Ice nucleation activity of antarctic marine microorganisms
Ice nucleation activity of antarctic marine microorganisms L.V. PARKER U.S. Army Cold Regions Research and Engineering Laboratory Hanover, New Hampshire 03755-1290 C.W. SULLIVAN
Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371 T.W. FOREST
Department of Mechanical Engineering University of Alberta Edmonton, Alberta, Canada T6G 2G8 S.F. ACKLEY
U.S. Army Cold Regions Research and Engineering Laboratory Hanover, New Hampshire 03755-1290
During several expeditions to investigate sea-ice properties in the Antarctic, researchers observed that large concentrations of biological material, primarily algae, were incorporated in the frazil sea ice (Ackley, Buck, and Taguchi 1979; Cow et al. 1982). Ackley (1982) proposed two possible mechanisms for the incorporation of this matter in sea ice. Incorporation could occur either by ice nucleation, if the frazil ice crystals preferentially nucleated on the suspended particles, or by scavenging. However, based on the number of crystals per unit volume of ice, Garrison, Ackley, and Buck (1983) concluded that for algae the principal mechanism for incorporation is scavenging. They found concentrations of algae with densities that were greater than what would be accounted for by preferential nucleation. They also noted the lack of any preferential concentration of any one species in the sea ice when compared with concentrations found in the water column. Bacteria are also found in sea ice (Sullivan and Palmisano 1981, 1984) and may be involved in its nucleation (Sullivan 1984). The role of ice nucleation active (INA) bacteria in frost damage is well documented in the literature. Schnell (1975) tested 23 marine phytoplankton cultures for ice-nucleation activity and found that one was especially active, Heterocapsa niei. Subsequently, Fall and Schnell (1985) isolated an INA bacterial strain from cultures of this dinoflagellate. They found this organism to be phenotypically similar to Pseudomonas fluorescens biotype C. They also searched for an INA pseudomonad in sea water from La Jolla, California but were unsuccessful. They did isolate several INA Erwinia, which they felt were most likely terrestrial in origin. Schnell (1975) proposed that marine INA organisms may be responsible for the bands of airborne ice nucleation activity found along latitudes of 400 to 55°S by Bigg (1973). In this paper, we present some initial studies on the 126
relative ability of melted sea ice and pure cultures of ice algae and ice bacteria to nucleate water droplets. Ice nucleation activity was determined using the droplet freezing technique of Vali (1977). The freezing point of the droplets was determined by measuring the surface temperature with a calibrated thermistor. The diatoms tested for ice nucleation activity were cultured in Guillard's F/2 medium and grown at 0°C. Ice nucleation tests were performed on cultures concentrated by centrifugation and resuspended in a small volume of the growth medium. The bacteria were cultured in Zobell's marine broth (2216E) at 0°C and were tested in the broth medium. Initial screening tests were performed by adjusting the optical density of bacterial suspensions at 660 nanometers to 0.05. Bacterial suspensions contained approximately 107 cells per milliliter. When looking for ice nucleation activity, we first tested a sample of sea ice that was rich in biological material. Figure 1 gives the freezing spectra for the sea-water blank and for samples that had been concentrated by factors of 2, 20, and 60. The results indicated an increased activity associated with the particulate material. However, it was not clear whether the active materials were algae or bacteria since many cell types of both were present in the sample (Clarke, Ackley, and Kumai 1984). We next tested several pure cultures of antarctic marine diatoms, which were provided to us by Greta Fryxell of Texas A&M University. The species we tested included Synedra sp., Chaetoceros dichaeta Ehrenberg, Chaetocerosfl.wsum Fryxell, Porosira glacialis (Grunow) Jorgensen, and Porosira pseudodenticulata (Hustedt) Jouse. The freezing spectra of these organisms showed no significant ice nucleation activity at temperatures higher than - 12°C. Figure 2 gives the freezing spectrum for C. flexuosum.
