Lake Kauhako is meromictic, and characterized by what appears to be permanent stratification into a shallow (ca. 2 – 3 m) oxic zone, overlaying some 245 m of anoxic, sulfide rich water. Water samples collected represented 3 distinct fractions of the water column, specifically the surface, the chemocline at 4 m, and a ‘deep’ sample from 30 m. Data determined in the field and subsequently in the laboratory confirmed the distinct chemical differences between these depths. For example, alkalinity more than doubled between the surface (2.26 meq/L) and 4 m (5.47 meq/L), and almost doubled again by 30 m (9.92 meq/L). Moreover, pH between the surface and 4 m (8.6 – 8.5), fell to only 6.8 by 30 m. We utilized such data by tailoring media to enrich specific microorganisms, particularly of those among the sulfate-reducing d-Proteobacteria. Water samples only were retrieved from Lake Kauhako during collection in June 2002. The bottom of the 850 ft. deep lake is covered by chaotic boulders from previous eruptions and erosion activity. Attempts to retrieve benthic sediments with a “Ponar” grab were frustrated by such features.

Kauhakō water chemistry figure

 

At least 14 Bacteria taxa were represented in the Lake Kauhako clone library (Table 1). As with clone libraries we constructed for other sites, the majority of OTUs belonged in the a- and g-Proteobacteria. Of particular note is the complete absence of clones that affiliate with the Firmicutes; this taxon is in fact represented in all clone and culture libraries for the other sites we have investigated under this proposal. Indeed, 13 distinct Firmicute cultures were isolated from the same Kauhako water samples used to compile the clone libraries (Table 1). Two cyanobacteria clones were determined, but neither affiliated with the Prochlorococcus, a genus previously detected in Lake Kauhako through the presence of divinyl chlorophyll a, a marker pigment for these bacteria, and by flow cytometry. Forty four phylogenetically distinct bacteria cultures were prepared from an initial screen of 95 isolates (Table 1). These distinct cultures belong in 7 major taxa, including the Actinobacteria, Bacteroidetes, a-, b-, g-, and d-Proteobacteria, and the Firmicutes. Eighteen cultures share <97% 16S rDNA sequence identity with their nearest neighbor in the public domain, indicating they may be novel species. With degrees of 16S rDNA identity ranging from 89.7%, new genera will have to be established to accommodate some of the cultures (Tables. 1, 2).