mmmmmmbbbbbbbbbbbbbbmm

 

Boiling Springs Lake Thermal Area (BSLTA) is a pristine, high-elevation (1805 m) designated wilderness area of LNVP that includes a large, acidic (pH 2), hot (52C) lake known as Boiling Springs Lake (BSL), several weakly pressurized fumaroles, and small hot springs and mud pots of variable T and pH (Fig. 1). BSL occurs in an area of weakened rock along a fault zone, and is geologically and chemically distinct from caldera-type volcanic lakes typified by the Uzon Caldera (Kamchatka, Russia) and Frying Pan Lake (New Zealand), and from high chloride, lower sulfate features characteristic of the liquid-dominated geothermal systems in Yellowstone National Park (YNP) (7, 14). The BSLTA was extensively studied by USGS geologists from the 1980’s to mid-1990’s (12, 16, 25, 26). In 1988, the lake area covered approximately 12,000 m2(25), and the entire BSLTA covered approximately 14,000 m2. Therefore, BSL is approximately 1/3 the size, and significantly more acidic than the world's largest hot spring - Frying Pan Lake (pH 3.5, 52C)

Since the summer of 1999, we have determined approximate lake area, T and pH along lake margins throughout the summer months (June-August), and these values agreed with previous estimates suggesting this is a relatively stable feature. BSL surface water has an average summer T of ~52 +/-3 C, and an average pH value of 2.0 +/- 0.4. Winter water temperatures may be significantly cooler. At the northeast end of the lake is a small outflow stream that is typically dry by mid-July. The southern end of the lake appears to be the hottest region of BSLTA, where actively bubbling springs and mud pots are continually present, and T ranges from 65-95 C, even though many of these features are in direct contact with lake water. On the west and east sides of the lake are a variety of springs ranging in T from 25-93C, pH from 1-4.5.

Geochemistry: While different thermal features within the same thermal region vary widely in chemical composition (14, 22, 24, 26), conductivity and total dissolved solids (3520 S cm-1, and 1071 ppm, respectively) fall within the middle range for other LVNP sites analyzed. Geochemical analyses of BSL water (from 2004) indicate a fairly high SO42- concentration of 636 ppm (~8 mM), and low Cl- concentrations of 0.32 ppm (~9 µM) (22-24). These data are similar to those reported for this site in the mid-1980’s (26). Fe, Mg, and Mn are present within BSL water at 29.8 ppm, 4 ppm, and 16ppm, respectively. All other metals (with the exception of Al and Si) were found at fairly low concentrations (data not shown). NH4 is present at 0.8 ppm (~44 µM), while NO3- is less than 0.02 ppm. Dissolved organic carbon (DOC) was measured at 1.2 mg/L (summer 2004). Gas analyses of features within the BSLTA from the mid-1990’s indicate that mole percent dry weight of the volcanic gases emitted from various sites within the BSLTA are ~88% CO2, 6% N2, 4.7% H2S, 0.28% H2, 0.12% Ar, 0.072% CH4, 0.0015% NH3, 0.008%O2, 0.0007% He, with a gas/steam molar ratio of 0.0024 (14).


Microbiology:


i. Prokaryotes – Siering & Wilson, and undergraduates in their laboratories, have generated 16S rRNA gene libraries from BSL site A (Fig. 1) sediment using three different primer sets (Table 1). In each of the 3 clone libraries, the most numerous clones exhibit low sequence identity (< 88%) with cultured organisms. The A1116R and the U1406R libraries primarily contain Archaeal phylotypes that share <85% identity with the closest described isolates. Some of these are more closely related (97-98% identity) to environmental sequences retrieved from a 60°C, pH 3 sulfate-chloride spring in YNP (13). The P1525R library contained primarily Bacterial phylotypes that share <88% identity with described isolates. Both the 1406R and 1525R libraries have a few clones that show high similarity (>97%) to common soil/water bacteria that are unexpected in the conditions prevailing at BSL. There was overlap between the detected phylotypes with the different primer sets, but each library contains sequences not detected in the other libraries, reflecting the biases inherent in this approach. Rarefaction analyses indicate that the A1116R and 1406R libraries were sampled sufficiently to determine the majority of predominant phylotypes in the library; analysis of the 1525R library is continuing. An 18S rRNA gene library created from Site A water using the primer set Ek82F/U1200R contained exclusively fungal sequences that shared 96-98% identity with described fungal species. 16S rRNA gene libraries have also been constructed from DNA extracts of site A and site D water samples,and sequencing of these libraries is in progress.