IN
0.8 C 0
60X/ /20X
C N
0 LL U,
0.4
Q. 0 C
2X
0.2 ea Water
0 1 -4 -8 -12 -16 -20 I
I
I
Temperature (°C) Figure 1. Freezing spectra for sea water and plankton. ANTARCTIC JOURNA
ft
ice nucleation activity at temperatures higher than - 10°C. We are currently conducting tests to identify HK-31. The authors wish to thank Diane Clarke, Melissa Hutt, and Pat Schumacher for technical assistance. This research was supported by a DA grant, 2-00145, to S.F. Ackley, D. Clarke, and L. Parker (In-House Laboratory Independent Research) on "Frazil Ice Nucleation From Bacterial Sources" and by National Science Foundation grant DPP 81-17237 to C.W. Sullivan.
0.8
C 0
0.8
0 C
C 0
9.-
0 0
C NJ 0
C
a) N4
LL
0
C),
LL
0.4
. 0.4
a)
0. 0 0
0.2
0.2 -2 -4 -6 Temperature (°C) Figure 3. Freezing spectrum for HK-31.
-10 -12 -14 -16 -18 Temperature (°C)
References
Figure 2. Freezing spectrum for C. flexuosum.
We also screened 11 strains of antarctic marine bacteria primarily isolated from sea ice from the McMurdo Sound area of Antarctica (Kobori, Sullivan, and Shizuya 1984). These organisms are psychrotrophic or psychrophilic and, therefore, are capable of growing at 0°C. We tested a number of sea-ice psychrotrophs, including HK-1, HK-10 and HK-31 (white pigmented), HK-4 (orange pigmented), HK-7, HK-54, HK-60 (all pink pigmented), and HK-64, (a yellow pigmented bacterium isolated from sediment). We also screened three sea-ice psychrophiles HK-16 (yellow pigmented), HK-21 (pink pigmented), and HK-44 (orange pigmented). HK-31 was ice nucleation active between —2.0 and —3.5°C. Figure 3 gives the freezing spectrum of this organism. The other organisms did not show any 1985 REVIEW
Ackley, S.F. 1982. Ice scavenging and nucleation: Two mechanisms for incorporation of algae into newly-forming sea ice. EQS. 63, 54. Ackley, S.F., K.R. Buck, and S. Taguchi. 1979. Standing crop of algae in the sea ice of the Weddell region. Deep Sea Research, 26A, 269 - 281. Bigg, E.K. 1973. Ice nucleus measurements in remote areas. Journal of Atmospheric Sciences, 30, 1153 - 57. Clarke, D.B., S.F. Ackley, and M. Kumai. 1984. Morphologyand ecology of diatoms in sea ice from the Weddell Sea. (CRREL Report 84-5.) Hanover, N.H.: U.S. Army Cold Regions Research and Engineering Laboratory. Fall, R., and R.C. Schnell. 1985. Association of an ice-nucleating pseudomonad with cultures of the marine dinoflagellate Heterocapsa niei. Journal of Marine Research, 43, 257 - 265. Garrison, D.L., S.F. Ackley, and K.R. Buck. 1983. A physical mechanism for establishing algal populations in frazil ice. Nature, 306, 363365.
127
Cow, A.J., S.F. Ackley, W.F. Weeks, and J.W. Govoni. 1982. Physical and structural characteristics of Antarctic sea ice. Annals of Glaciology, 3, 113-117. Korobri, H., C.W. Sullivan, and H. Schizuya. 1984. Bacterial plasmids in antarctic microbial assemblages. Applied and Environmental Microbiology, 48, 515 - 518. Schnell, R.C. 1975. Ice nuclei produced by laboratory cultured marine phytoplankton. Geophysical Research Letters, 2, 500 - 502. Sullivan, C.W. 1984. Bacteria associated with antarctic sea ice. Second
American conference on ice nucleating bacteria. Northern Arizona University, Flagstaff, Arizona. 6 - 9 June 1984. Sullivan, C.W., and A.C. Palmisano. 1981. Sea-ice microbial communities in McMurdo Sound, Antarctica. Antarctic Journal of the U.S., 16(5), 126 - 127. Sullivan, C.W., and A.C. Palmisano. 1984. Sea ice microbial communities: Distribution, abundance, and density of ice bacteria in McMurdo Sound, Antarctica, in 1980. Applied and Environmental Microbiology, 47, 788 - 796.
