1. Salt Plains Microbial Observatory and LExEn Research: Characterization of Halotolerant Microbes William Henley 1, Kelly Major 1,4, Mark Schneegurt 2, Mark Buchheim 3, Robert Miller 1 and Andy Potter 1 1 Oklahoma State University; 2 Wichita State University; 3 University of Tulsa; 4 University of South Alabama Abstract The Salt Plains National Wildlife Refuge in Oklahoma is a minimally studied terrestrial- aquatic ecotone featuring wide temporal swings in salinity and temperature and intense solar irradiation. The salt flats are perpetually moist from Permian brine diffusely seeping to the surface, which leaves a salt crust on the surface (Figs. 1, 2). We are isolating heterotrophic bacteria and archaea in media varying in salinity, Mg and temperature, and in media selective for diazotrophs and nitrifiers. Chlorophytes, diatoms and cyanobacteria are among the photoautotrophs isolated to date. All microbial isolates will be characterized phenetically, and RFLP and rDNA sequencing will be performed on selected unique isolates. We also will investigate the role of the recA gene in stress tolerance in bacteria. We have characterized two chlorophyte isolates with respect to salinity and thermal tolerance. At moderate temperatures, Dunaliella sp. tolerates saturated brine (>30% total salts) and grows best at about 10%, whereas Nannochloris sp. grows fastest between 0-5% and tolerates up to 15%. SPNWR Nannochloris is more broadly halotolerant than marine congeners. At 5%, Dunaliella and Nannochloris tolerate 2-h exposures to 41.5 and 45 °C, respectively. Both species exhibit greater thermotolerance at 10% compared to 2%. Supported by NSF-MCB grants 9978203, 0132097, 0132083 and 0131659. Introduction and Ecological Context Extremophiles are usually arbitrarily defined as those organisms inhabiting one particular environmental condition (e.g. temperature, salinity, pH) beyond the range typical of most organisms. Two often overlooked components of “extremeness” are a wide dynamic range of conditions rather than a relatively stable environment (Gorbushina & Krumbein 1999), and two or more concurrent stress factors. Most studies of halophiles have been in chronically hypersaline lakes (e.g. Dead Sea, Great Salt Lake) or coastal solar salterns. The Salt Plains National Wildlife Refuge (SPNWR) in Oklahoma is an unusual natural semiaquatic ecosystem where salinity of surface pools and interstitial water may vary greatly in space and time from nearly freshwater to saturated brine. It thus may represent a more extreme environment in the sense that resident organisms must tolerate widely varying rather than permanently high salinity. Through ongoing NSF Life in Extreme Environments research (www.okstate.edu/artsci/ biol3252/LExEn/LExEn.htm) and a new collaborative NSF Salt Plains Microbial Observatory project, we have begun to characterize the environmental conditions, and isolate and characterize the heterotrophic and photosynthetic microbes from the SPNWR. For example, widely fluctuating temperature (Fig. 3) and intense solar irradiation are potential stress factors in addition to variable salt stress. Preliminary evidence of a correlation between high NH 4 and soil chlorophyll biomass (Fig. 4) suggests that nutrients also may partly determine algal distribution. Mean soluble reactive PO 4 concentrations are extremely low (0.1-0.7 µm) at all stations (not shown). Although algal biomass appears to be relatively low, preliminary indications are that considerable diversity is present, at least in surface pools (Fig. 5). LExEn: Physiological Characterization of Algae We isolated two chlorophyte algae from the SPNWR for physiological characterization. A new isolate of the classic halophilic biflagellate genus Dunaliella (Fig. 6) does not appear to be a carotenoid accumulating strain. Originally isolated from saturated brine (>30%), it tolerates the full range of salinities encountered at the SPNWR, and is found in most soil and water samples. Nannochloris sp. (Fig. 7A) grows from 0 to 15% salinity in culture, whereas marine Nannochloris strains from UTEX do not grow at 10% salt (Fig. 7B). Limited observations suggest that it also may be relatively common at the SPNWR, although its small size and lack of morphological characters precludes definitive statements about its occurrence. Fig. 5. Examples of diatoms, chlorophytes and cyanobacteria from the SPNWR. Fig. 1. View of the SPNWR salt flats following a prolonged rain-free period. Fig. 2. Aerial photo of the SPNWR region (from www.mapquest.com). Note the reservoir, various creeks, and variable salt crust. Nannochloris growth rate (µ), light-saturated photosynthetic capacity (P max ), light- limited photosynthetic efficiency (  ), and chlorophyll content decrease with increasing