Crude estimates of diversity in Site A water and sediments were also made by studying Terminal Restriction Fragment Length Polymorphisms (T-RF). The sequence data from the clone libraries allows us to gauge the most effective restriction enzymes for discriminating between predominant phylotypes, and it also allows us to assign T-RFs of a particular fragment length to a tentative phylotype. The observed T-RF chromatograms were reproducible and similar to those predicted from the clone library analysis (data not shown). Fragments derived from the novel phylotypes detected in the clone library were also detected in the T-RF chromatograms, but peak height could not be predicted based on clone library abundance, presumably due to cloning biases and PCR specificity changes associated with the use of labeled primers in T-RF studies. The 515F/1406R T-RF profile produced using DNA extracted from the overlying water was distinct from but comparable to that produced using sediment DNA. Of the fourteen predominant fragments detected in the sediment sample, six were also detected in the water sample. One of the two remaining fragments in the water sample was predicted from the sediment clone library. The relative abundance of shared fragments differed between the profiles obtained with the two samples.
Acridine orange direct counts (AODC) indicate approximate cell concentrations of 2.1 x 108 cells/ml sediment, and 1.2 x 106 cells/ml in water (2002 data) (22, 24). A Humboldt State University (HSU) undergraduate student previously obtained isolates from BSL that were identified (16S rDNA sequence) as a Geobacillus spp., Sulfobacillus spp. and Alicyclobacillus spp.. Only the Sulfobacillus sp. had been identified by the cloning efforts. The Stedman lab has cultured Sulfolobus-like organisms from high-temperature, acidic springs on the east side of BSL, but none from the lake itself. During the summer of 2005, Siering and 2 undergraduates (supported by HHMI and REU summer research fellowships) initiated efforts to cultivate heterotrophic prokaryotes from BSL sediments and waters. Liquid and solid media were prepared using filtered, autoclaved, pH-adjusted site waters. Gelrite-solidified media were prepared at pH 2.9, 5, and 7. Agarose-solidified media were prepared at pH 2. Liquid media were also prepared at pH 1, 2, and 3. Incubations (+/- O2) were done at 30, 50, and 70??qC. We used 3 different concentrations of organic amendments: peptone and tryptone at 5, 25, 250 mg/L; yeast extract and glucose at 10, 50, 500 mg/L. Vitamins and trace minerals were also added to all media. We prepared frozen stocks of 98 putative isolates representing all isolation conditions, and are presently determining the identity (16S rRNA gene sequence), and initiating physiological studies on a subset of these in the Genetics Lab and Bacteriology classes taught by Wilson and Siering, respectively at HSU.


ii. Eukaryotes –Dr. Gordon Wolfe's lab at CSU, Chico has been characterizing eukaryotic SSU rRNA diversity in acidic, high T samples from various areas within LVNP, including the BSLTA, over a wide variety of temperatures. After optimizing nucleic acid extraction and amplification protocols to maximize apparent eukaryotic diversity (by 18S rRNA gene-targeted DGGE), we found autotrophic protists to be the dominant eukaryotes; heterotrophic protists and fungi are also present, especially at lower temperature sites. Most sequences have high (>95%) similarity to other acidophilic chlorophycean and stramenopile taxa, with alveolates and cercozoa also represented (5).
At BSL, we believe there is a significant, if limited, protist community. SSU rRNA analysis also suggests the presence of chlorophytes (Chlorogonium) and chrysophytes (Poteriochromonas) at BLSTA, but we believe the dominant alga is the acidophilic rhodophyte Cyanidioschyzon and its close relative Cyanidium. We have detected these in 18S rRNA clone libraries from lake waters and side pools, observed them in micrographs, and cultured them following enrichments. Curiously, we have not found Cyanidiaceae at other LVNP sites using 18S rRNA methods, despite prior reports of their enrichment from the park (4, 8).

While we suspect the Cyanidiales are likely the main photosynthetic eukaryotic taxa at BSL, it is not clear if they predominate as planktonic or benthic, what limits their productivity, and the extent to which they contribute to food web dynamics. While light penetration is low due to turbidity, BSL has several shallow near-shore shelves that have visible benthic photosynthetic mats, and acidic mining lakes can support considerable benthic primary production (15). Enrichment studies suggest possible CO2 limitation (Fig. 2), well documented for other acidic lake environments (19), and although most members of this group appear to grow well both auto- and heterotrophically (8), temperature likely imposes a strong limitation on their distributions as well. We suspect that populations may be higher during cooler water column temperatures presumably present in winter months.