Ecology of sea-ice microbial communities during the 1984 winterto-summer transition in McMurdo Sound, Antarctica
• How does the growth and metabolism of the sea-ice microbial community change during this seasonal transition? • What is the effect of salinity on metabolism of the sea-ice microbial community? • What are the dominant "cryopelagic" fauna (Golikov and Scarlato 1973) in McMurdo Sound and the trophodynamics of these organisms? Vertical profiles of temperature for sea ice in the light-perturbation experiment were obtained by freezing duplicate strings of thermocouples (copper constantan with Teflon insulators and oversheaths) in 4-inch diameter holes made by a Jiffy Drill. From early October to mid-December, sea ice without snow cover exhibited temperatures ranging from - 18° to - 1.9°C at 25 centimeters above the surface of congelation ice, - 24° to - 1.9°C at the surface of congelation ice, to - 1.9°C at the interface of the congelation/platelet ice layers, the platelet layer, and sea water beneath the sea ice (figure, A and B). During the same period, sea ice under a 1 meter of snow cover exhibited a range of temperatures from - 18° to + 4°C at 25 centimeters above and at the surface of the snow cover, - 4° to - 1.9°C at the surface of the congelation ice, to - 1.9°C at the interface of the congelation/ platelet ice layers and deeper. An RTD (copper constantan) was lowered into the seawater beneath the sea ice to determine its temperature. Seawater was isothermal down to 19 meters and warmed only slightly from —1.91 to - 1.76°C during the season. Because the majority of the sea-ice microbial community biomass is found in the bottom 20 centimeters of congelation ice (Palmisano and Sullivan 1983; Sullivan and Palmisano 1984) and in the platelet ice layer (Bunt and Wood 1963), growth and metabolism of the sea-ice microbial community occur at temperatures of - 1.9°C or lower. Diel patterns of surface and sub-ice photosynthetically active radiation (PAR) (400 to 700 nanometers) were determined during the seasonal transition. A hemispherical irradiance sensor (QSR-240, Biospherical Instruments) was mounted on the roof of our dive hut, a spherical profiling irradiance sensor (QSP-200, Biospherical Instruments) was moored beneath the platelet ice layer, and a spectroradiometer with a cosine collector (MER-1000, Biosphencal Instruments) was moored beneath the congelation ice and connected via cable to a DEC 350 Professional Computer in the heated dive hut to collect irradiance data. Preliminary analysis of the irradiance data indicates that peak surface irradiance increased from approximately 100 microEinstems per square meter per second in mid-September to more than 1,600 microEinsteins per square meter per second from
S.T. KOTTMEIER,
M.A. MILLER, M.P. LIZOTTE, L.L. CRAFT, and C.W. SULLIVAN
Marine Biology Research Section Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371
B. GULLIKSEN Marine Biological Station N 9001 Tromso, Norway
During the austral spring and summer, land-fast sea ice of McMurdo Sound supports the growth of rich and diverse seaice microbial communities composed of psychrophilic microalgae, bacteria, and protozoans (Bunt and Wood 1963; Palmisano and Sullivan 1983; Grossi, Kottmeier, and Sullivan 1984; Sullivan and Palmisano 1984). In addition, a cryofauna (animals associated with sea ice) consisting of pteropods, copepods, amphipods, fish, and seals has been described (Dayton, Robilliard, and DeVries 1969; Bunt and Lee 1970; Bradford 1978). Thus sea ice may be of importance not only in primary productivity but also in the secondary productivity of McMurdo Sound (Palmisano and Sullivan 1983; Sullivan and Palmisano 1984). Research during the 1984— 1985 season began at winter fly-in (last week of August). A light-perturbation experiment was initiated to study the effect of extremes in downwelling irradiance on the growth and development of the sea-ice microbial community. The following questions addressed the ecology of the sea-ice microbial community during the seasonal transition from winter (low irradiance) to summer (high irradiance): • What are the seasonal patterns of temperature gradients in sea ice under variable snow cover? • How does the spectral composition and total downwelling irradiance change during this seasonal transition? 128
