1. Conclusions
• Thus far, we have been able to amplify archaeal 16S rDNA
from samples taken from Eleuthera and a salt marsh in
Tuckerton, NJ (not shown). Preliminary DNA sequencing
analysis places the microbes from Eleuthera in the genus
Haloferax. Haloferax grow in a salinity range of 1.5 M-3.3 M.
Enrichment culture samples from which these haloarchaea
were identified were cultured in 1.5 M NaCl.
• Further analysis of cultivated sand samples from Eleuthera
contained organisms related to halophilic bacteria of the genus
Halomonas. Halomonas species are moderate halophiles
commonly found in marine environments.
• We have been able to isolate halophilic bacterial DNA from all
environments tested. This result is unsurprising as most
bacterial halophiles are moderate halophiles. Moderate
halophily is defined as organisms that grow in salinities
ranging from 0.8 M-2.5 M.
• Evidence supporting the existence of halophilic archaea in
seawater will be crucial in explaining how these organisms
began to occupy hypersaline environments.
• Identification of culturable haloarchaea in seawater will allow
us to begin elucidating the genetic and biochemical
adaptations that allow them to migrate from “low” salinity
(i.e., seawater) to higher salinities prevalent in salterns or
hypersaline seas.
• Data obtained from this research may suggest that the term
‘obligate halophile’ may need to be reexamined.
• We plan to take daily samples at each individual salinity
point during enrichment culturing. This will allow us to obtain
a comprehensive analysis of how these microbial
communities change over time in response to changing
salinity.
• Thus far, we have only examined a very small number of
samples from our enrichment culturing efforts. We anticipate
many other novel bacterial and archaeal species will be
revealed as a result of our work.
Significance
Works Cited
1. Cummings & Gilmore. (1995) The effect of NaCl on the growth of a Halomonas species: accumulation
and utilization of compatible solute. Microbiol. 141: 1413-1418.
2. Paul, S., et al. (2008). Molecular signature of hypersaline adaptation: Insights from genome and
proteome composition of halophilic prokaryotes. Genome Biol. 9(4) PAGES
3. Rodriguez-Valera et al. (1979) Isolation of extreme halophiles from seawater. App. Environ. Microbiol.
38: 164-165.
4. Ciaramella & Bidle. (2009) Cultivation of Halophilic Archaea and Bacteria from a coastal salt
marsh. Rider University Baccalaureate Honors Thesis.
5. Inoue, K., et al. (2011) Halomarina oriensis gen. nov., sp. nov., a halophilic archaeon isolated from a
seawater aquarium. Int. J. Sys. Evol. Microbiol. 61: 942-946.
6. Ruslan, Mamedov. Panorimo – photo of Bahamas Eleuthera Island. Pink Sand Beach.
http://www.panoramio.com/photo/84315288 {image}.
7. Burt, William. William Burt Photographer. http://www.williamburt.com/gallery.html {image}.
Acknowledgements
• Ronald E. McNair Program
• Independent College Fund of NJ
• Rider University Undergraduate Travel Grant Program
• ABRCMS Student Travel Award
• Dr. Paul Jivoff
Methods
Isolation and Cultivation of Halophilic Microorganisms from
Various Marine Environments
Brandon Enalls, Michael Maniscalco, Christopher Marrocco, and Kelly Bidle
Rider University, Department of Biology, Lawrenceville, NJ 08648
#F148
Introduction
Extremophiles are microorganisms that are able to withstand
environmental conditions that are too extreme to host most other
living things. These conditions can include, but are not limited to,
extremes of temperature, salinity, and/or pH. Of particular
interest in our research are halophiles, which are microbes that
live in environments of high salinity, such as the Dead Sea, the
Great Salt Lake, or solar salterns where NaCl levels can reach
upwards of 4 M (24%).
There are two distinct osmotic strategies employed by halophilic
microbes to thrive in hypersaline environments. Halophilic
bacteria accumulate compatible solutes, such as glycine betaine,
in their cytoplasm in order to keep excess salt from entering their
cells1. Conversely, halophilic archaea employ a “salt-in” strategy,
whereby they accumulate comparable levels of potassium ions in
their cytoplasm to match that of the sodium ions in the
surrounding environment. This reduces the osmotic gradient
between the cell and its environment2. In addition, haloarchaeal
proteins contain hydrophilic amino acids that help prevent
aggregation in their saline cytoplasms2. Many of these residues
are negatively charged and can form salt bridges with basic
residues2.
