Microbial populations and activity in an antarctic freshwater pond
ionized water in no-nutrient controls. The bicarbonate solution was made up of 3 milliliters carbon-14 bicarbonate (5 microcuries per milliliter) and 12 milliliters bicarbonate (5 microcuries per milliliter) and 12 milliliters bicarbonate buffer (10 milligrams sodium bicarbonate per 100 milliliters carbon-dioxide-free distilled deionized water). The vials were capped with parafilmcoated corks and incubated in a constant temperature (14°C) constant light (115 micromoles per square meter per second) water bath. The carbon-14 labeled lipids were extracted and analyzed as previously described (Vestal 1985). There was no significant enhancement of measured photosynthesis when nitrate, ammonia, phosphate, manganese or iron was added in test solutions at concentrations between 10 and 100 millimolar. Phosphate significantly inhibited photosynthesis over the whole range of nutrient additions. Dissolved iron both as ferrous iron and ferric iron had significant effects on community photosynthesis. Dissolved ferric iron inhibited photosynthesis at concentrations of 0.1 millimolar in the Linnaeus Terrace lichen community and in all three communities at 1 millimolar (see figure). Dissolved ferrous iron was inhibitory at 1 millimolar in the Linnaeus Terrace community and at 10 millimolar in all three communities studied. From these data, photosynthetic metabolism in the endolithic lichen and cyanobacterial communities did not appear to be limited by the usual limiting nutrients. Neither nitrogen as ammonia and nitrate nor phosphorus additions had any positive effect on light-driven carbon-14 bicarbonate incorporation. Incubations with added phosphate showed photosynthesis inhibition over the entire range of nutrient additions. This inhibition of photosynthetic carbon fixation may be indicative of phosphate limitation, because both nutrient uptake and carbon fixation require reducing power and adenosine tnphosphate (Lean and Pick 1981). To our surprise, dissolved iron as ferric iron inhibited photosynthesis at what may be within the natural concentration range in the lichen communities. The concentration of iron oxide in
colonized Linnaeus Terrace sandstone is of the order of 1 to 10 millimoles per gram outside the lichen zone (Friedmann 1982). Iron oxides could be a source of ferric iron possibly of ferrous iron as well. Lichens produce lichen substances, which leach metal compounds, leading to 100 times lower iron concentrations in the lichen zone than in the surrounding rock. The results suggest a close coupling between the inorganic iron geochemistry and photosynthetic activity in the endolithic lichen communities where significant iron concentrations occur. The metabolism of the cyanobacterial community from Battleship Promontory may not be coupled to iron geochemistry as in the lichen communities. In the rocks predominantly colonized by cyanobacteria, iron oxides are not apparent. Despite this lack of a natural source of reduced iron, the cyanobacterial community responds as do the lichen communities, showing inhibition of photosynthesis upon ferrous iron and ferric iron addition. The field research was supported by National Science Foundation grant DPP 83-14180 to E. Imre Friedman of Florida State University.
Microbial populations and activity in an antarctic freshwater pond
Smith 1985). However, despite their large numbers, investigations to determine the activities of microorganisms in these antarctic waters have been limited (Heywood 1984). Freshwater habitats ranging from meltwater pools to small lakes are abundant in the area of Arthur Harbor, Anvers Island, and some of their biological and physico-chemical characteristics have been the subject of previous investigations (e.g., Parker, Samsel, and Prescott 1972). To expand our understanding of the ecology of antarctic ponds, we studied the microbial community and its activity in a freshwater pond near Palmer Station on Anvers Island. Samples were collected from a shallow freshwater pond (maximum depth 0.6 meter) near Palmer Station (64°46'S 64°05'W), Anvers Island, Antarctica. Water samples were collected every 6 hours over a 24-hour period during February 1986 from both the surface microlayer (air/water interface) and a subsurface depth of 20 centimeters. Surface microlayer samples were collected using a glass plate sampler (Garrett and Duce 1980) while subsurface samples were collected by opening a hand-held sterile 1-liter polypropylene bottle at the correct depth. Air temperature was recorded from the weather station