ANTARCTIC JOURNAL
Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371 T.W. FOREST
Department of Mechanical Engineering University of Alberta Edmonton, Alberta, Canada T6G 2G8 S.F. ACKLEY
U.S. Army Cold Regions Research and Engineering Laboratory Hanover, New Hampshire 03755-1290
During several expeditions to investigate sea-ice properties in the Antarctic, researchers observed that large concentrations of biological material, primarily algae, were incorporated in the frazil sea ice (Ackley, Buck, and Taguchi 1979; Cow et al. 1982). Ackley (1982) proposed two possible mechanisms for the incorporation of this matter in sea ice. Incorporation could occur either by ice nucleation, if the frazil ice crystals preferentially nucleated on the suspended particles, or by scavenging. However, based on the number of crystals per unit volume of ice, Garrison, Ackley, and Buck (1983) concluded that for algae the principal mechanism for incorporation is scavenging. They found concentrations of algae with densities that were greater than what would be accounted for by preferential nucleation. They also noted the lack of any preferential concentration of any one species in the sea ice when compared with concentrations found in the water column. Bacteria are also found in sea ice (Sullivan and Palmisano 1981, 1984) and may be involved in its nucleation (Sullivan 1984). The role of ice nucleation active (INA) bacteria in frost damage is well documented in the literature. Schnell (1975) tested 23 marine phytoplankton cultures for ice-nucleation activity and found that one was especially active, Heterocapsa niei. Subsequently, Fall and Schnell (1985) isolated an INA bacterial strain from cultures of this dinoflagellate. They found this organism to be phenotypically similar to Pseudomonas fluorescens biotype C. They also searched for an INA pseudomonad in sea water from La Jolla, California but were unsuccessful. They did isolate several INA Erwinia, which they felt were most likely terrestrial in origin. Schnell (1975) proposed that marine INA organisms may be responsible for the bands of airborne ice nucleation activity found along latitudes of 400 to 55°S by Bigg (1973). In this paper, we present some initial studies on the 126
relative ability of melted sea ice and pure cultures of ice algae and ice bacteria to nucleate water droplets. Ice nucleation activity was determined using the droplet freezing technique of Vali (1977). The freezing point of the droplets was determined by measuring the surface temperature with a calibrated thermistor. The diatoms tested for ice nucleation activity were cultured in Guillard's F/2 medium and grown at 0°C. Ice nucleation tests were performed on cultures concentrated by centrifugation and resuspended in a small volume of the growth medium. The bacteria were cultured in Zobell's marine broth (2216E) at 0°C and were tested in the broth medium. Initial screening tests were performed by adjusting the optical density of bacterial suspensions at 660 nanometers to 0.05. Bacterial suspensions contained approximately 107 cells per milliliter. When looking for ice nucleation activity, we first tested a sample of sea ice that was rich in biological material. Figure 1 gives the freezing spectra for the sea-water blank and for samples that had been concentrated by factors of 2, 20, and 60. The results indicated an increased activity associated with the particulate material. However, it was not clear whether the active materials were algae or bacteria since many cell types of both were present in the sample (Clarke, Ackley, and Kumai 1984). We next tested several pure cultures of antarctic marine diatoms, which were provided to us by Greta Fryxell of Texas A&M University. The species we tested included Synedra sp., Chaetoceros dichaeta Ehrenberg, Chaetocerosfl.wsum Fryxell, Porosira glacialis (Grunow) Jorgensen, and Porosira pseudodenticulata (Hustedt) Jouse. The freezing spectra of these organisms showed no significant ice nucleation activity at temperatures higher than - 12°C. Figure 2 gives the freezing spectrum for C. flexuosum.
