Psychrotrophic Metal Tolerant Bacteria for Mobilisation of Metals in Antarctic Waters

By: Maria-Judith Gonsalves
Cold tolerant psychrotrophic bacteria abound in the Antarctic waters. While Antarctic krills are known to concentrate heavy metals at ppm levels, psychrotrophic bacteria from Antarctic fresh and marine waters have been reported to tolerate them. Of the 252 isolates from these waters varying degrees of resistance to metals like chromium, cadmium, zinc and mercury was observed. Some of the marine bacterial isolates showed 29 and 16 per cent resistance to 100 ppm of cadmium and chromium salt respectively. Tolerance to lower concentration (10 ppm) of mercury was observed in 68 per cent of the isolates. Surprisingly, the bacterial resistance shown by these bacteria were on similar levels to those reported from coastal waters. Some of the freshwater isolates had multiple metal resistance. is intrinsic resistance encountered in isolates from these pristine environments could be useful for orchestrating metal mobilisation like iron in these waters to promote in situ production for climate modulation. Alternatively, these native bacteria could be useful for harnessing mineral resources in the distant future.
Policy

Introduction

Antarctic has for a long time been viewed as a pristine environment as compared to other oceanic regions experiencing both local and global anthropogenic influences only over larger time scales (Bonner 1984, Evans et al., 2000). This remotest region of the earth is a model system where human assisted biological invasion can be understood (Rudolph and Benninghoff, 1977; Vincent, 1988). However, it is now being established that microbial diasporas are also reaching Antarctic from land masses (Walton, 1990). Heavy metals occur naturally in the ecosystem with large variations in concentration and are required by living organisms in varying amounts. These metals can be iron, cobalt, copper, manganese, molybdenum, and zinc. In modern times, the concentration of heavy metals, have been increasing in all ecosystems including the Antarctic (Hur et al., 2007). The krills from these regions harbour heavy metals at varying concentrations (Yamamoto et al., 1987). Petri and Zauke (1993) reported that metal concentrations show considerable interspecific heterogeneity. The cadmium (Cd) levels observed in caridean decapods (Chorismus antarcticus and Notocrangon antarcticus) were highest among marine crustaceans (13 mg/kg dry weight). Yet other crustaceans like amphipod Maxilliphimedia longipes and the isopod Aega antarctica (6-8 mg/kg) show that they do not have requirements for reduced metals. This led to the suggestion that in monitoring studies, Antarctic organisms may no longer serve as the basis for global reference levels (Nygaerd et al., 2001). Bioavailability of heavy metals and bioessential metals by natural leaching from the sediments may also add to the presence of these metals in the Antarctic region (Byerley and Scharer 1992). Marine bacteria are important models for studies of heavy metal toxicity. They not only represent the initial step in most food chains but are also able to adsorb, accumulate and transform heavy metals (Chan and Dean, 1988). With the present rate at which some of the heavy metals bioaccumulate, (Jerez et al., 2011; Guhathakurta and Kaviraj, 2000; Krishnamurti and Nair, 1999) it is pertinent to understand their effect at the primary microbial level in freshwater and seawater environments, where there is a paucity of such observations. The present study re-examines the previous work carried out by the author on some of the cold adapted/tolerant psychrotrophic Antarctic isolates and makes use of the ecological knowledge base to show how these indigenous bacteria could be used to augment primary production.

 

 

Material and methods

Site Description and Sampling

The present work is on the freshwater lakes which are situated in the Schirmacher Oasis around the east and west of the Indian Research Station Maitri and along the cruise track extending from 50°S and 18°E to 65°S and 30°E. Meteorological data show that the area has a dry polar climate. Environmental parameters from literature (Ingole and Parulekar, 1987) of the eastern and western lakes show that the western lakes harbour twice the organic carbon of the eastern. Also, there was a difference in salinity between the east and west lakes. The details of sampling and analyses are described elsewhere (Loka Bharathi et al., 2001).