J.S. MAKI
Laboratory of Microbial Ecology Division of Applied Sciences Harvard University Cambridge, Massachusetts 02138 R.P. HERWIG
Department of Microbiology and Immunology SC-42 University of Washington Seattle, Washington 98195
Numerous lakes and ponds of varying size, salinity, and trophic condition are found in Antarctica (Heywood 1984; 226
References Friedmann, E.I. 1982. Endolithic microorganisms in the Antarctic cold desert. Science, 215, 1045-1053. Kappen, L., and E. Friedmann. 1983. Ecophysiology of lichens in the dry valleys of Southern Victoria Land, Antarctica. ll. CO 2 gas exchange in cryptoendolithic lichens. Polar Biology, 1, 227-232. Kappen, L., E.I. Friedmann, and J . Garty. 1981. Ecophysiology of lichens in the dry valley of Antarctica. I. Microclimate of the cryptoendolithic lichen habitat. Flora, 171, 216-235. Lean, D.R.S., and F.R. Pick. 1981. Photosynthetic response of lake plankton to nutrient enrichment: A test for nutrient limitation. Limnology and Oceanography, 26, 1001-1019. Vestal, J.R. 1985. The effects of light intensity on the cryptoendolithic microbiota. Antarctic Journal of the U.S., 20(5), 181-182. Vestal, J.R., T.W. Federle, and E.I. Friedmann. 1984. The effects of light and temperature on antarctic microbiota in vitro. Antarctic Journal of the U. S., 19(5), 173-174.
ANTARCTIC JOURNAL
at Palmer Station, while water temperature and dissolved oxygen was measured using a Yellow Springs Instrument Co. Model 54 combination temperature/dissolved oxygen meter and probe. The pH of the subsurface sample was measured using a standard pH meter in the laboratory. Water samples were analyzed for the following microbial parameters: (1) acridine orange direct count by epifluorescence microscopy was used to estimate numbers of total bacteria (Hobbie, Daley, and Jasper 1977); (2) bacterial colony-forming units were estimated from spread plates of a peptone/yeast extract/glucose agar incubated for 14 days at 50 C; (3) chlorophyll a and phaeophytin a (Holm-Hansen and Riemann 1978) were measured; and (4) tritiated thymidine(i incorporation (Fuhrman and Azam 1982) was determined. Average air and water temperature, dissolved oxygen, and pH are presented in table 1. The pond was always well oxygenated, and the pH was near neutrality. The average water temperature was about twice the average air temperature. The mean numbers of total bacteria and bacterial colony-forming units, pigments, and thymidine incorporation rates are presented in table 2. The data indicate that surface microlayer samples (sample thickness approximately 34 micrometers) contained much higher numbers of total bacteria, bacterial colonyforming units, and pigments than did the subsurface samples. The relatively large standard deviations, which are also found with the surface microlayer samples, indicate a considerable fluctuation in the concentrations of each parameter while the subsurface samples were more stable. The ratio of phaeophytin a to chlorophyll a and the thymidine incorporation rates indicate that the surface microlayer microbial populations were not as healthy or as metabolically active as their subsurface counterparts. The numbers of bacteria and the concentration of chlorophyll a suggest the pond is more comparable to nutrient-enriched freshwaters (Ellis-Evans 1981b) than to oligotrophic waters (Ellis-Evans 1981a). However, any assumption made about the trophic condition of the pond will have to await further investigation. The data show that the surface microlayer of this antarctic pond accumulates large numbers of microorganisms similar to data from more temperature marine and inland waters (see review by Norkrans 1980). Furthermore, the data also indicate that the surface microlayer microbial populations were less active than those in the subsurface water. The activity of both bacteria and algae in antarctic inland waters are strongly influenced by temperature fluctuation (Heywood 1984) and this is particularly true of surface microlayer populations in general Table 1. Average value (± standard deviation) of air and water temperature, dissolved oxygen, and pH during sampling Parameter
