Psychrotrophic bacteria isolated from Antarctic ecosystems

A. Correa-Guimaraes1, J. Martín-Gil1, M. C. Ramos-Sánchez2, L. Vallejo-Pérez Department of Forestry, Agricultural and Environmental Engineering, ETSIA, Avenida de Madrid, 57, Palencia, Spain 2. Laboratory of Environmental Microbiology, Hospital Universitario Río Hortega, Dulzaina, 2, Valladolid, Spain

Key words: Psychrotrophic bacteria, Antarctic ecosystems, Shewanella putrefaciens; Pseudomonas maltophilia; Stenotrophomonas maltophilia; Sphingomonas paucimobilis

Abstract Microbial research on bacterial communities was carried out on ice samples collected from the field stations Hannah Point (Livingston Is.), Gabriel de Castilla (Deception Is.), Arctowsky (25 de Mayo Is.), at different depths, and ice floating on the ocean near the Almirante Brown station (Antarctic Peninsula), during an Antarctic travel in the first days of December 2004 on the “Grigoriy Mikheev” research vessel. Bacterial densities evaluated on 2216 Difco marine broth and after incubation at 37ºC for 2 days respectively ranged from 0 to 7.9 x 10(2) CFU/ml for heterotrophic bacteria. The qualitative composition of heterotrophic bacterial communities was studied through morphological and biochemical characteristics of 10 strains isolated from the stations. Almost all the heterotrophic, psychrotrophic isolates were non fermentative Gram-negative rods, belonging to the genera Pseudomonas (Sphingomonas spp and Stenotrophomonas spp), Alcaligenes, Flavobacterium and Cytophaga. Sphingomonas paucimobilis has been characterised in coastal Antarctic sites, from both marine environments and glacial ices. They form coloured colonies, consistent with pigment production providing protection from solar irradiation during airborne transport and subsequent exposure on the glacier surface. Stenotrophomonas maltophilia was characterised in floating ice in an oceanic zone near the Antarctic Peninsula. Although the bacterial communities in the two habitats investigated were clearly different, both have in common to be located in the West Antarctic zone, on great Fe-Mn deposits. This finding reinforces a previous observation on the Fe(III)/Mn(IV)-reducing bacteria Shewanella putrefaciens (a bacteria adapted to cold, isolated from the rusted Prestige tanker came from the Baltic Sea which destroyed the northern coast of Spain in 2002) that supports the hypothesis (shared with another authors) that if these micro-organisms can exist in glacial ice on Fe-Mn deposits in the Earth, they can also exist in Martian permafrost (Mars´polar caps) and in certain regions of Jupiter's ice-covered moons Europa, Callisto, and Ganymede.

Introduction Specific terms are used for the temperature ranges in which microbes can grow. Organisms that have such temperature range between -15 and +15 ºC are termed psychrophiles (Herbert, 1986; Russell and Hamamoto, 1998). Most of the Earth’s surface is covered by seawater and the vast majority of this, in the Polar zones, is at a temperature of 4°C. It should not be surprising then that the vast majority of microbes grow at medium to low temperatures. Psychrophiles are most often found in the constantly cold environments of the Arctic and Antarctic and in the deep sea. In this paper, microbial research on bacterial psychrotrophic communities was carried out both on ice floating on the ocean near the Almirante Brown station and the Cuverville Island (Antarctic Peninsula) and on ice collected from field stations in the Deception, Half Moon, Livingston and 25 de Mayo Islands. Lying off of the tip of the Antarctic Peninsula, these islands are part of an archipelago known as the South Shetland Islands. The studied area is located between 62º-65º S and 58º-63º W.

Material and methods Bacterial densities evaluated on 2216 Difco marine broth and after incubation at 37 ºC for 2 days respectively ranged from 0 to 7.9 x 10(2) CFU/ml for heterotrophic bacteria. The qualitative composition of heterotrophic bacterial communities was studied through morphological and biochemical characteristics of 10 strains isolated from the stations.

