CAN. J. MICROBIOL.
VOL. 22, 1976
Temperature optima for bacteria and yeasts from cold-mountain habitats1
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
JERRYL. MOSSER,GARYM . HERDRICH, A N D THOMAS D. BROCK Deprrr!r?len!r,fBac!eriology. Uni\,ersi!y of Wisconsitl, marl is or^, Wisconsin 53706 Accepted October 17, 1975
J . L., G. M. H E R D R I Cand H , T . D. BROCK.1976. Temperature optima for bacteria and MOSSER, yeasts from cold-mountain habitats. Can. J . Microbioi. 22: 324-325. Optimal temperatures for the growth of bacteria and yeasts isolated from several coldmountain habitats were determined. The lowest optimal temperatures encountered were in the 10-15 % range, even though most of the isolates were obtained from sites at or near 0 "C. MOSSER,J . L., G. M. H E R D R I CetH T . D. BROCK.1976. Temperature optima for bacteria and yeasts from cold-mountain habitats. Can. J . Microbiol. 22: 324-325. Les temperatures optimales de croissance des bacterieset des levures isolees de divers ha b'ltats montagneux et froids furent determinkes. Les temperatures optimales les plus basses si situent entre 10 et 15 "C. bien que la plupart des isolatsfurent obtenus de sites ou la temperature etait prks de, ou de 0 "C. [Traduit par Ie journal]
During a general study of the microorganisms inhabiting high mountain snowfields, glaciers, and streams, we observed large numbers of bacteria and yeasts associated with snow algae and other eucaryotic algae. T o ascertain the extent to which the bacteria and yeasts were adapted t o the low temperatures of their environment, we isolated a number of them and determined their optimal temperatures for growth. Samples of snow containing red snow algae and of benthic algal mats in streams draining melting snowfields and high mountain lakes were collected during July and August, 1973, in the Beartooth Mountains, located on the MontanaWyoming boundary. Sampling sites were within the area bounded by latitudes44'57' and 45'2'30'' N and longitudes 109'30' and 109'37' Wand were a t elevations ranging from 2900 to 3350 m. The region is generally alpine in character; tree-line is at approximately 3000 m. One sample of water containing silt and gravel was obtained a t the ice surface of the Galena Creek rock-covered glacier in Wyoming (44'38'30" N, 109"47'30" W) (4). The glacier was reached by digging through about 60 cm of rock chips and was found to have an 8-cm layer of water flowing downslope on the ice surface. All samples were collected aseptically in sterile Whirl-Pak bags, immediately placed in ice, and transported to a field laboratory in West Yellowstone, Montana. None of the samples 'Received June 2, 1975.
were permitted t o warm up; snow samples had not melted when cultures were started from them. The temperature of the surface of snow was invariably 0 "C, that at the surface of the Galena Creek glacier was 1 "C, and those of the streams sampled ranged from 1 to 15 "C. Cultures were obtained by incubating a t 4 'C tubes of medium D (l), pH 7, containing 0.1% glucose and 0.1 "j, yeast extract (Difco) or plates of the same medium containing 1.5% Bacto-agar (Difco), after inoculation with portions of the samples. Bacteria that grew in the liquid medium were streaked o n plates. Colonies on plates were successively picked and restreaked until pure cultures were obtained. Different isolates were distinguished o n the basis of morphology, motility, and colony type. Great care was taken t o avoid exposing cultures to temperatures greater than 4 "C, and, during manipulations, media and utensils were prechilled to 0 "C o r 4 'C before use. Optimal temperatures for growth were determined by incubating replicate liquid cultures of the isolates (50 ml in unshaken 250-ml Delong flasks) in each of a series of water baths with carefully controlled temperatures ranging from 0 to 50 "C.Periodic microscopic examination revealed that cell size did not change duringgrowth of the cultures, permitting the use of optical density to assess growth. Optimal density a t 650 nm was used t o monitor growth, and :emperatures a t which growth rates were the fastest were considered optimal. At o r near the optimal
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
NOTES
