The chemical profiles and initial microbiological data collected previously in Lake Bonney (Priscu, Ward, and Downes, Antarctic Journal, in this issue) identified several interesting features in the nitrogen cycle of the lake, raising unusual questions. First, nitrate (NO3) is present in high concentrations in the east lobe of the lake, indicating that although the water column is apparently anoxic below the chemocline, denitrification does not occur. By contrast, nitrate is depleted in the anoxic bottom waters of the west lobe, indicating that denitrification does occur there. Second, nitrous oxide (N20) is present at extremely high levels just below the chemocline in the east lobe yet much lower concentrations are present in the west lobe. N2O accumulation at low (but nonzero) oxygen levels is an indication of nitrification (Cohen and Gordon 1978; Goreau et al. 1980). Finally, bacterial numbers in the east lobe show a discrete maximum near 25 meters (m), below the oxic/anoxic interface, a feature not evident in the west lobe of the lake (figure 1A and B). The distribution of bacterial activity in the east lobe measured by uptake of tritiated-thymidine (a measure of heterotrophic bacterial activity) shows a somewhat typical depth profile, in that it decreases with increasing depth and is almost undetectable at the chemocline. Thus, the peak in bacterial numbers does not coincide with the peak in heterotrophic activity, resulting in extremely low specific activity (activity per cell) below the chemocline. (figure 1C).
What could be so different about the two lobes of the same lake as to result in different microbiology and nitrogen cycling? Why is nitrification occurring to the exclusion of denitrification in the east lobe? Are the bacteria in the abundance maximum of the the east lobe primarily nitrifying bacteria?
These observations led to a series of hypotheses (see Priscu et al., Antarctic Journal, in this issue). Briefly, we suggest that bacteria in the abundance maximum below the interface in the east lobe are either moribund and inactive or their metabolism is not heterotrophic. The abundance maximum could be due to nitrifying bacteria, autotrophs which would not incorporate thymidine (Johnstone and Jones 1989). We also propose that denitrification occurs in the west lobe but not in the east lobe, and the difference is reflected in differences in the distribution of denitrifying bacteria and other indicators of denitrification between the two lobes.
One aspect of our effort to reconcile the unusual nitrogen profiles in the two lobes of Lake Bonney (see Priscu et al., Antarctic Journal, in this issue) involves the localization of bacterial strains or types responsible for those chemical signals. Because nitrification and denitrification are the primary processes known to influence nitrogen concentrations in aphotic waters, we have focused our efforts on nitrifying and denitrifying bacteria. Other aspects of the project involving direct measurements of the transformations are reported elsewhere (Woolston and Priscu; Bartlett, Woolston, and Priscu, Antarctic Journal, in this issue).
To test the hypotheses concerning nitrifying bacteria, we are using radiotracer assays for ribulose bisphosphate carboxylase (Glover 1983), a key enzyme in the pathway used by nitrifiers and many other autotrophs to fix carbon dioxide. We are also developing DNA probes for the rubisco gene from autotrophic bacteria which will be used to detect the genetic potential for autotrophy. The third approach for the detection of nitrifying bacteria employs the polymerase chain reaction (PCR) to amplify ribosomal RNA genes from nitrifiers. A set of primers developed in our lab for the Nirtosomonas group of ammonium oxidizing nitrifiers is being used to analyze DNA collected from the lake during our first field season, austral spring of 1992.
We will use a second set of probes for the detection of denitrifying bacteria to investigate the denitrification question. Using samples collected in 1991, several denitrifying strains were isolated from Lake Bonney. One strain (ELB17) was used to produce polyclonal antibodies and to develop an immunofluorescent assay for cell enumeration, similar to those used in the past for both nitrifying and denitrifying bacteria (Ward and Carlucci 1985; Ward and Cockcroft 1993). This assay was then used to enumerate the strain in lake samples collected in 1992. As a more general probe, we have a DNA probe which recognizes a portion of the nitrite reductase gene, a key enzyme in the denitrification pathway (Ward, Cockcroft, and Kilpatrick 1993). The probe hybridizes with the ELB17 and has detected homology in samples of DNA extracted from lake samples.
Bacterial numbers (total, enumerated by DAPI or acridine fluorescence) showed a maximum (at about 25 m) below the oxic/anoxic interface (20 m) in the east lobe (figure 1A); bacterial activity was maximal at 13 m, the region of maximum primary production (J.C. Priscu unpublished data). Bacterial activity in the west lobe (figure 1B) coincided with the primary production maximum at 13 m.
Results from newly developed methods to study nitrifiers are still preliminary at this time. Preliminary results from the PCR amplification of nitrifying bacterial DNA from lake samples produced positive results only at 22 m in the east lobe. Although we have many more samples to analyze, including replicates, this preliminary finding is consistent with the concentration of nitrifying bacteria in this interval. Our hypothesis would be further supported by finding nitrifiers in high concentrations at the bacterial abundance maximum in the east lobe but by finding no such elevated concentrations in the west lobe.
The distribution of the ELB17 strain of denitrifying bacteria as enumerated by immunofluorescence is consistent with the apparent occurrence of bulk denitrification in the west lobe of the lake and not in the east lobe: ELB17 is present in the west lobe in much higher numbers than in the east lobe at comparable depths in the oxygen depleted deep waters (figure 2). ELB17 shows a distribution pattern that is quite different from the bacterial population as a whole, albeit at much lower abundance levels.
Before our next field season, we plan to analyze the approximately 100 DNA extracts we collected in 1992 (using tangential flow filtration to concentrate 4-liter samples). We are also planning to produce more polyclonal antisera to other Lake Bonney denitrifier isolates and to characterize these isolates as to their temperature and salinity ranges and optima. We have been unable so far to isolate any nitrifying strains from lake samples. Our nitrifier probes, however, are ready for application in next year's program.
This research was supported by National Science Foundation grant OPP 91-17907.
References
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