Results Ice samples on soil and from a lake in Arctowsky station (25 de Mayo Island) were microbiologically sterile. Also an ice core to a depth of 2 m from the undisturbed snow surface in Almirante Brown station result sterile. The rest of ice samples harbour their own microbiological ecosystems. Almost all the heterotrophic, psychrotrophic isolates were non fermentative Gramnegative rods, belonging to the genera Pseudomonas, Alcaligenes, Flavobacterium and Cytophaga (in agreement to Bowman et al., 1997). Species sufficiently well characterised were Sphingomonas paucimobilis and Stenotrophomonas maltophilia. Sphingomonas paucimobilis is characterized by medium to long rods that form large, occasionally mucoid colonies exhibiting a non-diffusible yellow carotenoid pigment. Motility (by a single polar flagellum) may be slow. It is, as Shewanella putrefaciens, an oxidase-positive, indole-negative, saccharolitic, nonferment. Although S. paucimobilis is isolated frequently in medical laboratories, clinically significant strains constitute a minority. Sphingomonas paucimobilis is a dominant member of the picoplankton population in Antarctic marine environments because their ability to utilize a wide range of organic compounds and their ability to grow and survive under low-nutrient or starvation conditions. Previous to this report, five isolates of Stenotrophomonas maltophilia have been reported by S. Shivaji and D. Delille, 1996 in the open Austral oceanic zone (South of Kerguelen archipelago), and S. Vazquez et al., 2005 have suggest that the Antarctic isolates could be adapted to cold by means of synthesising more enzymes with high activity but that the proteases they produce are not truly cold-active, being more similar to mesophilic enzymes. Sphingomonas shows remarkable biodegradative and biosynthetic capabilities, which have been utilized for a wide range of biotechnological applications, from bioremediation of environmental contaminants to production of extracellular polymers such as sphingans used extensively in the food and other industries. Stenotrophomonas maltophilia is a short to medium-sized straight gram-negative rod with a polar tuft of flagella. It forms large, smooth, glistening colonies with uneven margins and lavender-green to light purple pigmentation. In heart infusion agar with tyrosine, a water-soluble brown pigment, and on blood agar, a greenish discoloration appears underneath the growth. The smell of ammonia may be pronounced. With a few exceptions, most strains require methionine (or cystine plus glycine) for growth. The organism does not produce oxidase; oxidizes maltose faster than glucose; decarboxylates lysine; and hydrolizes esculin, gelatine and Tween 80. Stenotrophomonas maltophilia is ubiquous in nature and also been isolated from the hospital environment. It is frequently resistant to antimicrobial agents. Most strains are still to trimetroprim-sulfamethoxazole, moxalactam, doxycycline, and chloramphenicol. Stenotrophomonas maltophilia is a species in relationship to ice and Fe-Mn (Northup et al., 2003). We can to find it in artificial environments (e.g., in ice-making machines) but their natural niche are freezed oligotrophic subterranean environments that contains an abundance of low-density ferromanganese deposits (Somoza et al., 1994; Rey et al., 1997). When was discovered that the Antarctic krill (Euphausia superba Dana) use Stenotrophomonas maltophilia as proteolytic aims, it has been proposed to use this bacterium in the industrial crustacean chitin/chitosan extraction (Shimahara et al., 1982; Denner et al., 2001)

