A novel thermoalkaliphilic bacterium, strain Eh-2, was isolated from a hot spring at Lake Shala in Ethiopia. Eh-2 grows optimally at 55°C and pH 7.5-8.9 and can tolerate up to 6% salinity. Based on 16S rRNA sequencing, Eh-2 is most closely related to Natronicola peptidovorans and Anaerovirgula multivorans but differs significantly in physiology. Eh-2 represents a potential new species or genus. Thermostable enzymes from Eh-2 may have industrial applications and it could serve as a model organism for studying life in extreme environments.

1. Characterization of a Novel Thermoalkaliphilic Bacterium
Isolated from a Hot Spring at Lake Shala, Ethiopia
Jessica Hardwicke, Yitai Liu, Anna-Louise Reysenbach
Biology Department, Center for Life in Extreme Environments, Portland State University, Portland, OR 97201, USA
Background
Methods
Results
Conclusions References
Lake Shala is a highly alkaline lake
located in the East African Rift
Valley of Ethiopia.
Volcanic activity in the area forms
hot springs that feed into the
lake, creating a unique niche for
thermoalkaliphiles to inhabit.
The microorganisms thrive at
temperatures above 40°C and pH
values of 8.0 and higher (Le Turdu
et al., 1999).
Acknowledgments
Here we report the description
of a novel thermoalkaliphile
from the thermal springs on
the bank of Lake Shala. Studies
such as this are necessary in
order to understand the unique
environmental adaptations
these extremophiles possess.
• Strain Eh-2 grows in a wide range of conditions,
with an optimal at 55°C and pH between 7.5-8.9.
It is halotolerant and can grow in up to 6.0% (w/v)
salinity, likely higher.
• Based on its 16S rRNA sequence there are only
two organisms currently described that have
significant phylogenetic similarity: Natronicola
peptidovorans (95.04%) and Anaerovirgula
multivorans (94.76%).
• Using the standard species-level cutoff of 99% 16S
rRNA sequence identity and genus-level cutoff of
95%, Eh-2 represents a novel species. Considering
the substantial metabolic and physiological
differences it is likely a candidate novel genus.
Applications
• Thermostable and alkaline-stable enzymes
present in Eh-2 my be valuable for industrial
applications including detergents, biofuels and
biopolymers (Kwon et al., 2013).
• Researching extremophiles contributes to
understanding how life might arise beyond
Earth, and what precautions should be taken
when exploring our solar system.
• There is a lack of genetic data available for
thermoalkaliphiles, and Eh-2 may provide an
ideal model organism for study
This research was funded by NSF grant DEB-1134877
to ALR. Support for this project was also funded by the
Ronald R. McNair Scholars program and the Oregon NASA/
Oregon Space grant Consortium, grant NNX10AK68H.
Kristen Brileya is thanked for her guidance in the project
and Rick Davis for his assistance in the phylogenetic
analyses.
Kwon, J. C. M., & Young, H. (2013). Isolation and characterization of a
metagenome-derived thermoalkaliphilic esterase with high stability over a
broad pH range. Extremophiles, 17(6), 1013–1021.
Le Turdu, C., Tiercelin, J. J., Gibert, E., Travi, Y., Lezzar, K. E.,
Richert, J. P., … Taieb, M. (1999). The Ziway-Shala lake basin system,
Main Ethiopian Rift: Influence of volcanism, tectonics, and climatic forcing
on basin formation and sedimentation. Palaeogeography, Palaeoclimatology,
Palaeoecology, 150(3-4), 135–177.