temperature, cultures reached stationary phase (after four to six doublings) within 1 day, whereas a t other temperatures growth was monitored for 2 or 3 days. A total of 29 bacterial and 5 yeast isolates was obtained. The bacteria were all rod-shaped, but otherwise varied considerably with regard t o size, colony appearance, pigmentation, and motility, whereas the yeasts all appeared to be of the one type characteristically seen in samples of snow algae. Further identification of these organisms was beyond the scope of this survey and was not attempted. Two of the bacterial isolates (both from the surface of snow) had optimal temperatures of 10-15 "C, and five others (three from the surface of snow, one from the Galena Creek glacier, and one from the outlet of Night Lake, which was 15 "C at the time of sampling) exhibited broad optimal temperatures that extended downward to 10-15 "C. The optima for all other bacterial isolates, whether from the snow surface or from streams, were between 20 and 30°C. Two yeast isolates from snow had optima of 10-15 " C , whereas 3 others, 2 from snow and 1 from a stream at 15 "C, grew best at 20-30 "C. We had anticipated that since bacteria and yeasts seemed to be thriving in snow and water at or near 0 "C, they might have adapted to low environmental temperatures to the extent that they exhibited growth optima at or near these temperatures. This is apparently not the case, however. Straka and Stokes (7) obtained similar results with soil, feces, and other material from Antarctica; most of their isolates had growth optima of 20-30 "C. In the latter study, however, a possible shortcoming was that the samples were stored at - 10 "C for about 2 years before culturing was and in addition the samples air might have been exposed peratures. Such exposure might be expected to be deleterious to extremely- -psychrophilic organisms (2). We took great care in our studies to use freshly collected samples that had not been exposed to temperatures higher than the site temperature. It may be that the cold habitats investigated are too unstable to permit organisms with lower optimal temperatures to develop. Snow algal populations dense support heterotrophic bacteria develop in the study area only in
325
mid or late summer and many snowfields completely melt by the end of the summer before they are repienished by fresh snows. Inocula of algae (6) and presumably also of bacteria are transported to the snow in the air and are therefore exposed, at least transiently, to ambient air temperature. The annual period of growth may be too short to permit development of psychrophilic populations optimally adapted to low temperatures. However, one isolate was obtained from a stream which must be permanently cold, since even in mid-August the water temperature was 1 " C , and this isolate did not have an optimal temperature lower than the other bacteria. Perhaps reflecting the greater age a n d long-term stability of some Antarctic lakes. bacteria with generally lower optimal temperatures (down to 5 "C) were isolated by Stanley and Rose (5). A limitation of this and the ~ r e v i o u swork is that important psychrophilic organisms would be missed if they could not be cultured under the particular conditions used. Using a radioisotope incorporation technique, Morita et al. (3) have obtained preliminary evidence of maximal microbial activity at 3 "C in Antarctic Sea water. It is possible that such assays for microbial activity applied to sufficiently stable habitats might reveal the existence of organisms with lower optimal temperatures.
Acknowledgment This work was supported by The College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by a contract from the Atomic Energy Commission (COO-21 61-21). I . CASTENHOLL. R. W. 1969. Therrnophilic blue-green algae and the thermal environment. Bacteriol. Rev. 33: 476-504. 2. MORITA, R. Y . 1966. Marine psychrophilic bacteria. Oceanogr. Mar. Biol. Ann. Rev. 4: 105-121. 3. MORITA, R. Y., P. A. G I L L E S P Iand E , L. P. JONES. 1971. Microbiology of Antarctic Seawater. Antarct. J. U.S. 6: 157. 4. POTTER.N . 1972. Ice-cored rock glacier, Galena Creek, Northern Absaroka Mountains, Wyoming. Geol, Sot, A m , Bull. 83: 3025-3058, 5. S T A N L E Y S.. 0..and A. H. ROSE.1967. Bacteriaand yeast from lakes on Deception Island. Philos. Trans. R . Soc. London, Ser. B, 252: 199-207. 6. S T E I NJ. . R.. and C . C. AMUNDSEN. 1967. Studieson snow algae and fungi from the front range ofcolorado. Can. J. Bot. 45: 2033-2045. 7. S T R A K A R., P.,and J. L.. STOKES.1960. Psychrophilic bacteria from Antarctica. J. Bacterial. 80: 622-625.
