LExEn: Response of Photosynthetic Microbes of the Salt Plains

National Wildlife Refuge to Dynamic Extreme Conditions

William J. Henley

Department of Botany, Oklahoma State University

This project is supported by the National Science Foundation, more specifically the Life in Extreme Environments Program (grant MCB-9978203). It would not be possible without the cooperation (access permission) and material assistance (use of ATVs, trailer and fuel) of Salt Plains NWR staff, part of the U.S. Fish & Wildlife Service. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation (NSF), the U.S. Fish & Wildlife Service, or the Salt Plains NWR.

Also visit our NSF Salt Plains Microbial Observatory.

PROJECT SUMMARY

Extreme environments are often defined as those chronically exhibiting an arbitrarily high or low level of a particular physical or chemical factor. Extremophilic organisms tolerant of such conditions have yielded valuable insight into the functioning of organisms inhabiting more typical conditions, including crop plants. Few studies have addressed multiple variable stress factors likely to be even more adverse to life. Evaporitic salt flats, such as at the Salt Plains National Wildlife Refuge in Oklahoma, represent such a dynamically stressful habitat: direct sunlight, up to 20-30° C diel (60-70° C annual) surface temperature range, episodically varying interstitial and surface pool salinities from near 0 to over 300 g/L, and potentially wide diel fluctuation in pH, redox potential, and dissolved gases and nutrients. Thus, the mostly undescribed photosynthetic and heterotrophic microbes in this habitat may provide unique insights into the evolution of life beyond the limits of tolerance for virtually all species on Earth. The proposed general hypothesis is that interacting stress factors determine survival, productivity and competitive outcomes in hypersaline photoautotrophs, and that halophiles are tolerant of wide fluctuations in physiocochemical conditions in addition to steady-state extreme conditions. The objectives are: (1) isolation and physiological characterization of salt flat cyanobacteria and algae; (2) temporal and spatial characterization of chlorophyll biomass in relation to the dynamic salt flat physical/chemical conditions (aided by a nearby Oklahoma Mesonet climatological station); (3) in situ manipulations of physical and chemical conditions to identify limiting and stress factors; and (4) laboratory culture experiments to test specific a priori hypotheses and others inspired by objectives 1-3. Comparisons will be made of the physiological responses to temperature, nutrients, visible and ultraviolet irradiance, and constant versus variable salinity. Laboratory analytical techniques include photosynthesis and respiration rate measurements (O₂ exchange) and chlorophyll fluorescence analysis to diagnose acute and chronic stress effects on the photosynthetic apparatus.

SALT PLAINS IMAGES

SPNWR location:

Salt flats photo:

Aerial photo of the Salt Plains region (from Mapquest.com)

FIELD MONITORING

One of our goals is to characterize the range of physical and chemical conditions on the flats and in shallow ephemeral pools. For example:

One remarkable feature of the salt flats is extremely high daytime soil surface temperatures in summer, and a huge day/night temperature range, as seen in this continuous temperature trace in June/July 2001. Surface temperatures may be 10° C or more above air temperature on sunny days, unless the wind is strong enough for evaporative cooling of the moist soil surface. We are trying to determine if algae and cyanobacteria can survive in the soil under such extreme conditions, or if they are restricted to shallow pools (which are probably slightly cooler) near the intermittent creeks that episodically flood the flats surface.

We have set up three stations, each with three shallow (~1 m) wells to help us sample the near-surface groundwater:

Groundwater samples are analyzed for salinity, nitrate, ammonium, and orthophosphate. We are also attempting to relate soil chlorophyll biomass (which, not surprisingly, appears to be very low) to such chemical and physical factors. [Chlorophyll, a universal photosynthetic pigment, is an indicator of the amount of algae and cyanobacteria present.]

IN SITU MANIPULATIVE EXPERIMENTS

In addition, we are conducting in situ manipulations to determine what factors significantly influence chlorophyll biomass. Initially, we are shading the surface (upper left photo) and adding specific nutrients to semi-enclosed plots (upper right photo) and looking for a change in chlorophyll biomass in response to these treatments.

LABORATORY EXPERIMENTS

Laboratory experiments are being conducted to determine the physiological response of individual algal isolates (mainly Nannochloris and Dunaliella so far) to various controlled conditions of salinity, photon fluence rate (“light intensity”), and high temperature stress, either singly or in combination. Physiological variables include specific growth rate, chlorophyll content, photosynthesis and respiration rates, and variable fluorescence analyses. One interesting finding to date is that growth at moderately high salinities increases short-term thermal tolerance, even if cells have reduced growth rates at the higher salinity. Algae grow well under moderately bright fluorescent lights at room temperature, but we also use a microprocessor-controlled incubator with programmable diel temperature in addition to photoperiod, and a room equipped with two 1000-W metal halide lamps that attain close to full sunlight over a limited area:

PERSONNEL

Much of the work on this project is currently being conducted by:

Postdoctoral Associate Dr. Kelly Major, now an Assistant Professor at the University of South Alabama

Undergraduate student technician Andy Potter

Undergraduate student technician Marga Mlenek

High school student Laura Ward

Undergraduate student technician Allyson Fry

Others who have participated in this project in the past include graduate students Janice Hironaka and Dan Ratcliff, undergraduate research students Tambra Stevenson, George Heine (now a graduate student at Penn. State), Cathy Sonleitner, and high school student Julie Ward (now an undergraduate student at Univ. of Tulsa).

ALGAL ISOLATES

Although our primary goal is ecophysiological rather than to survey or isolate numerous algae from this habitat, graduate student Janice Hironaka isolated a few strains for use in physiological experiments. One appears to be a new species of the coccoid chlorophyte Nannochloris, which tolerates salinities from 0 to 150 g/L, i.e. >4X seawater. Another is the common genus of halophytic chlorophyte flagellate Dunaliella, which tolerates saturated brine (>300 g/L). We also have in culture, but have not yet characterized, a filamentous cyanobacterium resembling Oscillatoria. There are certainly many other algae and cyanobacteria present, at least in the moderate salinity areas. A few examples are shown below.

Dunaliella	Nannochloris	Oscillatoria-like cyanobacterium	Spirulina-like cyanobacterium	pennate diatom, etc.

PUBLICATIONS

Henley,W.J., K.M. Major and J.L. Hironaka. 2002. Response to salinity and heat stress in two halotolerant chlorophyte algae. J. Phycol. 38:757-766.

Henley,W.J. 2001. Algae and cyanobacteria under desiccation and ionic stress. Nova Hedwigia 123:443-452.

Henley,W.J., J.L. Hironaka, M.A. Buchheim, J.A. Buchheim, M.W. Fawley & K.P. Fawley. 2004. Phylogenetic analysis of the “Nannochloris-like” algae and diagnoses of Picochlorum oklahomensis gen. et sp. nov. (Trebouxiophyceae, Chlorophyta). Phycologia 43:641-652.

Kvíderová, J. & W.J. Henley. 2005. The effect of ampicillin and streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae. J. Appl. Phycol. 17:301-307.

Major, K.M., A.E. Kirkwood, C.S. Major, J.W. McCreadie & W.J. Henley. 2006. In situ studies of algal biomass in relation to physicochemical characteristics of the Great Salt Plains, Oklahoma, USA. Saline Systems 1:11.