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
4
of Tulsa; University of South Alabama
Abstract
LExEn: Physiological Characterization of Algae
Microbial Observatory: Microbial Isolation & Characterization
The Salt Plains National Wildlife Refuge in Oklahoma is a minimally studied terrestrialaquatic 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.
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.
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 (RodriguezValera 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.
Fig. 6. Dunaliella sp.
Length ~5-7 µm.
Fig. 7A. Nannochloris sp.
Length ~2-3 µm.
Fig. 7B. Week-old 10% salt cultures of
3 marine Nannochloris UTEX strains
(left) and our SPNWR isolate (right).
Fig. 11. Presumed Archaea
isolates from the SPNWR.
This domain assignment
requires confirmation.
Nannochloris growth rate (µ), light-saturated photosynthetic capacity (Pmax), lightlimited photosynthetic efficiency (a), and chlorophyll content decrease with increasing salinity
above 25 ppt (Fig. 8), indicating that this species is halotolerant rather than halophilic.
Fig. 2. Aerial photo of the SPNWR
region (from www.mapquest.com).
Note the reservoir, various creeks,
and variable salt crust.
Fig. 1. View of the SPNWR salt flats
following a prolonged rain-free period.
Fig. 8. A. Net photosynthetic capacity
(Pmax), dark respiration (Rd) and
photosynthetic efficiency (a); 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, a, 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).
SCD 2001
Temperature (°C)
50
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.
40
30
Isolate
Gram Morphology
nd
-
F1C-y
F1C-y2
F1S-y
GC-o
GC-p
GC-w
GC-y
HS-o
HD-w
HD-y
P1C-p
P1C-tr
P1C-y
P1D-y
P2C-p
P2C-yw
P1S-a
P1S-b
P1S-p
P1S-p1
SD
SS-w
SS-y
HD-o low
HS low
faint +/faint faint +
+/+/-
SD-sm low
SS-o low
SS-p low
SS-w low
+/+
faint -
Mot Oxid- Catal-ility ase
ase
nd
+
+
+
indeterminate
coccobacilli, freq.
filaments
very small rods
+
small rods
very small rods
very small cocci
indeterminate
+
indeterminate
nd
indeterminate
nd
very small cocci
long large rods
large rods
small coccobacilli
cocci
very small cocci
cocci, in clusters of 8-10
very small cocci
small rods
small cocci
rods
cocci
rods, diplobacilli
rods, some short filaments cocci
short rods
coccobacilli, longer rods
very small rods, some
filaments
short rods, gas-vacuoles
short rods & coccobacilli
+
very small rods
+
very small rods
+
+
+
+
+
nd
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
nd
nd
nd
nd
+
+
+
+
+
-
+
+
+
+
+
nd
nd
+
+
nd
+
+
nd
Gelatin Starch Lipid Ferm- H2S
hydrol. hydrol. hydr. ent. Prod.
+
+
nd
nd
nd
nd
weak +
+
nd
nd
nd
weak +
nd
+
+
nd
nd
+
+
+
nd
nd
nd
+
+
+
nd
nd
+
nd
nd
+
+
+
+
+
nd
nd
nd
+
nd
nd
+
+
+
+
weak
+
+
+
+
+
+
+
+
+
+
+
+
+
nd
+
nd
-
nd
nd
nd
nd
nd
nd
+
-
nd
nd
nd
nd
+
weak
-
-
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.
19 July
14 July
9 July
4 July
29 June
24 June
20
19 June
Table 1. Preliminary phenetic characterization of bacterial isolates from SPNWR.
F1C-p
F1C-p2
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 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).
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. 3. Soil surface temperature on the SPNWR
salt flats.
Fig. 4. Groundwater NH4, (NO3 + NO2)
and soil chlorophyll biomass at three
SPNWR sites.
Fig. 5. Examples of diatoms, chlorophytes and cyanobacteria from the 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.
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. 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.