VOL. 22, 1976
Temperature optima for bacteria and yeasts from cold-mountain habitats1
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
JERRYL. MOSSER,GARYM . HERDRICH, A N D THOMAS D. BROCK Deprrr!r?len!r,fBac!eriology. Uni\,ersi!y of Wisconsitl, marl is or^, Wisconsin 53706 Accepted October 17, 1975
J . L., G. M. H E R D R I Cand H , T . D. BROCK.1976. Temperature optima for bacteria and MOSSER, yeasts from cold-mountain habitats. Can. J . Microbioi. 22: 324-325. Optimal temperatures for the growth of bacteria and yeasts isolated from several coldmountain habitats were determined. The lowest optimal temperatures encountered were in the 10-15 % range, even though most of the isolates were obtained from sites at or near 0 "C. MOSSER,J . L., G. M. H E R D R I CetH T . D. BROCK.1976. Temperature optima for bacteria and yeasts from cold-mountain habitats. Can. J . Microbiol. 22: 324-325. Les temperatures optimales de croissance des bacterieset des levures isolees de divers ha b'ltats montagneux et froids furent determinkes. Les temperatures optimales les plus basses si situent entre 10 et 15 "C. bien que la plupart des isolatsfurent obtenus de sites ou la temperature etait prks de, ou de 0 "C. [Traduit par Ie journal]
During a general study of the microorganisms inhabiting high mountain snowfields, glaciers, and streams, we observed large numbers of bacteria and yeasts associated with snow algae and other eucaryotic algae. T o ascertain the extent to which the bacteria and yeasts were adapted t o the low temperatures of their environment, we isolated a number of them and determined their optimal temperatures for growth. Samples of snow containing red snow algae and of benthic algal mats in streams draining melting snowfields and high mountain lakes were collected during July and August, 1973, in the Beartooth Mountains, located on the MontanaWyoming boundary. Sampling sites were within the area bounded by latitudes44'57' and 45'2'30'' N and longitudes 109'30' and 109'37' Wand were a t elevations ranging from 2900 to 3350 m. The region is generally alpine in character; tree-line is at approximately 3000 m. One sample of water containing silt and gravel was obtained a t the ice surface of the Galena Creek rock-covered glacier in Wyoming (44'38'30" N, 109"47'30" W) (4). The glacier was reached by digging through about 60 cm of rock chips and was found to have an 8-cm layer of water flowing downslope on the ice surface. All samples were collected aseptically in sterile Whirl-Pak bags, immediately placed in ice, and transported to a field laboratory in West Yellowstone, Montana. None of the samples 'Received June 2, 1975.
were permitted t o warm up; snow samples had not melted when cultures were started from them. The temperature of the surface of snow was invariably 0 "C, that at the surface of the Galena Creek glacier was 1 "C, and those of the streams sampled ranged from 1 to 15 "C. Cultures were obtained by incubating a t 4 'C tubes of medium D (l), pH 7, containing 0.1% glucose and 0.1 "j, yeast extract (Difco) or plates of the same medium containing 1.5% Bacto-agar (Difco), after inoculation with portions of the samples. Bacteria that grew in the liquid medium were streaked o n plates. Colonies on plates were successively picked and restreaked until pure cultures were obtained. Different isolates were distinguished o n the basis of morphology, motility, and colony type. Great care was taken t o avoid exposing cultures to temperatures greater than 4 "C, and, during manipulations, media and utensils were prechilled to 0 "C o r 4 'C before use. Optimal temperatures for growth were determined by incubating replicate liquid cultures of the isolates (50 ml in unshaken 250-ml Delong flasks) in each of a series of water baths with carefully controlled temperatures ranging from 0 to 50 "C.Periodic microscopic examination revealed that cell size did not change duringgrowth of the cultures, permitting the use of optical density to assess growth. Optimal density a t 650 nm was used t o monitor growth, and :emperatures a t which growth rates were the fastest were considered optimal. At o r near the optimal
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of North Dakota on 12/21/14 For personal use only.