Although Cyanidiales physiology, genetics, and taxonomy have been well studied (6, 20 , 21), their ecology, and especially their interactions with prokaryotes, grazers, and viruses are still virtually unknown. We have not yet identified any heterotrophic protists in BSL waters. However, cloning has revealed 18S rRNA genes for acidophilic and thermophilic ciliates and cercozoans at other sites in LVNP that have similar pH and T ranges to BSL (5), and cooler acidic mine lakes can support populations of ciliates and other heterotrophic protists (17). The Cyanidiales can also excrete large amounts of organic matter to fuel acidophilic prokaryote or fungal growth (3). Both Wolfe’s and Siering & Wilson’s groups (11) have detected fungal SSU rRNA sequences at BSL, and speculate these may be fueled by both algal excretion and DOC from allochthonous forest leaf litter. In preliminary studies, fungi dominate enrichments from leaf and wood litter in BSL water incubated at room temperature, while in situ temperatures favor prokaryotes.

iii. Viruses. Dr. Ken Stedman’s lab at Portland State University has sampled extensively at high T (>70 C) and low pH (<4) locations elsewhere in LVNP as part of the NSF Microbial Observatories grant “Viruses from Yellowstone Thermal Acid Environments”. Recently they have isolated Sulfolobus-like organisms and have fusellovirus-like (SSV-like) sequences from peripheral springs at BSL (unpublished). The vast majority of BSL is too cold for Sulfolobus (<70C), so other viruses are likely to be found there. We have collected ca. 30 L samples of BSL water for microscopic analysis. Algal Phychodnaviruses or their relatives should be easy to identify because of their large icosahedral morphology (27, 28). Direct virus counts in BSL water have been low (103 ml-1, Stedman and Rohwer, unpublished) but similar to other high-T low pH systems.

 


