured Article:
Without Sicily, Italy leaves no image in the soul.Sicily is the key to everything. - Johann Wolfgang von Goethe
Harold C. (Hal) Helgeson was, until the day he died, a practicing theoretical geochemist. While he truly cherished the results from an elegant computation, he also appreciated the messy reality of chaotic field data and the need for carefully crafted laboratory experiments. Helgeson was an ardent supporter of the notion put forth in 1943 by the microbial ecologist Claude ZoBell of “bacteria as geologic agents” (ZOBELL, 1943). This respect for the outcrop (where Helgeson claimed he could see the chemical potential axis coming at him) and an infatuation with the geo-bio interface conspired in 1991 to beckon Helgeson (with me in tow) to the hydrothermal system of Vulcano Island (Italy). During the 1980s, another microbial ecologist - Karl Stetter from the University of Regensburg in Germany - cultured many heat-loving microorganisms from Vulcano, discovering some of the highest temperature archaea and bacteria in the process. For decades, a cadre of geochemists had monitored and modeled the volcanic activity in southern Italy, especially at Mt. Vesuvius, Mt. Etna, and the Aeolian Islands. These predominantly Italian geochemists included Marco Leone, Marcello Carapezza, Mariano Valenza, Mario Nuccio, and Sergio Gurrieri, and they were close personal friends to Helgeson from an ‘earlier life’.
The geochemists, however, were unaware of the thermophilic microorganisms in their volcanic back yard, and the microbiologists were equally unaware of the geochemistry that defined the thermophile habitats. Helgeson tried to change all that. He introduced his Sicilian friends to the literature of Stetter, John Baross, Wolfram Zillig, and other thermophile enthusiasts; and he visited Stetter in Bavaria to lecture him on the importance of geochemistry in microbiology - that meeting did not go well - Helgeson told anyone who would listen that the best, and perhaps only, way to really understand these extremophilic organisms in their natural environments, required an integration of quantitative geochemical methods with the myriad molecular methods that were revolutionizing (geo)biology. And for reasons scientific, pragmatic, historical, geographic, and culinary, Vulcano was the site of choice. What follows here is an introduction for some, an update for others, and perhaps a lesson for all of Vulcano’s unique place in the geobiology and geochemistry of hot life.
Vulcano is the southern-most of the seven islands that, together with several seamounts, form the ~200 km long Aeolian arc in the Tyrrhenian Sea north of Sicily. This arc and associated back-arc basin are part of a complex that includes the subducting African plate under the southern part of the Italian peninsula (BARBERI et al., 1973; BARBERI et al., 1974; DE ASTIS et al., 1997; ELLAM et al., 1989; KELLER, 1974). Structurally, Vulcano and the islands of Lipari and Salina form a ridge inside a roughly NW-SE trending graben that dissects the volcanic arc (BARBERI et al., 1994). This ridge and graben system is part of the Tindari-Letojanni right-lateral strike-slip fault system that dominates the regional tectonics in northern Sicily (MAZZUOLI et al., 1995; VENTURA et al., 1999). Along the entire Aeolian arc, shallow-sea hydrothermal sources are ubiquitous (GUGLIANDOLO et al., 1999), and at Vulcano, can be readily accessed in geothermal wells, heated sediment seeps, and two-phase submarine springs. Subaerial volcanic activity in this region is limited to Stromboli and Vulcano. At Stromboli, this is manifested as small, short-lived explosions of viscous lava (“strombolian”), and at Vulcano, as high temperature fumaroles around the rim and on the flank of La Fossa caldera. Vulcano, in particular, has experienced a complex volcanic evolution that has produced lavas ranging in composition from tephrites and shoshonites to trachytes and rhyolites (DE ASTIS et al., 1997; KELLER, 1980), with the oldest lavas geochemically dated to only 120 ka (DE ASTIS et al., 1989) and the youngest recorded in black-and-white photography.