Air temperature (°C) Water temperature (°C) (milligrams per liter) Dissolved oxygen (mgi-1) pH
1986 REVIEW
Value
3.7 (± 1.9) 7.5 (± 1.8) 12.9 (± 0.6) 6.8 (± 0.1)
Table 2. Mean concentrations (± standard deviation) of total bacteria, bacterial colony-forming units, pigments, and tritiated thymidine incorporation for surface microlayer and subsurface samples Parameter
Surface microlayer Subsurface
Total bacteria x 106 per milliliter) 7.9 (± 8.0) 3.3 (± 0.5) Bacterial colony-forming units x 105 per milliliter) 5.7 (± 2.8) 4.1 (± 1.9) Chlorophyll a (milligrams per cubic meter) 2.5 (i 1.3) 0.6 (± 0.1) Phaeophytin a (milligrams per cubic meter) 2.6 (± 1.8) 0.2 (± 0.1) Phaeophytin a 1.1 (± 0.9) 0.4 (± 0.2) Chlorophyll a 1.3 (± 0.5) 2.0 (± 0.2) tritiated thymidine incorporation (moles x 10" per hour)
(Norkrans 1980). An additional factor affecting surface microlayer microorganisms is high light intensity (Norkans 1980). We suspect that both temperature fluctuation and high light intensity are the major factors influencing the lower activity in the surface microlayer. We thank the staff of Palmer Station, Anvers Island, for their excellent assistance during this and other studies. This research was supported in part by National Science Foundation grant DPP 84-15069. References Ellis-Evans, J.C. 1981a. Freshwater microbiology in the Antarctic: I. Microbial numbers and activity in oligotrophic Moss Lake, Signy Island. British Antarctic Survey Bulletin, 54, 85-104. Ellis-Evans, J.C. 1981b. Freshwater microbiology in the Antarctic: II. Microbial numbers and activity in nutrient-enriched Heywood Lake, Signy Island. British Antarctic Survey Bulletin, 54, 105-121/ Fuhrman, J.A., and F Azam. 1982. Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: Evaluation and field results. Marine Biology, 66, 109-120. Garrett, W.D., and R.A. Duce. 1980. Surface microlayer samplers. In F. Dobson, L. Hasse, and R. Davis, (Eds.), Air-sea interaction. New York: Plenum Publishing. Heywood, R.B. 1984. Antarctic inland waters. In R.M. Laws, (Ed.) Antarctic Ecology, (Vol. 1). London: Academic Press. 1-lobbie, J.E., R.J. Daley, and S. Jasper. 1977. Use of nuclepore filters for counting bacteria by epifluorescence microscopy. Applied and Environmental Microbiology, 33, 1225-1228. Holm-Hansen, 0., and B. Riemann. 1978. Chlorophyll adetermination: Improvements in methodology. Qikos, 30, 438-448. Norkrans, B. 1980. Surface microlayers in aquatic environments. Advances in Microbial Ecology, 4, 51-85. Parker, B.C., G. L. Samsel, and G.W. Prescott. 1972. Fresh-water algae of the Antarctic Peninsula 1. Systematics and ecology in the U.S. Palmer Station area. In G.L. Llano (Ed.), Antarctic Research Series, Antarctic Terrestrial Biology, 20, 69-81.
Smith, R.I.L. 1985. Nutrient cycling in relation to biological productivity in Antarctic and Sub-Antarctic terrestrial and freshwater ecosystems. In W.R. Siegfried, P.R. Condy, and R.M. Laws (Eds.), Antarctic nutrient cycles and food webs. Berlin: Springer-Verlag.
227
colonized Linnaeus Terrace sandstone is of the order of 1 to 10 millimoles per gram outside the lichen zone (Friedmann 1982). Iron oxides could be a source of ferric iron possibly of ferrous iron as well. Lichens produce lichen substances, which leach metal compounds, leading to 100 times lower iron concentrations in the lichen zone than in the surrounding rock. The results suggest a close coupling between the inorganic iron geochemistry and photosynthetic activity in the endolithic lichen communities where significant iron concentrations occur. The metabolism of the cyanobacterial community from Battleship Promontory may not be coupled to iron geochemistry as in the lichen communities. In the rocks predominantly colonized by cyanobacteria, iron oxides are not apparent. Despite this lack of a natural source of reduced iron, the cyanobacterial community responds as do the lichen communities, showing inhibition of photosynthesis upon ferrous iron and ferric iron addition. The field research was supported by National Science Foundation grant DPP 83-14180 to E. Imre Friedman of Florida State University.