 

Isolation

In this study 209 psychrotrophic isolates retrieved from nutrient agar medium prepared in freshwater, and 43 seawater isolates retrieved from ZoBell’s medium, which showed psychrotrophic properties were used. The isolates were identified based on taxonomic, physiological and biochemical properties detailed by Oliver and Smith, 1982.

 

Test for metal resistance

The test for metal resistance has been carried out as outlined in Nair et al., (1992). Briefly, working cultures were maintained in VNSS medium (Hermansson et al., 1987). Metal resistance was examined on VNSS agar medium containing one of the test metals (10 ppm of HgCl2, 100ppm of CdCl2, ZnCl2 and K2CrO4) in Muller Hinton agar (Muller and Hinton, 1941) which comply with the WHO (1961, 1977) and DIN Norm 5. Appropriate controls were maintained for all experiments.

 

 

Results

Of the 209 bacterial isolates from Antarctic freshwater lakes, 20 per cent were pigmented, of which half were associated with multiple metal resistance at 10 ppm to 100 ppm. Fourteen percent of the pigmented bacteria showed multiple resistance to all the metals tested (Figure 1). Examples of the most dominant groups of bacteria showing multiple metal resistance inclusive of mercury belonged to freshwater lakes east of Maitri. Conversely, there were bacteria that were resistant to only one or two metals excluding Hg and belonged to lakes west of Maitri. Interestingly, bacteria from water samples which were able to show resistance to multiple metals were invariably sensitive to mercury. The isolates from the eastern lakes showed comparatively higher multiple metal resistance compared to the western lakes. Thirty-seven and 25 isolates from the waters of the eastern lakes showed resistance to 3-4 metals respectively. In general 12 per cent of the isolates were resistant to all the metals tested and about 33 per cent of the isolates could express for single metal resistance (Figure 1). 5 Theme C article_Maria-Judith Gonsalves 1Taxonomic identification of the isolates revealed 18 groups of bacteria of which 14 were gram-negative, and 4 were gram-positive. The multiple metal resistant isolates were also reported to be antibiotic resistant (De Souza et al., 2006, 2007). These resistant bacteria predominantly belonged to Gammaproteobacteria (Pseudomonas, Alcaligenes, Enterobacteriace Vibrio, Aeromonas, Xanthomonas, Lucibacterium, Photobacterium and Moraxella). Besides other groups like Bacteriodetes (Flavobacterium, Flexibacter), Actinobacteria (Coryneforms, Micrococcus, Acetinobacter), Firmicutes (Staphylococcus, Bacillus), Alphaproteobacteria (Agrobacterium) and Betaproteobacteria (Spirillum) were also encountered. In the marine environment of the Antarctic region the bacterial abundance was between 108 to 109l-1 with the retrievable counts showing 102-105|-l. Results show that the Antarctic waters from the Indian side are not exempt to the spread of metal resistant bacteria. Of the 43 isolates screened, about 29 per cent and 16 per cent were resistant to 100 ppm of cadmium and chromium salt respectively. While tolerance to lower concentration (10 ppm) of mercury (Hg) was observed in 68 per cent of the isolates.

 

 

Discussion

Occurrence of resistance

Heavy metals occur naturally in the ecosystem with large variations in concentration and are required by living organisms in varying amounts. These metals can be iron, cobalt, copper, manganese, molybdenum and zinc. In modern times, the concentrations of heavy metals have been increasing in all ecosystems including the Antarctic. Consequently there are reports (De Souza et al., 2006, 2007) on bacteria from the Antarctic freshwater and seawater that are tolerant to these metals. These psychrotrophic bacteria are therefore useful in any cold environment. The growth of psychrophilic heterotrophs is fuelled by large pools of dissolved organic matter consisting largely of carbohydrates produced by the death and lysis of sea-ice organisms, as well as by the exudation of organic polymers by algae and bacteria (Thomas et al., 2001). Some of these organisms are resistant to heavy metals, a finding which could be surprising in pristine environments. In fact a high organic loading in the lakes on the west of Maitri may be one of the factors that lead to isolates from western and eastern lakes showing distinct geographical differences in properties of metal resistance.