NOTES
temperature, cultures reached stationary phase (after four to six doublings) within 1 day, whereas a t other temperatures growth was monitored for 2 or 3 days. A total of 29 bacterial and 5 yeast isolates was obtained. The bacteria were all rod-shaped, but otherwise varied considerably with regard t o size, colony appearance, pigmentation, and motility, whereas the yeasts all appeared to be of the one type characteristically seen in samples of snow algae. Further identification of these organisms was beyond the scope of this survey and was not attempted. Two of the bacterial isolates (both from the surface of snow) had optimal temperatures of 10-15 "C, and five others (three from the surface of snow, one from the Galena Creek glacier, and one from the outlet of Night Lake, which was 15 "C at the time of sampling) exhibited broad optimal temperatures that extended downward to 10-15 "C. The optima for all other bacterial isolates, whether from the snow surface or from streams, were between 20 and 30°C. Two yeast isolates from snow had optima of 10-15 " C , whereas 3 others, 2 from snow and 1 from a stream at 15 "C, grew best at 20-30 "C. We had anticipated that since bacteria and yeasts seemed to be thriving in snow and water at or near 0 "C, they might have adapted to low environmental temperatures to the extent that they exhibited growth optima at or near these temperatures. This is apparently not the case, however. Straka and Stokes (7) obtained similar results with soil, feces, and other material from Antarctica; most of their isolates had growth optima of 20-30 "C. In the latter study, however, a possible shortcoming was that the samples were stored at - 10 "C for about 2 years before culturing was and in addition the samples air might have been exposed peratures. Such exposure might be expected to be deleterious to extremely- -psychrophilic organisms (2). We took great care in our studies to use freshly collected samples that had not been exposed to temperatures higher than the site temperature. It may be that the cold habitats investigated are too unstable to permit organisms with lower optimal temperatures to develop. Snow algal populations dense support heterotrophic bacteria develop in the study area only in
325
mid or late summer and many snowfields completely melt by the end of the summer before they are repienished by fresh snows. Inocula of algae (6) and presumably also of bacteria are transported to the snow in the air and are therefore exposed, at least transiently, to ambient air temperature. The annual period of growth may be too short to permit development of psychrophilic populations optimally adapted to low temperatures. However, one isolate was obtained from a stream which must be permanently cold, since even in mid-August the water temperature was 1 " C , and this isolate did not have an optimal temperature lower than the other bacteria. Perhaps reflecting the greater age a n d long-term stability of some Antarctic lakes. bacteria with generally lower optimal temperatures (down to 5 "C) were isolated by Stanley and Rose (5). A limitation of this and the ~ r e v i o u swork is that important psychrophilic organisms would be missed if they could not be cultured under the particular conditions used. Using a radioisotope incorporation technique, Morita et al. (3) have obtained preliminary evidence of maximal microbial activity at 3 "C in Antarctic Sea water. It is possible that such assays for microbial activity applied to sufficiently stable habitats might reveal the existence of organisms with lower optimal temperatures.
Acknowledgment This work was supported by The College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by a contract from the Atomic Energy Commission (COO-21 61-21). I . CASTENHOLL. R. W. 1969. Therrnophilic blue-green algae and the thermal environment. Bacteriol. Rev. 33: 476-504. 2. MORITA, R. Y . 1966. Marine psychrophilic bacteria. Oceanogr. Mar. Biol. Ann. Rev. 4: 105-121. 3. MORITA, R. Y., P. A. G I L L E S P Iand E , L. P. JONES. 1971. Microbiology of Antarctic Seawater. Antarct. J. U.S. 6: 157. 4. POTTER.N . 1972. Ice-cored rock glacier, Galena Creek, Northern Absaroka Mountains, Wyoming. Geol, Sot, A m , Bull. 83: 3025-3058, 5. S T A N L E Y S.. 0..and A. H. ROSE.1967. Bacteriaand yeast from lakes on Deception Island. Philos. Trans. R . Soc. London, Ser. B, 252: 199-207. 6. S T E I NJ. . R.. and C . C. AMUNDSEN. 1967. Studieson snow algae and fungi from the front range ofcolorado. Can. J. Bot. 45: 2033-2045. 7. S T R A K A R., P.,and J. L.. STOKES.1960. Psychrophilic bacteria from Antarctica. J. Bacterial. 80: 622-625.
