Microbial respiration potential in Lake Bonney using a ...

Microbial respiration potential in Lake Bonney using a novel tetrazolium-reduction method JAMES I. SMITH, Department of Microbiology, Montana State University, Bozeman, Montana 59717 JOHN C. PRIscu, Department of Biology, Montana State University, Bozeman, Montana 59717

he permanently ice-covered lakes of the McMurdo Dry to east-lobe samples where respiring cell numbers decreased T Valleys region of Antarctica present unique environments from 60 to 45 percent between 13 and 17 rn, then increased to for study of nutrient cycling (Priscu, Ward, and Downes, a maximum of 73 percent at 30 rn (figure 2). In addition, Antarctic Journal, in this issue). respiring fractions were higher in 13-rn samples compared to Annual hydrologic recharge from streams contains negli17-rn samples in both lobes. gible amounts of nutrients (Greene, Angle, and Chave 1988), These results indicate that the fraction of microorganand annual phytoplankton blooms constitute the major isms maintaining a low cellular reduction potential via respisource of organic matter for subsequent heterotrophic breakration (using tetrazoliurn salt reduction as an indicator) is down. A lack of vertical mixing has resulted in highly stratified much lower in the west lobe of Lake Bonney than in the east zones of phytoplankton and bacterial productivity (Ward, lobe (particularly below 17 in). It should be noted that the Cockcroft, and Priscu, Antarctic Journal, in this issue). Little is CTC method yields the fraction of organisms showing respiraknown, however, about the stratification and degree of hettion potential; absolute respiratory activity remains unknown. erotrophic breakdown in these systems (Smith and Howes Detailed determinations of microbial activity are required to 1990). In this study, 5-cyano-2,3-ditolyl tetrazolium chloride elucidate the types of organisms (for example, photoau(GIG) was used to estimate the fraction of phytoplankton and totrophs, chemoautotrophs, and heterotrophs) responsible bacterioplankton showing respiration potential at selected for the respiration potential we observed with the GIG depths in both lobes of Lake Bonney. CTC forms intracellular, method. fluorescent- formazan deposits upon reduction by active succinate dehydrogenase, mdicating respiratory activity. Cells were counterstained with 4,6-diamidino-2-phenylin- 12 dole (DAPI) and enumerated for CTC-deposit K>1 H3 HIH HIH containing, and total microorganisms by epi-14 fluorescence microscopy. One thousand cells were counted from at least 10 fields for each 16 sample. Samples were collected using a 1-liter 18 Niskin sampler on 8 December 1992 from the east and west lobes of Lake Bonney at depths 20 of 13, 17, 25, and 30 meters (m) (hereafter E designated as "E" or "W" for east and west lobes, respectively). Nine-milliliter subsarn- ., ples were incubated with 5 millimolar GIG 24 (as described by Rodriguez et al. 1992), for 8 i•ist HIF hours in the dark. Samples E13, E17, and E25 26 were incubated at 5°C; E30 and W13 were incubated at 2°C; and W17, W25, and W30 28 were incubated at -2°C. These temperatures / were near those from which the samples were 30 o1 . HH collected. Total microbial numbers ranged 32 between 105 and 106 per milliliter in both 106 lobes at all depths (figure 1). Although total cell numbers remained relatively constant for Cells per milliliter west-lobe samples, the fraction indicating respiratory activity decreased with depth Figure 1. Total and CTC-reduction positive cell numbers in the east and west lobes of from 35 percent at 13 in 10 percent at Lake Bonney. Squares indicate west-lobe samples; circles indicate east-lobe samples. 25-30 in 1 and 2). This is in contrast Closed symbols denote total cells numbers; open symbols denote CTC-reduction positive cell numbers.

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We would like to thank Barb Kelley, Rich Bartlett, and VXE-6 for their assistance in this project. This research was supported by National Science Foundation grant OPP 9117907 to John C. Priscu.

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References

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Greene, W.J., M.P. Angle, and K.E. Chave. 1988. The geochemistry of antarctic streams and their role in the evolution of four lakes of the McMurdo Dry Valleys. Geochimica et Cosmochimica Acta, 52(5), 1247-1265. Priscu, J.C., B.B. Ward, and M.T. Downes. 1993. Water column transformations of nitrogen in Lake Bonney, a perennially ice-covered antarctic lake. Antarctic Journal of the U.S., 28(5). Rodriguez, G.G., D. Phipps, K. Ishiguro, and H.F. Ridgeway. 1992. Use of a fluorescent redox probe for direct visualization of actively respir-

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ing bacteria. Applied and Environmental Micro-

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Percent CTC Positive Cells Figure 2. Percentage fractions of microorganisms showing respiratory potential in the east (closed circles) and west (open circles) lobes of Lake Bonney as indicated by CTC-reduction (see text).

biology, 58(6), 1801-1808. Sharp, T.R., and J.C. Priscu. 1990. Ambient nutrient levels and the effects of nutrient enrichment on primary productivity in Lake Bonney. Antarctic Journal of the U.S., 25(5), 226-227. Smith, R.L., and B.L. Howes. 1990. Bacterial biomass and heterotrophic activity in the water column of an amictic antarctic lake. Antarctic Journal of the U.S., 25(5), 233-235. Ward, B.B., A.R. Cockcroft, and J.C. Priscu. 1993. Nitrifying and denitrifying bacteria in Lake Bonney. Antarctic Journal of the U.S., 28(5).

Influence of high salinity levels on ambient inorganic nitrogen and nitrogen- 15 extraction efficiency in Lake Bonney, Antarctica RICHARD D. BARTLETT, JOHN C. PRIscu, and CHRISTOPHER D. WOOLSTON, Department of Biological Sciences, Montana State University, Bozeman, Montana 59717

ke Bonney, which is characteristic of many of the lakes in L he antarctic dry valleys with respect to salinity profiles, derives its primary hydraulic input from glacial streams during the austral summer. A wide variety of salts are introduced into the lake through this inflow. Because of continuous ablation and no outflow, salinities in Lake Bonney range from near 0 parts per thousand (ppt) below the ice cap to 247 ppt (nearly 7 times open ocean) at 35 meters (m) (Spigel et al. 1991). Our current research project on Lake Bonney (see Priscu, Ward, and Downes, Antarctic Journal, in this issue) utilizes the stable isotope nitrogen-15 to measure rates of nitrogen transformation directly. Many of our experiments using this isotope require ammonium (NH 4 ) or nitrite (NO2j to be extracted from solution. The wide range of salinities

encountered at our experimental depths in Lake Bonney prompted us to test extraction efficiencies of nitrogen and potential isotopic discrimination as a function of salinity. Such tests are necessary if accurate measurements of nitrogen transformations are to be obtained. We used a modification of the NO 2- extraction protocol of Olson (1981) that is based on chemical complexation of sample NO2- with aniline sulfate under acidic conditions. This reaction yields a diazonium salt that is condensed with alkaline beta-naphthol to form a base-soluble azo dye which is partitioned from the aqueous phase through repeated extractions in a nonpolar solvent. After the absorbance [at a wavelength of 500 nanometers (nm)] of the solvent-dye mixture was measured, the solvent-dye mixture was dried and

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