Accumulation of metals in the polar regions

Considering that the ocean is contiguous and the polar regions are also influenced by atmospheric processes, it is not surprising that the background metal concentration can vary significantly (Zauke et al., 2003; Petri and Zauke 1993). Global atmospheric processes can contribute heavy metals to the Antarctic in the form of aerosols and are a natural source of heavy metals (Marteel et al., 2008; Hur et al., 2007; Pongratz and Heumann, 1999). However, through human activities the heavy metal content of aerosols has increased. Atmospheric inputs other than incineration smoke from a base cannot be controlled, and have been shown to be highly variable depending on the metal, geographical location, altitude and other factors. In fact, comparing results of different studies is difficult because of inherent variability due to season, chemical composition and amount of aerosols, coastal vs. inland sites, and different sampling and analysis procedures for each study. Therefore, quoting numerical results will not form an adequate picture of the issue, and so it is appropriate to approach this via a more qualitative rather than quantitative manner (Emnet, 2009). Isotope ratios of lead in Antarctic sea waters showed significant anthropogenic inputs, but levels remain low due to biological scavenging (Flegal et al., 1993).

Application of resistant bacteria in metal mobilisation

Consequently, bacterial resistance and interactions with metals is therefore an environmentally important phenomena. Tolerance to metals may occur from non-specific mechanisms such as impermeability to the cell or it may be due to specific resistance transfer factors. Under natural field conditions, microbial mobilisation of metals is difficult to quantify. Microorganisms can mobilise metals through autotrophic and heterotrophic leaching and redox transformations. Besides, there are various ways and means by which metal-mobilisation can occur. The way a particular metal becomes mobilised depends on the metal’s chemical and physical characteristics and the physico-chemical conditions that exist. Geochemically related metals for example Mn/Co have been reported to have the same mobilisation patterns around 2ppb.

 

 

Conclusion

Mechanism of tolerance

These patterns of response of metal tolerant bacteria differ depending on the environment. They overcome the toxicity by forming metal complexes or they catalyse its precipitation as oxides under oxic conditions or as sulphides under anoxic conditions. Alternatively the dissolved metals can get adsorbed to dead cells or suspended particles. It is documented that the genes coding for metal, antibiotic, pollutant detoxification cluster together (Domínguez, 2000). Thus, those capable of mobilising heavy metals can also mobilise iron (Gadd, 2001). This increased bioavailability of iron in turn could improve primary production. Iron is essential for the growth of nearly all microorganisms; yet it is only sparingly soluble near the neutral pH at aerobic conditions under which many microorganisms grow. The higher pH of sea water lowers the concentration of soluble/bio-available iron. To compound the problem the availability of iron is generally low in surface ocean water.

Hence, it is proposed that this property of the indigenous bacteria to catalyse primary production in natural Antarctic ecosystems/macro/mesocosms could be effectively used to mobilise iron intrinsically available, but locked and not bioavailable in systems. The assumption is that the mobilisation of iron will be much higher than other heavy metals from the mineral given that iron is the 4th most abundant element in the earth’s crust (Zheng, 2010). Consequently it would lead to CO2 sequestration by increased primary production which could either sink to the bottom or float. Alternatively this can be harvested for various applications. The approach that could be used would be appropriate choice of enclosed water body. Suspension of appropriate minerals/rocks at different depth intervals is recommended after prior seeding with suitable bacterial isolates. Time schedule could cover end of winter to the end of summer. Those bacteria capable of mobilising iron would do so during winter. The increased ambient concentration of iron would thus be bioavailable to both stimulate and improve primary production with the onset of summer. Increased primary production will thus increase the CO2 sequestered in these environments. The promotion of right indigenous primary producer could lead to desirable products e.g. lipids, biofuel, neutraceuticals etc. The harnessing of these products could be done in cycles to sychronise with their growth patterns. Finally the indigenous microflora and substrates could be used for a dual purpose for orchestrating metal mobilisation like iron in these waters to promote primary production that can modulate the climate locally. The primary producer could then be subsequently harvested for biotechnological applications. Alternatively, these native bacteria could be useful for harnessing mineral resources in the distant future.