In fact, for the past circa 2500 years, Vulcano’s eruptive history has been recorded directly by human observation, starting with the Greeks who settled these islands around 580 BC. Highlights of this recent history include the first eruption of the current active cone of La Fossa around 475 BC; the emergence from the sea of the small edifice of Vulcanello in 183 BC, followed by many hundreds of years of relative quiescence punctuated by occasional explosive activity from Vulcanello and La Fossa; the formation of an isthmus that joined Vulcano and Vulcanello in 1550, thereby creating the famous Baia di Levante (see below); and the final eruptive period that ended 22 March 1890. Vulcano so impressed the ancient Greeks that they considered it the home of Hephaistos - god of fire, son of Hera, husband of Aphrodite, master smith, and patron of craftsmen. In his fiery home beneath Vulcano, Hephaistos designed an invisible net that entangled his wife with her lover Ares, he made the armor for the warrior Achilles, and hammered out lightning bolts for Zeus. Greek mythology also tells of King Aeolus - for whom these islands are named - who was appointed by Zeus as keeper of all the winds. It was Aeolus who presented Odysseus with a bag of wind for his voyage home to Ithaca after the Trojan War.
Vulcano is famous as the namesake of all the world’s volcanoes, as a centerpiece in Greek (and Roman) mythology, and, because of the more than ten thousand tourists who arrive every summer, as arguably Italy’s most dangerous volcano. But in biogeochemistry, Vulcano is famous as home to more cultured hyperthermophiles than any other place on Earth. Hyperthermophiles are organisms with optimum growth temperatures of at least 80 °C. In fact, the first isolated organism to thrive at temperatures above 100 °C was the sulfur reducing archaeon Pyrodictium occultum (STETTER, 1982). Later, the furious fireball, Pyrococcus furiosus, was cultured and characterized; it is the source of a very thermostable, commercially available DNA polymerase (FIALA and STETTER, 1986). Archaeoglobus fulgidus still holds the record as the highest temperature marine sulfate reducer (STETTER, 1988; STETTER et al., 1987), and Ferroglobus placidus couples nitrate reduction with iron oxidation to drive its biochemical machinery (HAFENBRADL et al., 1996). Two of the deepest, most slowly evolving, and highest temperature lineages in the domain bacteria are Thermotoga maritima (HUBER et al., 1986) and Aquifex aeolicus (DECKERT et al., 1998) - their genomes were among the first from any organism to be fully sequenced. Aquifex, Latin for the ‘water maker’, can extract energy from the Knallgas reaction, in which molecular hydrogen and molecular oxygen are combined with a bang (‘Knall’ in German). An isolate from a geothermal well, Palaeococcus helgesonii, may provide insight into the shallow subsurface biosphere (AMEND et al., 2003a), and it is also one of the rare oxygen tolerant hyperthermophiles. These, and other thermophilic and hyperthermophilic strains, were discovered, and in some cases found only at Vulcano. In other words, the hydrothermal system at the Baia di Levante could be considered the type locality of these heat-loving archaea and bacteria.
The thermophile diversity at Vulcano is, of course, far greater than that represented by these cultivated strains. Sequences of DNA extracted from water and sediment samples documented that most of the archaea and bacteria belong to phylogenetic groups made up entirely of uncultured organisms (ROGERS, 2006). As just one example, the archaeal DNA from a geothermal well (Pozzo Istmo) points to more than a dozen crenarchaeal, euryarchaeal, and korarchaeal lineages belonging to phylogenetic groups that have no cultured representatives at all (ROGERS and AMEND, 2005). It was, however, comforting to observe that one of the sequences from Pozzo Istmo was nearly identical (99%) to Palaeococcus helgesonii, the euryarchaeon isolated previously from this well (AMEND et al., 2003a).