IN
0.8 C 0
60X/ /20X
C N
0 LL U,
0.4
Q. 0 C
2X
0.2 ea Water
0 1 -4 -8 -12 -16 -20 I
I
I
Temperature (°C) Figure 1. Freezing spectra for sea water and plankton. ANTARCTIC JOURNA
ft
ice nucleation activity at temperatures higher than - 10°C. We are currently conducting tests to identify HK-31. The authors wish to thank Diane Clarke, Melissa Hutt, and Pat Schumacher for technical assistance. This research was supported by a DA grant, 2-00145, to S.F. Ackley, D. Clarke, and L. Parker (In-House Laboratory Independent Research) on "Frazil Ice Nucleation From Bacterial Sources" and by National Science Foundation grant DPP 81-17237 to C.W. Sullivan.
0.8
C 0
0.8
0 C
C 0
9.-
0 0
C NJ 0
C
a) N4
LL
0
C),
LL
0.4
. 0.4
a)
0. 0 0
0.2
0.2 -2 -4 -6 Temperature (°C) Figure 3. Freezing spectrum for HK-31.
-10 -12 -14 -16 -18 Temperature (°C)
References
Figure 2. Freezing spectrum for C. flexuosum.
We also screened 11 strains of antarctic marine bacteria primarily isolated from sea ice from the McMurdo Sound area of Antarctica (Kobori, Sullivan, and Shizuya 1984). These organisms are psychrotrophic or psychrophilic and, therefore, are capable of growing at 0°C. We tested a number of sea-ice psychrotrophs, including HK-1, HK-10 and HK-31 (white pigmented), HK-4 (orange pigmented), HK-7, HK-54, HK-60 (all pink pigmented), and HK-64, (a yellow pigmented bacterium isolated from sediment). We also screened three sea-ice psychrophiles HK-16 (yellow pigmented), HK-21 (pink pigmented), and HK-44 (orange pigmented). HK-31 was ice nucleation active between —2.0 and —3.5°C. Figure 3 gives the freezing spectrum of this organism. The other organisms did not show any 1985 REVIEW
Ackley, S.F. 1982. Ice scavenging and nucleation: Two mechanisms for incorporation of algae into newly-forming sea ice. EQS. 63, 54. Ackley, S.F., K.R. Buck, and S. Taguchi. 1979. Standing crop of algae in the sea ice of the Weddell region. Deep Sea Research, 26A, 269 - 281. Bigg, E.K. 1973. Ice nucleus measurements in remote areas. Journal of Atmospheric Sciences, 30, 1153 - 57. Clarke, D.B., S.F. Ackley, and M. Kumai. 1984. Morphologyand ecology of diatoms in sea ice from the Weddell Sea. (CRREL Report 84-5.) Hanover, N.H.: U.S. Army Cold Regions Research and Engineering Laboratory. Fall, R., and R.C. Schnell. 1985. Association of an ice-nucleating pseudomonad with cultures of the marine dinoflagellate Heterocapsa niei. Journal of Marine Research, 43, 257 - 265. Garrison, D.L., S.F. Ackley, and K.R. Buck. 1983. A physical mechanism for establishing algal populations in frazil ice. Nature, 306, 363365.
127
Cow, A.J., S.F. Ackley, W.F. Weeks, and J.W. Govoni. 1982. Physical and structural characteristics of Antarctic sea ice. Annals of Glaciology, 3, 113-117. Korobri, H., C.W. Sullivan, and H. Schizuya. 1984. Bacterial plasmids in antarctic microbial assemblages. Applied and Environmental Microbiology, 48, 515 - 518. Schnell, R.C. 1975. Ice nuclei produced by laboratory cultured marine phytoplankton. Geophysical Research Letters, 2, 500 - 502. Sullivan, C.W. 1984. Bacteria associated with antarctic sea ice. Second
American conference on ice nucleating bacteria. Northern Arizona University, Flagstaff, Arizona. 6 - 9 June 1984. Sullivan, C.W., and A.C. Palmisano. 1981. Sea-ice microbial communities in McMurdo Sound, Antarctica. Antarctic Journal of the U.S., 16(5), 126 - 127. Sullivan, C.W., and A.C. Palmisano. 1984. Sea ice microbial communities: Distribution, abundance, and density of ice bacteria in McMurdo Sound, Antarctica, in 1980. Applied and Environmental Microbiology, 47, 788 - 796.