 

 

Acknowledgements

The author acknowledges the support of Dr. Shetye, Director, NIO for carrying out this work. She also records her thankfulness to Dr. P. A. LokaBharathi for useful discussions on this subject. This is NIO contribution no. 5348

 

 

References

  1. Bonner, W.N. (1984), ‘Conservation and the Antarctic’, Laws RM (ed), Antarctic Ecology, 2, Academic Press: London, 821-850.
  2. Byerley, J.J, Scharer, J.M. (1992), ‘Natural release of copper and zinc into the aquatic environment’, Hydrometallurgy, 30, 107-126.
  3. Chan, K.Y, Dean, A.C.R. (1988), ‘Effects of cadmium and lead on growth, respiration and enzyme activity of the marine bacterium Pseudomonas marina’, Chemosphere, 17, 597-607.
  4. De Souza, M.J, Loka Bharathi, P.A., Nair, S., Chandramohan, D. (2006), ‘Metal and antibiotic- resistance of psychrotrophic bacteria from Antarctic marine waters’, Ecotoxicology, 15: 379-384.
  5. _________ (2007), ‘Tradeoff in Antarctic bacteria-Limnetic psychrotrophs concede multiple enzyme expression for multiple metal resistance’, Biometals: 20: 6, 821-828.
  6. Evans, C.W., Hills, J.M., Dickson, J.M.J. (2000), ‘Heavy metal pollution in Antarctica: a molecular ecotoxicological approach to exposure assessment’, Journal of Fish Biology, 57, 8-19.
  7. Flegal, A.R, Maring, H., Nlemeyer, S. (1993), ‘Anthropogenic lead in Antarctic sea water’, Letters to Nature, 365, 242-244.
  8. Domínguez, M.G., Lopez, L.M., Florencio, F.J., Reyes, J.C. (2000), ‘A gene cluster involved in metal homeostasis in the Cyanobacterium Synechocystis sp. strain PCC 6803’, Journal of Bacteriology, 182:6, 1507-1514.
  9. Gadd, G.M. (2001), ‘Microbial Metal Transformations’, The Journal of Microbiology, 39:2, 83-88.
  10. Guhathakurta, H., Kaviraj, A. (2000), ‘Heavy metal concentration in water, sediment, shrimp (Penaeus monodon) and mullet (Liza parsia) in some brackish water ponds of Sunderban, India’, Marine Pollution Bulletin, 40, 914-920.
  11. Hermansson, M., Jones, G. W., Kjelleberg, S. (1987), ‘Frequency of antibiotic and heavy metal resistance, pigmentation and plasmids in bacteria of marine air-water interface’, Applied Environmental Microbiology, 53: 2338-2342.
  12. Hur, S.D., Cunde, X., Hong, S., Barbante, C., Gabrielli, P., Lee, K., Boutron, C.F., Ming, Y. (2007), ‘Seasonal patterns of heavy metal deposition to the snow on Lambert Glacier basin, East Antarctica’, Atmospheric Environment, 41, 8567-8578.
  13. Ingole, B.S, Parulekar, A.H. (1987), ‘Microfauna of Schirmacher Oasis, Antarctica’, Water-moss communities: Scientific Report of Indian Expedition to Antarctica, Technical Publication: 4, 139-148
  14. Jerez ,S., Motas, M., Palacios, M.J., Valera, F., Cuervo, J.J., Barbosa, A. (2011), ‘Concentration of trace elements in feathers of three Antarctic penguins: Geographical and interspecific differences’, Environmental Pollution, 159: 2412-2419.
  15. Krishnamurti, A.J, Nair, V.R. (1999), ‘Concentration of metals in shrimps and crabs from Thane- Basin creek system Maharashtra’, Indian Journal of Material Science, 28, 92-95.