Other studies further demonstrated the phylogenetic diversity at Vulcano, but also provided insight into the catabolic diversity. Using fluorescent labels to tag specific subgroups of archaeal and bacterial cells in Vulcano sediments, all of the key thermophilic and hyperthermophilic groups (except methanogens) were identified. These included the fermenting Thermococcales and Thermotogales, and the sulfate reducing Archaeoglobales (RUSCH and AMEND, 2004). These results matched beautifully with a laboratory incubation study, where 14C-labeled substrates were fermented by the naturally occurring hyperthermophile community to yield carboxylic acids and H2 (TOR et al., 2003). These fermentation products then served as key reactants in sulfate reduction, the predominant terminal electron accepting process in the heated Vulcano sediments. Carbon-14 labeled methane was not detected, which is entirely consistent with the absence of methanogens in the culturing trials, gene surveys, and fluorescent labeling studies mentioned above.
Why does Vulcano harbor so many thermophiles?
The expansive thermophile diversity at Vulcano may be enabled by the vast menu of available redox reactions. Life, whether microbial or macrobial, photosynthetic or chemosynthetic, lithotrophic or heterotrophic, runs on energy harvested from electron transfer. Redox disequilibria equate to potential catabolic energy. The heated sediments, shallow-sea vents, and geothermal wells in and around the Baia di Levante are characterized by hundreds, perhaps thousands, of such redox disequilibria among organic and inorganic aqueous compounds, gases, and minerals (AMEND et al., 2004; AMEND et al., 2003b; ROGERS and AMEND, 2005; ROGERS and AMEND, 2006; ROGERS et al., 2007; SKOOG et al., 2007). Many such reactions are now well-documented catabolisms, including aerobic respiration, sulfur and sulfate reduction, the Knallgas reaction, fermentation, and iron redox.
However, many other redox reactions that are also energy-yielding at Vulcano, are completely unknown as catabolic strategies. Thus, they can be viewed as potential catabolisms.Their potential as microbial food is based on thermodynamic principles, i.e., a negative Gibbs energy of reaction (ΔGr) at the temperature, pressure, and chemical composition that obtain at the site of interest. While some will cringe at the thought of using thermodynamics to predict biochemistry (‘isn’t it all about kinetics?’), it should be reaffirmed that such an approach has a long and successful track record. For example, the existence of anaerobic ammonia oxidation (anammox) was hypothesized from negative Gibbs energies (BRODA, 1977) nearly 20 years prior to its experimental confirmation (VAN DE GRAAF et al., 1995). It took even longer to demonstrate the anaerobic oxidation of methane (AOM) (BOETIUS et al., 2000; HINRICHS et al., 1999; ORPHAN et al., 2001), which, also had been predicted on thermodynamic grounds (BARNES and GOLDBERG, 1976). Simply put, a redox reaction with a negative ΔGr is in the running as a potential energy source for life, but one with a positive ΔGr is not.
It is certainly not surprising that the reduction of elemental sulfur (S0), written as
H2(aq) + S0 ? H2S(aq), (1)
is exergonic (energy-yielding) at Vulcano. After all, the widespread yellow sulfur crystals and pervasive sulfidic odor hint instantaneously at its occurrence. The knowledge that numerous Vulcano thermophiles make their living by mediating this seemingly simple reaction lends further support (FISCHER et al., 1983; STETTER, 1982; STETTER et al., 1983). However, it is perhaps surprising how little energy is released from this apparently ecologically important process. Gibbs energy calculations (AMEND et al., 2004; AMEND et al., 2003b; ROGERS et al., 2007) show that in the hydrothermal sites around the Baia di Levante, reaction (1) yields only 8-21 kJ per mole of electrons transferred. The energy yield is even less impressive from lithotrophic sulfate reduction (3-18 kJ/mol e-),
SO42- + 4H2(aq) + 2H+ ? H2S(aq) + 4H2O, (2)
and sulfur disproportionation (<12 kJ/mol e-),
4S0 + 4H2O ? SO42- + 3H2S(aq) + 2H+, (3)
to name but two documented catabolisms in Vulcano thermophiles. Compared with the aerobic respiration of glucose, which yields about 115 kJ/mol e-, these chemolithotrophic energy sources are truly anemic.