Ecology of sea-ice microbial communities during the 1984 winterto-summer transition in McMurdo Sound, Antarctica
• How does the growth and metabolism of the sea-ice microbial community change during this seasonal transition? • What is the effect of salinity on metabolism of the sea-ice microbial community? • What are the dominant "cryopelagic" fauna (Golikov and Scarlato 1973) in McMurdo Sound and the trophodynamics of these organisms? Vertical profiles of temperature for sea ice in the light-perturbation experiment were obtained by freezing duplicate strings of thermocouples (copper constantan with Teflon insulators and oversheaths) in 4-inch diameter holes made by a Jiffy Drill. From early October to mid-December, sea ice without snow cover exhibited temperatures ranging from - 18° to - 1.9°C at 25 centimeters above the surface of congelation ice, - 24° to - 1.9°C at the surface of congelation ice, to - 1.9°C at the interface of the congelation/platelet ice layers, the platelet layer, and sea water beneath the sea ice (figure, A and B). During the same period, sea ice under a 1 meter of snow cover exhibited a range of temperatures from - 18° to + 4°C at 25 centimeters above and at the surface of the snow cover, - 4° to - 1.9°C at the surface of the congelation ice, to - 1.9°C at the interface of the congelation/ platelet ice layers and deeper. An RTD (copper constantan) was lowered into the seawater beneath the sea ice to determine its temperature. Seawater was isothermal down to 19 meters and warmed only slightly from —1.91 to - 1.76°C during the season. Because the majority of the sea-ice microbial community biomass is found in the bottom 20 centimeters of congelation ice (Palmisano and Sullivan 1983; Sullivan and Palmisano 1984) and in the platelet ice layer (Bunt and Wood 1963), growth and metabolism of the sea-ice microbial community occur at temperatures of - 1.9°C or lower. Diel patterns of surface and sub-ice photosynthetically active radiation (PAR) (400 to 700 nanometers) were determined during the seasonal transition. A hemispherical irradiance sensor (QSR-240, Biospherical Instruments) was mounted on the roof of our dive hut, a spherical profiling irradiance sensor (QSP-200, Biospherical Instruments) was moored beneath the platelet ice layer, and a spectroradiometer with a cosine collector (MER-1000, Biosphencal Instruments) was moored beneath the congelation ice and connected via cable to a DEC 350 Professional Computer in the heated dive hut to collect irradiance data. Preliminary analysis of the irradiance data indicates that peak surface irradiance increased from approximately 100 microEinstems per square meter per second in mid-September to more than 1,600 microEinsteins per square meter per second from
S.T. KOTTMEIER,
M.A. MILLER, M.P. LIZOTTE, L.L. CRAFT, and C.W. SULLIVAN
Marine Biology Research Section Department of Biological Sciences University of Southern California Los Angeles, California 90089-0371
B. GULLIKSEN Marine Biological Station N 9001 Tromso, Norway
During the austral spring and summer, land-fast sea ice of McMurdo Sound supports the growth of rich and diverse seaice microbial communities composed of psychrophilic microalgae, bacteria, and protozoans (Bunt and Wood 1963; Palmisano and Sullivan 1983; Grossi, Kottmeier, and Sullivan 1984; Sullivan and Palmisano 1984). In addition, a cryofauna (animals associated with sea ice) consisting of pteropods, copepods, amphipods, fish, and seals has been described (Dayton, Robilliard, and DeVries 1969; Bunt and Lee 1970; Bradford 1978). Thus sea ice may be of importance not only in primary productivity but also in the secondary productivity of McMurdo Sound (Palmisano and Sullivan 1983; Sullivan and Palmisano 1984). Research during the 1984— 1985 season began at winter fly-in (last week of August). A light-perturbation experiment was initiated to study the effect of extremes in downwelling irradiance on the growth and development of the sea-ice microbial community. The following questions addressed the ecology of the sea-ice microbial community during the seasonal transition from winter (low irradiance) to summer (high irradiance): • What are the seasonal patterns of temperature gradients in sea ice under variable snow cover? • How does the spectral composition and total downwelling irradiance change during this seasonal transition? 128
ANTARCTIC JOURNAL