salinity above 25 ppt (Fig. 8), indicating that this species is halotolerant rather than halophilic. Fig. 8. A. Net photosynthetic capacity (P max ), dark respiration (R d ) and photosynthetic efficiency (  ); and B. growth rate (µ), chl a + b and chl b:a ratio (mean ± SD, n = 3) of Nannochloris sp. as a function of salinity in AS medium. Under moderate light and temperature, both Nannochloris and Dunaliella maintain high photosystem II photochemical efficiency regardless of growth salinity from 2 to 10% (Fig. 9), despite a decrease in µ, , P max and R d of Nannochloris (Fig. 8) over the same salinity range. However, both species exhibit a distinct salinity-dependent heat stress sensitivity; cells are more heat resistant and recover faster at higher salinities as determined by variable fluorescence yield (Fig. 9) and low temperature fluorescence emission spectra (Fig. 10). These heat treatments are well within the range of soil surface temperatures at the SPNWR (Fig. 3). Fig. 9. Photosynthetic efficiency (variable fluorescence yield measured with a Walz PAM fluorometer) in Nannochloris and Dunaliella grown and heated at three salinities (SP medium), during 2 h sublethal heat treatments and subsequent recovery. Fig. 10. Low temperature (77 K) fluorescence emission spectra of Nannochloris and Dunaliella following 2 h sublethal heat treatments and subsequent recovery at 20 and 100 ppt salinity (SP medium). Results for 50 ppt salinity (not shown) were intermediate between 20 and 100 ppt. Fig. 3. Soil surface temperature on the SPNWR salt flats. Fig. 4. Groundwater NH 4, (NO 3 + NO 2 ) and soil chlorophyll biomass at three SPNWR sites. Fig. 7A. Nannochloris sp. Length ~2-3 µm. Fig. 6. Dunaliella sp. Length ~5-7 µm. Fig. 7B. Week-old 10% salt cultures of 3 marine Nannochloris UTEX strains (left) and our SPNWR isolate (right). Microbial Observatory: Microbial Isolation & Characterization We established enrichment cultures using soil samples from the SPNWR. Surface samples (top cm) and deeper soil samples (10 cm) were collected from salt-crusted areas of the plains. Rich complex media were prepared following published protocols (Rodriguez- Valera et al. 1981, 1985) that contained 10% or 18% salts (mainly NaCl) with glucose, peptone, and yeast extract. A medium with 25% salts and higher magnesium concentrations was used to select for Archaea. Liquid shake-flask cultures were maintained at room temperature or at 37° C after inoculation with soil. The soils also were directly applied to agar plates. Liquid cultures were grown for several days and serially diluted and plated. Colonies that differed in appearance were collected and sequentially streaked on fresh plates at least eight times before being considered pure isolates. Table 1. Preliminary phenetic characterization of bacterial isolates from SPNWR. References Gorbushina, A.A. & W.E. Krumbein. 1999. Poikilotrophic response of microorganisms to shifting alkalinity, salinity, temperature and water potential. In: Oren, A. [Ed.] Microbiology and Biogeochemistry of Hypersaline Environments. CRC Press, Boca Raton, pp. 75-86. Rodriguez-Valera, F., A. Ventosa & J.F. Imhoff. 1985. Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern. Microbial Ecol. 11:107-15. Rodriguez-Valera, F., F. Ruiz-Berraquero & A. Ramos-Cormenzana. 1981. Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. Microbial Ecol. 7:235-43. At this time, approximately 70 morphologically distinct isolates have been obtained from the initial round of enrichments. Phenetic characterization of the isolates is underway. Selected biochemical and physiological results from 31 isolates are given in Table 1. These isolates are all believed to be Bacteria. More than a dozen other isolates are believed at this time to be Archaea (Fig. 11). A small group of fungi have also been isolated and are being identified and characterized. Fig. 11. Presumed Archaea isolates from the SPNWR. This domain assignment requires confirmation. A great majority (77%) of the isolates obtained from enrichment cultures of salt plains soils are Gram negative. Most are non-motile (71%). Oxidase and catalase activities were widespread (71% and 48%, respectively). It is interesting to note that the majority of the isolates (61%) are facultative organisms that can ferment one or more substrate (sucrose, glucose, lactose). Phenetic characterization will be used to identify the isolates to the genus level. Selected organisms will be subjected to phylogenetic analysis by cloning and sequencing rRNA genes. Growth curves of selected isolates are shown in Figure 12. Fig. 12. Growth curves of bacterial isolates from the SPMO grown in Rich complex medium as described above. Numbers in parentheses are generation times. Generation time range from 87 to 165 min.