  16. Loka Bharathi, P.A., Nair, S., De Souza, M.J.B.D., Chandramohan, D. (2001), ‘Assessment of viability in the bacterial standing stock of the Antarctic sea from the Indian side’, Oceanologica Acta, 24: 577-580.
  17. Marteel, A., Boutron, C.F., Barbante, C., Gabrielli, P., Cozzi, G., Gaspari, V., Cescon, P., Ferrari, C.P., Dommergue, A., Rosman, K., Hong, S., Hur, S.D. (2008), ‘Changes in atmospheric heavy metals and metalloids in Dome C (East Antarctica) ice back to 672.0 kyr BP’: Marine Isotope Stages 16.2, Earth and Planetary Science Letters, 272, 579-590.
  18. Mueller, H.J., Hinton, J. (1941), ‘A protein-free medium for primary isolation of the Gonococcus and Meningococcus’, Proceedings of the Society of Experimental Biological Medicine, 48: 330-333.
  19. Nair, S., Chandramohan, D., LokaBharathi, P.A. (1992), ‘Differential sensitivity of pigmented and non-pigmented marine bacteria to metals and antibiotics’, Water Research, 26: 431-434.
  20. Nygaerd, T., Lie, E., Roev, N., and Steinnes, E. (2001), ‘Metal Dynamics in an Antarctic Food Chain’, Marine Pollution Bulletin, 42: 598-602.
  21. Oliver, J.D, Smith, J.F. (1982), ‘Intestinal microflora of deep sea animal: A taxonomic study’, Deep Sea Research, 29: 785-794
  22. Petri, G., Zauke, G.P. (1993), ‘Trace metals in crustaceans in the Antarctic Ocean’, AMBIO, 22: 529-536.
  23. Emnet, P. (2009), ‘Heavy metal: A heavy burden on the icy continent’, ANTA: Literature Review, 502.
  24. Pongratz, R., Heumann, K.G. (1999), ‘Production of methylated mercury, lead, and cadmium by marine bacteria as a significant natural source for atmospheric heavy metals in polar regions’, Chemosphere, 39:1, 89-102.
  25. Rudolph E, Benninghof, W.S. (1977), ‘Competitive and adaptive responses of invading versus indigenous biotas in Antarctica—A plea for organized monitoring; Clano GA (eds): Adaptations within Antarctic ecosystems, Smithsonian Institute, Washington DC, 1211-1225.
  26. Thomas, D.N., Kattner, G., Engbrodt, R., Giannelli, V., Kennedy, H., Haas, C., and Dieckmann, G.S. (2001), ‘Dissolved organic matter in Antarctic sea ice’, Annals of Glaciology,33:297-303.
  27. Vincent, W.F. (1988), ‘Microbial ecosystems of Antarctica’, Journal of the Marine Biological Association of the United Kingdom, Cambridge University Press, 69:2, 304.
  28. Walton, D.W.H. (1990), ‘Colonisation of terrestrial habitats-organisms, opportunities and occurrence’, In: Kerry KR, Hempel G (eds) Antarctic Ecosystems: Ecosystems, ecological change and conservation, Springer Berlin Heidelbergp: 51-60, doi: 10.1007/978-3-642-84074-6_5.
  29. Yamamoto, Y., Honda, K., Tatsukawa, R. (1987), ‘Heavy metal accumulation in Antarctic krill Euphausia superba’, Proceedings of the NIPR Symposium on Polar Biology, National Instiute of Polar Research, Tokyo: Japan, 1, 198-204
  30. Zauke, G.P., Clason, B., Savinov, V.M., Savinov, T. (2003), ‘Heavy metals of inshore benthic invertebrates from the Barents Sea’, Science of the total environment, 306; 99-110.
  31. Zheng, S.J. (2010), ‘Iron homeostasis and iron acquisition in plants: maintenance, functions and consequences’, Annals of Botany, 105: 5, 799-800, doi: 10.1093/aob/mcq082.

Leave a Reply

Your email address will not be published. Required fields are marked *