References cited

  1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
  2. Baker, G. C., J. J. Smith, and D. A. Cowan. 2003. Review and analysis of domain-specific 16S primers. J. Microbiol. Methods 55:541-555.
  3. Belly, R. T., M. R. Tansey, and T. D. Brock. 1973. Algal excretion of 14C-labeled compounds and microbial interactions in Cyanidium caldarium mats. J. Phycol. 9:123-127.
  4. Brock, T. D. 1978. Thermophilic Microorganisms and Life at High Temperatures. Springer-Verlag, New York.
  5. Brown, P. B. 2005. A survey of protist genetic diversity in the hydrothermal environments of Lassen Volcanic National Park. M.S.Thesis. California State University, Chico. Chico, California.
  6. Ciniglia, C., H. S. Yoon, A. Pollio, G. Pinto, and D. Bhattacharya. 2004. Hidden biodiversity of the extremophilic Cyanidiales. Molec. Ecol. 13:1827-1838.
  7. Clynne, M. A. 2004. Volcano Hazards Team, United States Geological Survey. Menlo Park, CA. Personal Communication.
  8. Gross, W. 1999. Revision of comparative traits for the acido- and thermophilic red algae Cyanidium and Galdieria, p. 439-446. In J. Seckbach (ed.), Enigmatic Microorganisms and Life in Extreme Environments, vol. 1. Kluwer Academic Publishers, Dordrecht.
  9. Gross, W., I. Heilmann, D. Lenze, and K. Schnarrenberger. 2001. Biogeography of the Cyanidiaceae (Rhodophyta) based on 18S ribosomal RNA sequence data. Eur. J. Phycol. 36:275-280.
  10. Hamm, L. S., and P. L. Siering. 2005. Culture-independent measurement of microbial abundance in environmental samples from Boiling Springs Lake., Molecular and Cellular Systems Biology - CSU Biotechnology Symposium, Los Angeles, CA. January 14-16.
  11. Hauser, M. E., P. L. Siering, and M. S. Wilson. 2005. Molecular analysis of fungal diversity in a hot, acidic lake at Lassen Volcanic National Park., Molecular and Cellular Systems Biology - CSU Biotechnology Symposium, Los Angeles, CA. January 14-16.
  12. Ingebritsen, S. E., and M. L. Sorey. 1985. A quantitative analysis of the Lassen hydrothermal system, north central California. Water Resour. Res. 21:853-868.
  13. Jackson, C. R., H. W. Langner, J. Donahoe-Christiansen, W. P. Inskeep, and T. R. McDermott. 2001. Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring. Environ. Microbiol. 3:532-542.
  14. Janik, C. 2003. Volcano Hazards Team, United States Geological Survey. Menlo Park, CA. Personal Communication.
  15. Koschorreck, M., and J. Tittel. 2002. Benthic photosynthesis in an acidic mining lake (pH 2.6). Limnol. Ocean. 47:1197-1201.
  16. Muffler, L. J. P., N. L. Nehring, A. H. Truesdale, C. J. Janik, M. A. Clynee, and J. M. Thompson. 1982. The Lassen Geothermal System. Open-file Report 82-926. U.S. Geological Survey.
  17. Packroff, G. 2000. Protozooplankton in acidic mining lakes with special respect to ciliates. Hydrobiologia 433:157-166.
  18. Reysenbach, A.-L., and N. R. Pace. 1995. Reliable amplification of hyperthermophilic Archaeal 16S rRNA genes by the polymerase chain reaction, p. 101-106. In F. T. Robb and A. R. Place (ed.), Archaea - a laboratory manual (thermophiles). Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  19. Satake, K., and Y. Saijo. 1974. Carbon dioxide content and metabolic activity of microorganisms in some acid lakes in Japan. Limnol. Ocean. 19:331-338.
  20. Seckbach, J. 1999. The Cyanidiophyceae: hot spring acidophilic algae, p. 427-435. In J. Seckbach (ed.), Enigmatic Microorganisms and Life in Extreme Environments, vol. 1. Kluwer Academic Publishers, Dordrecht.
  21. Seckbach, J. (ed.). 1994. Evolutionary Pathways and Enigmatic Algae. Kluwer Academic Publishers, Dordrecht.
  22. Siering, P. L., J. M. Clarke, and M. S. Wilson. Accepted for publication, in revision. Geochemical and Biological Diversity of Acidic, Hot Springs in Lassen Volcanic National Park. Geomicrobiology Journal GJ(C)05-17.
  23. Siering, P. L., and M. S. Wilson. 2004. Geochemical and biological diversity in near-boiling acidic hotsprings., Abst. N271, In 104th General Meeting of the American Society for Microbiology, May 22-27, New Orleans, LA.
  24. Siering, P. L., and M. S. Wilson. 2001. Investigation of environmental and microbial diversity in high temperature, low pH geothermal features at Lassen Volcanic National Park., Abstr. N197 In 101st General Meeting of the American Society for Microbiology, May 19-23, Orlando, FL.
  25. Sorey, M. I., and E. M. Colvard. 1994. Measurements of heat and mass flow from thermal areas in Lassen Volcanic National Park, California, 1984-1993. Water Resources Investigations Report 94-4180-A. U.S. Geological Survey.
  26. Thompson, J. M. 1985. Chemistry of thermal and nonthermal springs in the vicinity of Lassen Volcanic National Park. J. Volcanol. and Geotherm. Energy 25:81-104.
  27. Van Etten, J. L., M. V. Graves, D. G. Mueller, W. Boland, and N. Delaroque. 2002. Phycodnaviridae – large DNA algal viruses. Arch. Virol. 147:1479-1516.
  28. Van Etten, J. L., and R. H. Meints. 1999. Giant viruses infecting algae. Ann. Rev. Microbiol. 53:447-494.
  29. Whitaker, R. J., D. W. Grogan, and J. W. Taylor. 2003. Geographic barriers isolate endemic populations of hyperthermophilic Archaea. Science 301:976-978.
  30. Wilson, M. S., A. S. Carter, and P. L. Siering. 2004. Molecular analysis of microbial communities in a 55??qC pH 1.7 lake in Lassen Volcanic National Park., Abst. N276, In 104th General Meeting of the American Society for Microbiology, May 22-27, New Orleans, LA.
  31. Wilson, M. S., R. M. Lehman, and F. S. Colwell. 1999. Comparison of culture and non-culture based approaches for quantifying methanotrophic bacteria in deep groundwater from the Snake River Plain Aquifer., In International Symposium on Subsurface Microbiology, Aug. 22-27., Vail, CO.