Salt Plains Microbial Observatory and LExEn Research: Characterization of Halotolerant Microbes

1
Salt Plains Microbial Observatory and LExEn
Research Characterization of Halotolerant
Microbes William Henley1, Kelly Major1,4,
Mark Schneegurt2, Mark Buchheim3, Robert Miller1
and Andy Potter1 1 Oklahoma State University
2 Wichita State University 3 University of
Tulsa 4 University of South Alabama
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.
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.
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.
Nannochloris growth rate (µ), light-saturated
photosynthetic capacity (Pmax), light-limited
photosynthetic efficiency (a), and chlorophyll
content decrease with increasing salinity above
25 ppt (Fig. 8), indicating that this species is
halotolerant rather than halophilic.
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.
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/art
sci/ 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 NH4 and soil chlorophyll biomass
(Fig. 4) suggests that nutrients also may partly
determine algal distribution. Mean soluble
reactive PO4 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).
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, ?, Pmax and Rd 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).
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.
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.
11107-15. 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.
7235-43.