Psychrotrophic bacteria isolated from Antarctic ecosystems

A. Correa-Guimaraes1, J. Martn-Gil1, M. C. Ramos-Snchez2, L. Vallejo-Prez

1.
Department of Forestry, Agricultural and Environmental Engineering, ETSIA, Avenida
de Madrid, 57, Palencia, Spain
2.
Laboratory of Environmental Microbiology, Hospital Universitario Ro 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 37C 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 (Marspolar 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 C are termed
psychrophiles (Herbert, 1986; Russell and Hamamoto, 1998). Most of the Earths surface
is covered by seawater and the vast majority of this, in the Polar zones, is at a temperature
of 4C. 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 62-65 S and 58-63 W.

Material and methods

Bacterial densities evaluated on 2216 Difco marine broth and after incubation at 37 C 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 Gram-
negative 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) (Martn-Gil et al., 2004) that metal-
reducing 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 p-
phenylphenol 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.

References

Anderson, C.G., DePablo, N., and Romo, C.R. 1978. Antarctic Krill (Euphausia superba) as a source of
chitin and chitosan. In Proceeding of the First International Conference on Chitin /Chitosan, R.A.A.
Muzarrelli and E.R. Pariser (Ed.), p.54. MIT Sea Grant Program, Cambridge, Project 2409-1
Beliaev, A.S., and Saffarini, D.A. 1998. Shewanella putrefaciens mtrB encodes an outer membrane protein
required for Fe(III) and Mn(IV) reduction, J. Bacteriol., 180:62926297.
Bowman J.P., McCammon S.A., Brown MV, Nichols D., McMeekin T.A. 1997. Diversity and
association of psychrophilic bacteria in Antarctic sea ice. Appl Environ Microbiol, 63(8):3068-78.
Cairns-Smith, A.G., Hall A.J., and Russell, M.R. 1992. Mineral theories of the origin of life and an iron
sulfide example Orig. Life Evol. Biosphere, 22, pp. 161180.
Davies, P. L. & Sykes, B. D. 1997. Antifreeze proteins. Curr Opin Struct Biol, 7, 828834.
Denner E.B, Mark B, Busse H.J, Turkiewicz M., Lubitz W. 2001. Psychrobacter proteolyticus sp. nov., a
psychrotrophic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting
a cold-adapted metalloprotease. Syst Appl Microbiol, 24(1):44-53.
DiChristina, T.J and DeLong, E.F. 1993. Design and application of rRNA-targeted oligonucleotide probes
for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens. Appl Environ
Microbiol,.59(12):4152-60.
Herbert, R. A. 1986. The ecology and physiology of psychrophilic microorganisms. In Microbes in Extreme
Environments. Special Publication of the Society for General Microbiology. Herbert, R.A. and Codd,
G.A.(Eds.). London: Academic Press. 1986.
Imai Y., Matsunaga I., Kusunose E. and Ichihara K. 2000. Unique heme environment at the putative
distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis
(peroxygenase P450(SPalpha). J Biochem (Tokyo), 128(2):189-94.
Ivanov V, Stabnikov, V., Zhuang, W.K., Tay, J.H. and Tay, S.T.L. 2005. Phosphate removal from the
returned liquor of municipal wastewater treatment plant using iron-reducing bacteria. Journal of Applied
Microbiology, 98(5):1152.
Lovley, D. R. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev, 55:259287.
Lovley D.R, Holmes D.E, Nevin K.P. 2004. Dissimilatory Fe(III) and Mn(IV) reduction. Adv Microb
Physiol, 49:219-86
Luning Prak, D.J.; Pritchard, P.H. 2002. Degradation of polycyclic aromatic hydrocarbons dissolved in
Tween 80 surfactant solutions by Sphingomonas paucimobilis EPA 505. Canadian Journal of Microbiology,
48(2):151-158.
Martn-Gil, J., Ramos-Snchez, M.C. and Martn-Gil, F.J. 2004. Shewanella putrefaciens in a fuel-in-
water emulsion from the Prestige oil spill. Antonie van Leeuwenhoek, 86(3):283-285
Myers, C. R., and J. M. Myers. 1993. Ferric reductase is associated with the membranes of anaerobically
grown Shewanella putrefaciens MR-1 FEMS Microbiol Lett, vol. 108, pp. 1522.
Myers, C. R., and J. M. Myers. 1997. Cloning and sequencing of cymA, a gene encoding a tetraheme
cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens strain
MR-1 J. Bacteriol., 179:11431152.
Myers CR, Myers JM. 2004. Shewanella oneidensis MR-1 restores menaquinone synthesis to a
menaquinone-negative mutant. J Appl Microbiol, 70(9):5415-25
Northup, D.E., Barns, S.M., Yu, L.E. et al. 2003. Diverse microbial communities inhabiting
ferromanganese deposits in Lechuguilla and Spider Caves. Environ Microbiol, 5(11):1701.
Price, P.B.. 2007. Microbial life in glacial ice and implications for a cold origin of life (minireview). FEMS
Microbiology Ecology, 59:217.
Rey, J., Somoza, L., Martnez-Fras, J., Benito, R., Martn-Alfageme, S., 1997. Deception Island
(Antarctica): A new target for exploration of Fe-Mn mineralization? In: Nicholson, K., Hein, J.R., Buhn, B.,
Dasgupta, S. (Eds.), Manganese Mineralization: Geochemistry and Mineralogy of Terrestrial and Marine
Deposits. Geological Society London, Special Publication 119, pp. 239251.
Russell, N. J. and Hamamoto, T. 1998. Psychrophiles. In Extremophiles: Microbial Life in Extreme
Environments, pp. 2545. K. Horikoshi and W. D. Grant (Eds.). Chichester: Wiley-Liss.
Shimahara, K., Ohkouchi, K., and Ikeda, M. 1982. A new isolation method of crustacean chitin using a
proteolytic bacterium, Pseudomonas maltophilia. In Proceeding of the Second International Conference on
Chitin and Chitosan; S. Hirano and S. Tokura (Eds.), p.10. The Japan Society of Chitin and Chitosan,
Sapporo, Japan
Shivaji S. and Delille D. 1996. Biodiversity and Functions of Bacteria from Antarctica. Biodiversity and
Conservation, 5:1505-1523
Somoza, L., Rey, J., Martinez-Fras, J. and Benito, R., 1994. Fe-Mn distribution patterns on sea-floor
sediments related to the discovery of submarine volcanic cones, mounds and vent structures (Deception
Island, Antarctica). European J. Min., 6:342-344.
Story, S. P.; Parker, S. H.; Hayasaka, S. S.; Riley, M. B.; Kline, E. L. 2001. Convergent and divergent
points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and
phenanthrene by Sphingomonas paucimobilis var. EPA505. Journal of Industrial Microbiology &
Biotechnology, 26(6):369-382.
Vazquez S., Ruberto L. and Mac Cormack W. 2005. Properties of extracellular proteases from three
psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil. Polar Biology, 28(4):319-325
Walker, J. C. G. 1980. Atmospheric constraints on the evolution of metabolism. Origins of Life, 10:93104.