This raises the question if all heat-loving microbial residents of the Baia di Levante survive on such paltry fuel supplies? And further, if perhaps a large array of redox reactions, each with low energy-yields, are a prerequisite for the broad diversity observed at Vulcano? The answers are clearly ‘no’. In situ, the aforementioned Aquifex could gain ~100 kJ/mol e- from the Knallgas reaction, and Ferroglobus might extract as much as 55 kJ/mol e- from the oxidation of ferrous iron with nitrate (AMEND et al., 2003b; ROGERS et al., 2007). This is not to mention the energy from aerobic respiration of organic compounds, which releases as much 120 kJ/mol e- (ROGERS and AMEND, 2006). Of the many hundreds of potential catabolisms at Vulcano (redox reactions with negative ΔGr), only a small subset are currently known as actual catabolisms. However, recall that despite the success in culturing heat-loving archaea and bacteria from Vulcano, most of the microbial diversity still is known only as gene sequences; there is no physiologic information for most of the microbial community. Perhaps culturing these “unculturables” requires media recipes that rely less on what has worked previously in microbiology, and more on what is plausible from geochemistry arguments.
Perhaps some of the “unculturables” extract energy from these potential catabolisms. For example, anaerobic oxidation of sulfides with nitrate or nitrite is unknown in Vulcano thermophily, but organisms with the proper biochemistries could liberate 85-105 kJ/mol e- from these redox couples at the various hydrothermal locations of the Baia di Levante. A second example, and perhaps one of the most intriguing scenarios, relies on ferric iron as terminal electron acceptor in the oxidation of reduced N- and S-compounds. Especially in the acidic parts of the Vulcano hydrothermal system, these redox reactions are highly exergonic, yielding >60 kJ/mol e-. And since FeIII is predominantly in solid form, commonly as (oxy)hydroxides, cells might be physically attached to the terminal electron acceptor while patiently waiting for the electron donor to flow past in the fluid phase. Of course, these few examples barely scratch the surface of plausible catabolisms; heterotrophy, both the respiration and fermentation of organic compounds, introduces near endless uninvestigated possibilities.
Vulcano is a special place, but I do not want to imply that it is uniquely suited for expansive thermophile diversity. Other places have similarly appealing attributes for hot life. For example, the hot springs of Yellowstone National Park (USA) boast an impressive number of exergonic redox reactions (MEYER-DOMBARD, 2004). At one locale, Obsidian Pool, the Gibbs energies of 182 potential chemolithotrophic catabolisms were quantified, with energy yields ranging from near 0 to >100 kJ/mol e- (SHOCK et al., 2005). And, like at Vulcano, gene surveys at Yellowstone have shown that the phylogenetic diversity (and arguably the catabolic diversity) far exceeds what is known based on culturing work (MEYER-DOMBARD, 2004; MEYER-DOMBARD et al., 2005). Deep-sea vent systems are also famous thermophile hunting grounds, but the range of energy-yields from the broad spectrum of redox couples there is generally less than that in shallow-sea and continental hydrothermal systems (AMEND et al., 2004). Deep-sea vent samples are also far more difficult to collect than their surface counterparts, and thus, detailed fluid analyses, including speciation of organic molecules and redox sensitive inorganic compounds, are relatively few. Consequently, redox energy calculations are also relatively few (MCCOLLOM and SHOCK, 1997).
Vulcano is worth a visit. It has subaerial and submarine volcanic activity, it features tremendous geochemical diversity, its broad microbial community structure is documented with culture-dependent and culture-independent methods, its hydrothermal fluids and sediments are easily sampled, and several exploratory geothermal wells drilled in the 1950’s provide access to the shallow subsurface. Helgeson recognized in Vulcano a place where geochemistry and microbiology were inseparable, and where geochemists and microbiologists could in collaboration - and over lunch of insalata di polipo and a bottle of Regaleali - probe the geochemical limits of life and interrogate bacteria (and archaea) as geologic agents of the first magnitude. The ancient Greeks anointed Vulcano with mythical status, and based on high temperature biogeochemical investigations, this stinky little island certainly deserves its special place.
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Jan P. Amend
Department of Earth and Planetary Sciences
St. Louis, MO 63130, USA