Contact
• Jessica Hardwicke
jhardwic@uoregon.edu
• Dr. Anna-Louise Reysenbach
bwar@pdx.edu
www.arlab.pdx.edu
Characteristic Eh-2 N. peptidovorans A. multivorans
Gram stain positive positive positive
Cell length (µm) 2-5 1.5-8.0 2.5-5
pH for growth
Optimal 7.8-9.6 8.4-8.8 8.5
Range 6.5-11.0 7.5-10.2 6.7-10.0
Temperature for growth (°C)
Optimal 55 35-37 35
Range 35-63 15-40 10-45
NaCl for growth (% w/v)
Optimal 1.0 ND 2.0
Range 0-6.0+ 0-7 0.5-9.0
Characteristic Eh-2 N. peptidovorans A. multivorans
Yeast extract + + +
Peptone + + +
Casamino acids + + +
Glucose + - +
Sucrose + - +
Fructose + - +
Acetate - ND -
Cellobiose - - +
Crotonate - - -
Ethanol - - -
Formate - ND -
Methanol - - +
Ribose - - +
Starch - ND +
Eh-2_16SrDNA
gi|664616069|emb|LK391562.1| Clostridiales bacterium S55_27_1 partial 16S rRNA gene, isolate S55_27_1
gi|636559943|ref|NR_116003.1| Natronincola peptidivorans strain Z-7031 16S ribosomal RNA gene, partial sequence
gi|343200604|ref|NR_041291.1| Anaerovirgula multivorans strain SCA 16S ribosomal RNA gene, partial sequence
gi|645320525|ref|NR_117711.1| Clostridium formicaceticum strain DSM 92 16S ribosomal RNA gene, partial sequence
gi|636560428|ref|NR_116488.1| Natronincola ferrireducens strain Z-0511 16S ribosomal RNA gene, partial sequence
gi|219846863|ref|NR_026455.1| Natronincola histidinovorans strain Z-7940 16S ribosomal RNA gene, partial sequence
gi|219857574|ref|NR_025162.1| Tindallia californiensis strain APO 16S ribosomal RNA gene, partial sequence
gi|1684664|emb|Z69927.1| C.paradoxum 16S rRNA gene (clone para1)
gi|444304030|ref|NR_074454.1| Clostridium difficile 630 strain 630 16S ribosomal RNA, complete sequence
gi|659364726|ref|NR_121725.1| Eubacterium acidaminophilum strain a1-2 16S ribosomal RNA gene, complete sequence
gi|183986843|gb|EU652084.1| Thermaerobacter subterraneus strain mt-14 16S ribosomal RNA gene, partial sequence
0.02
Clostridiales bacterium S55_27_1 [LK391562] (Soda lake, Hungary)
Natronicola peptidovorans [NR_116003.1] (Soda lake, Russia)
Anaerovirgula multivorans [NR_041291.1] (Soda lake, USA)
Clostridium formicaceticum [NR_117711.1] (Sewage plant)
Natronicola ferrireducens [NR_026455.1] (Soda lake, Russia)
Natronicola histidinovorans [NR_026455.1] (Soda lake, Kenya)
Tindallia californiensis [NR_025162.1] (Soda lake, USA)
Clostridium paradoxum [Z69927.1] (Sewage plant)
Clostridium difficile [NR_074454.1] (Human gut)
Eubacterium acidaminophilum [NR_121725.1] (Sewage plant)
Thermaerobacter subterraneus [EU652084.1] (Hot spring, China)
Eh-2
Fig. 2: Partial map of Lake Shala
system, including hot spring inlets
denoted by * (Le Turdu et al., 1999).
Fig. 1: Collecting samples in hot springs at the
banks of Lake Shala. Courtesy of Anna-Louise
Reysenbach.
Fig. 5: Maximum likelihood 16S rRNA phylogenetic tree of close relatives of Eh-2. Included are
[ascension number] and (environment, location).
Table 2: Characteristic comparisons of Eh-2 and related taxa. ND, Not determined.
Table 1: Substrate utilization of strain Eh-2 and its phylogenetically closest related taxa, Natronicola
peptidovorans & Anaerovirgula multivorans. +, Positive; -, Negative; ND, Not determined.
Fig. 3: Scanning Electron Micrograph (SEM) of
Eh-2 cells, exponential growth phase at 60°C.
Fig. 4: SEM of Eh-2 cells, stationary phase at
60°C.