Microbial populations and activity in an antarctic freshwater pond
Smith 1985). However, despite their large numbers, investigations to determine the activities of microorganisms in these antarctic waters have been limited (Heywood 1984). Freshwater habitats ranging from meltwater pools to small lakes are abundant in the area of Arthur Harbor, Anvers Island, and some of their biological and physico-chemical characteristics have been the subject of previous investigations (e.g., Parker, Samsel, and Prescott 1972). To expand our understanding of the ecology of antarctic ponds, we studied the microbial community and its activity in a freshwater pond near Palmer Station on Anvers Island. Samples were collected from a shallow freshwater pond (maximum depth 0.6 meter) near Palmer Station (64°46'S 64°05'W), Anvers Island, Antarctica. Water samples were collected every 6 hours over a 24-hour period during February 1986 from both the surface microlayer (air/water interface) and a subsurface depth of 20 centimeters. Surface microlayer samples were collected using a glass plate sampler (Garrett and Duce 1980) while subsurface samples were collected by opening a hand-held sterile 1-liter polypropylene bottle at the correct depth. Air temperature was recorded from the weather station
J.S. MAKI
Laboratory of Microbial Ecology Division of Applied Sciences Harvard University Cambridge, Massachusetts 02138 R.P. HERWIG
Department of Microbiology and Immunology SC-42 University of Washington Seattle, Washington 98195
Numerous lakes and ponds of varying size, salinity, and trophic condition are found in Antarctica (Heywood 1984; 226
References Friedmann, E.I. 1982. Endolithic microorganisms in the Antarctic cold desert. Science, 215, 1045-1053. Kappen, L., and E. Friedmann. 1983. Ecophysiology of lichens in the dry valleys of Southern Victoria Land, Antarctica. ll. CO 2 gas exchange in cryptoendolithic lichens. Polar Biology, 1, 227-232. Kappen, L., E.I. Friedmann, and J . Garty. 1981. Ecophysiology of lichens in the dry valley of Antarctica. I. Microclimate of the cryptoendolithic lichen habitat. Flora, 171, 216-235. Lean, D.R.S., and F.R. Pick. 1981. Photosynthetic response of lake plankton to nutrient enrichment: A test for nutrient limitation. Limnology and Oceanography, 26, 1001-1019. Vestal, J.R. 1985. The effects of light intensity on the cryptoendolithic microbiota. Antarctic Journal of the U.S., 20(5), 181-182. Vestal, J.R., T.W. Federle, and E.I. Friedmann. 1984. The effects of light and temperature on antarctic microbiota in vitro. Antarctic Journal of the U. S., 19(5), 173-174.
ANTARCTIC JOURNAL
at Palmer Station, while water temperature and dissolved oxygen was measured using a Yellow Springs Instrument Co. Model 54 combination temperature/dissolved oxygen meter and probe. The pH of the subsurface sample was measured using a standard pH meter in the laboratory. Water samples were analyzed for the following microbial parameters: (1) acridine orange direct count by epifluorescence microscopy was used to estimate numbers of total bacteria (Hobbie, Daley, and Jasper 1977); (2) bacterial colony-forming units were estimated from spread plates of a peptone/yeast extract/glucose agar incubated for 14 days at 50 C; (3) chlorophyll a and phaeophytin a (Holm-Hansen and Riemann 1978) were measured; and (4) tritiated thymidine(i incorporation (Fuhrman and Azam 1982) was determined. Average air and water temperature, dissolved oxygen, and pH are presented in table 1. The pond was always well oxygenated, and the pH was near neutrality. The average water temperature was about twice the average air temperature. The mean numbers of total bacteria and bacterial colony-forming units, pigments, and thymidine incorporation rates are presented in table 2. The data indicate that surface microlayer samples (sample thickness approximately 34 micrometers) contained much higher numbers of total bacteria, bacterial colonyforming units, and pigments than did the subsurface samples. The relatively large standard deviations, which are also found with the surface microlayer samples, indicate a considerable fluctuation in the concentrations of each parameter while the subsurface samples were more