Discussion on the Fe/Mn-ice availability for bacterial species The finding in the Antarctic of bacteria that have in common to be located on great Fe-Mn deposits supports the hypothesis (previously formulated for Shewanella putrefaciens, a bacteria adapted to cold, isolated from the rusted Prestige tanker came from the Baltic Sea which destroyed the northern coast of Spain in 2002) (Martín-Gil et al., 2004) that metalreducing bacteria are important components in the overall biogeochemical cycling of iron, manganese and other elements in seasonally anoxic freshwater basins (DiChristina and DeLong, 1993) and/or glacial ice. The habitats for life at low temperatures benefit from two unusual properties of ice. First, almost all ionic impurities are insoluble in the crystal structure of ice, which leads to a network of micron-diameter veins in which microorganisms may utilize ions for metabolism. Second, ice in contact with mineral surfaces develops a nanometre-thick film of unfrozen water that provides a second habitat that may allow microorganisms to extract energy from redox reactions with ions in the water film or ions in the mineral structure (Price, 2007). Dissimilatory Fe(III) and Mn(IV)-reducing microorganisms. Dissimilatory Fe(III) reduction is the process in which microorganisms transfer electrons to external ferric iron [Fe(III)], reducing it to ferrous iron [Fe(II)] without assimilating the iron. Most microorganisms that reduce Fe(III) also can transfer electrons to Mn(IV), reducing it to Mn(II). Detailed reviews of the literature covering many of these aspects of Fe(III) and Mn(IV) reduction are available (Lovley et al, 2004). Most of the Fe(II) and Mn(II) produced from microbial Fe(III) and Mn(IV) reduction is found in solid phases, often in the form of Fe(II) and Mn(II) minerals of geochemical significance as magnetite (Fe3O4) and greigite (Fe3S4). Formation of magnetite may contribute to the magnetic remanence of soils and sediments. The magnetic anomalies that aid in the localization of subsurface hydrocarbon deposits may result from the activity of hydrocarbon-degrading Fe(III) reducers. Magnetite is found in both freshwater and marine environments, while greigite appear to be unique to marine systems Dissimilatory Fe(III)- and Mn(IV)-reducing microorganisms can be separated into two major groups, those that support growth by conserving energy from electron transfer to Fe(III) and Mn(IV) and those in which Fe(III) and Mn(IV) reduction are linked to respiratory systems capable of ATP generation (abbreviated as FMR). Geobacter species, require direct contact with Fe(III) oxides in order to reduce them. In contrast, Shewanella and Geothrix species produce chelators that solubilize Fe(III) and use electron-shuttling compounds (humics and other extracellular quinones) that transfer electrons from the cell surface to the surface of Fe(III) oxides not in direct contact with the cells (Lovley et al. 2004). Although Fe(III)-reductase activity was primarily localized in the membrane of Fe(III)and Mn(IV)-reducing microorganisms such as S. putrefaciens (Myers and Myers, 1993), recently, these same authors have discovered that Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant, challenging the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration(Myers and Myers, 2004). Thus, the involvement of cytochromes of the c-type has been maintained in electron transport to Fe(III) for Shewanella species (Myers and Myers, 1997; Beliaev and Saffarini, 1998; Imao et al., 2000).

In other hand, the strain Stenotrophomonas maltophilia BK showed the ability to reduce Fe(III) using xenobiotics as diphenylamine, m-cresol, 2,4-dichlorphenol and pphenylphenol as sole sources of carbon under anaerobic conditions (Ivanov et al., 2005). Experiments with Sphingomonas paucimobilis var. EPA505 on fluoranthene, naphthalene, anthracene and phenanthrene have been carried out (Story et al, 2001) A paper suggested that the surfactant Tween 80 enhances its biodegradation by increasing competition among polycyclic aromatic hydrocarbons for the same enzymatic sites (Luning Prak and Pritchard, 2002).. Implications for a cold origin of life. The concept that Fe(III) reduction is an early form of respiration agrees with geological scenarios that suggest the presence of large quantities of Fe(III) on prebiotic Earth (Cairns-Smith et al., 1992) and elevated hydrogen levels (Walker, 1980)—conditions that would be conducive to the evolution of a hydrogen oxidizing, Fe(III)-reducing microorganism. The large accumulations of magnetite in the Precambrian iron formations (discussed above) indicate that the accumulation of Fe(III) on prebiotic Earth was biologically reduced early in the evolution of life on Earth. This and other geochemical considerations suggest that Fe(III) reduction was the first globally significant mechanism for organic matter oxidation (Lovley, 1991). A study published in the February 22, 2002, issue of Science shows that even common bacteria are viable under high-pressure conditions equivalent to about 50 km beneath the Earth's crust or 160 km in a hypothetical sea. This finding may expand the habitable zone for life within the solar system and it opens new doors for looking for life much deeper inside planetary bodies than previously considered. The scientific team headed by S. and J. Scott at the Geophysical Laboratory of the Carnegie Institution of Washington adapted the tools of high-pressure physics to microbiology by using diamond anvil cells to subject the metal reducing Shewanella oneidensis to pressures up to 16 thousand times the pressure found at sea level. Shewanella uses formate in their metabolic processes in the absence of oxygen. Optical observations on stained bacteria further confirmed their viability and found that they can survive pressures far beyond those of deep ocean trenches and in the deep crust. These studies suggests that as far as pressure goes, the subduction zones on Earth and deep water/ice structures, such as those found on the moons Europa, Callisto, and Ganymede, might be environments that could harbour life.

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