stable. The ratio of phaeophytin a to chlorophyll a and the thymidine incorporation rates indicate that the surface microlayer microbial populations were not as healthy or as metabolically active as their subsurface counterparts. The numbers of bacteria and the concentration of chlorophyll a suggest the pond is more comparable to nutrient-enriched freshwaters (Ellis-Evans 1981b) than to oligotrophic waters (Ellis-Evans 1981a). However, any assumption made about the trophic condition of the pond will have to await further investigation. The data show that the surface microlayer of this antarctic pond accumulates large numbers of microorganisms similar to data from more temperature marine and inland waters (see review by Norkrans 1980). Furthermore, the data also indicate that the surface microlayer microbial populations were less active than those in the subsurface water. The activity of both bacteria and algae in antarctic inland waters are strongly influenced by temperature fluctuation (Heywood 1984) and this is particularly true of surface microlayer populations in general Table 1. Average value (± standard deviation) of air and water temperature, dissolved oxygen, and pH during sampling Parameter
Air temperature (°C) Water temperature (°C) (milligrams per liter) Dissolved oxygen (mgi-1) pH
1986 REVIEW
Value
3.7 (± 1.9) 7.5 (± 1.8) 12.9 (± 0.6) 6.8 (± 0.1)
Table 2. Mean concentrations (± standard deviation) of total bacteria, bacterial colony-forming units, pigments, and tritiated thymidine incorporation for surface microlayer and subsurface samples Parameter
Surface microlayer Subsurface
Total bacteria x 106 per milliliter) 7.9 (± 8.0) 3.3 (± 0.5) Bacterial colony-forming units x 105 per milliliter) 5.7 (± 2.8) 4.1 (± 1.9) Chlorophyll a (milligrams per cubic meter) 2.5 (i 1.3) 0.6 (± 0.1) Phaeophytin a (milligrams per cubic meter) 2.6 (± 1.8) 0.2 (± 0.1) Phaeophytin a 1.1 (± 0.9) 0.4 (± 0.2) Chlorophyll a 1.3 (± 0.5) 2.0 (± 0.2) tritiated thymidine incorporation (moles x 10" per hour)
(Norkrans 1980). An additional factor affecting surface microlayer microorganisms is high light intensity (Norkans 1980). We suspect that both temperature fluctuation and high light intensity are the major factors influencing the lower activity in the surface microlayer. We thank the staff of Palmer Station, Anvers Island, for their excellent assistance during this and other studies. This research was supported in part by National Science Foundation grant DPP 84-15069. References Ellis-Evans, J.C. 1981a. Freshwater microbiology in the Antarctic: I. Microbial numbers and activity in oligotrophic Moss Lake, Signy Island. British Antarctic Survey Bulletin, 54, 85-104. Ellis-Evans, J.C. 1981b. Freshwater microbiology in the Antarctic: II. Microbial numbers and activity in nutrient-enriched Heywood Lake, Signy Island. British Antarctic Survey Bulletin, 54, 105-121/ Fuhrman, J.A., and F Azam. 1982. Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: Evaluation and field results. Marine Biology, 66, 109-120. Garrett, W.D., and R.A. Duce. 1980. Surface microlayer samplers. In F. Dobson, L. Hasse, and R. Davis, (Eds.), Air-sea interaction. New York: Plenum Publishing. Heywood, R.B. 1984. Antarctic inland waters. In R.M. Laws, (Ed.) Antarctic Ecology, (Vol. 1). London: Academic Press. 1-lobbie, J.E., R.J. Daley, and S. Jasper. 1977. Use of nuclepore filters for counting bacteria by epifluorescence microscopy. Applied and Environmental Microbiology, 33, 1225-1228. Holm-Hansen, 0., and B. Riemann. 1978. Chlorophyll adetermination: Improvements in methodology. Qikos, 30, 438-448. Norkrans, B. 1980. Surface microlayers in aquatic environments. Advances in Microbial Ecology, 4, 51-85. Parker, B.C., G. L. Samsel, and G.W. Prescott. 1972. Fresh-water algae of the Antarctic Peninsula 1. Systematics and ecology in the U.S. Palmer Station area. In G.L. Llano (Ed.), Antarctic Research Series, Antarctic Terrestrial Biology, 20, 69-81.
Smith, R.I.L. 1985. Nutrient cycling in relation to biological productivity in Antarctic and Sub-Antarctic terrestrial and freshwater ecosystems. In W.R. Siegfried, P.R. Condy, and R.M. Laws (Eds.), Antarctic nutrient cycles and food webs. Berlin: Springer-Verlag.
227