The majority of cultivated halophiles come from the domain
Archaea, the second major prokaryotic domain of life.
Haloarchaea are obligate halophiles, meaning they cannot grow
in environments containing salinity less than ~1 M. (5.8%). This
raises an interesting question. Given that seawater is ~330 mM
(3%) in terms of total salts, how do the haloarchaea transport
between environments?
Previously, genes of halophilic lineage have been isolated from
water off of the coast of Spain3. Archaea from the genus
Haloarcula have been isolated from a coastal salt marsh in NJ4. A
novel haloarchaeon has been isolated from the Pacific Ocean
near Tokyo5. Taken together, these results suggests it is possible
to isolate extreme halophiles from marine water. This research
aims to identify additional marine environments that are
potential habitats for extreme halophiles.
Figure 1. Pink beach sand from island of Eleuthera, Bahamas6. Salt marsh in Tuckerton, NJ7.
To accomplish our goals, we performed an enrichment culturing
strategy using increasing NaCl conditions to identify the presence
of halophilic organisms in seawater taken from a variety of
different saline environments. Ocean water samples were
obtained from Tuckerton, NJ (a coastal salt marsh). Beach sand
from the Bahamian island of Eleuthera was also obtained for this
study. PCR analyses were performed using domain-specific 16S
rDNA primers to identity samples containing halophilic microbes.
Positive amplicons were sequenced and phylogenetic trees were
constructed showcasing the relationships between the organisms
isolated in this study and their closest relatives.
• Growth experiments were conducted at 37°C in 2216 marine medium
(Difco) with moderate shaking
• Daily increments of 0.5 M NaCl added to growing cultures to enrich for
growth of halophilic microorganisms and limit growth of non-halophiles
• A parallel approach was also used that included the addition of the
antibiotic kanamycin to select against bacterial growth in order to select
for haloarchaea
• From these enrichments, a two-pronged approach was performed (see
details below)
1500
1000
500
1 2 3 4 5 6 7 8 9 10 11
Figure 4. PCR products from bacterial 16S rDNA
amplification of enrichment samples from colonies
obtained after enrichment culturing at 3 M of samples
from Eleuthera. Predicted amplicon is 1.5 kb. Lane 1:
100 bp ladder, Lanes 2-11: DNA amplified from colony
library grown on 2216 medium supplemented with 3M
NaCl.
Figure 5. PCR products from archaeal 16S rDNA direct
amplification of enrichment cultures from Eleuthera
samples grown at 1.5 M NaCl. Predicted amplicon is 900
bp. Lane 1: 100 bp ladder, Lanes 2: Positive control for
archaeal DNA, Lane 3: Negative control, Lane 4: DNA
amplified from 1.5M liquid cultures.
Direct DNA Amplification of liquid cultures
16s rDNA Archaeal
primers
16s rDNA Bacterial
primers
Develop Clone Library
Sequence DNA
Culture Independent PCR Culture Dependent PCR
Plate cultures directly on medium supplemented
with appropriate salinities
PCR
16s rDNA Archaeal
primers
16s rDNA Bacterial
primers
Select morphologically distinct colonies
Sequence DNA
Enrichment culture strategy
Original
sample
0.33 M 1.0 M 1.5 M 2.0 M 2.5 M 3.0 M 3.5 M 4.0 M
Results
Strategy for identifying halophiles found in environmental samples following enrichment culture
Future Directions
Figure 2. Phylogenetic tree
showing isolated bacterial
organisms (BE-1, BE-2, BE-
3, BE-4) from 3 M
enrichment cultures plated
onto 3 M NaCl medium.
Individual clones were
chosen for sequence
analysis. Tree shows the
relationship between clones
and their closest relatives.
Figure 3. Phylogenetic tree
showing isolated archaeal
species (BE-5) from 1.5 M NaCl
enrichment culture using the
culture-independent approach.
This isolate appears closely
related to the genus Haloferax,
a moderately halophilic
archaeon.
BE-1
UBC BF5_1538
BE-3
Halomonas sp. GX18B9-2
522
477
UBC BF5_1524
247
Proteobacterium 14AC15
Halomonas sp. whb43696
Halomonas sp. wbh35
191
UBC BF5_1513
105
Halomonas sp. whb32
209
UBC AND GV0309_D2.0.1_5S1
355
Halomonas cupida strain NBRC 100992
Halomonas sp. NBRC 1000984
409
760
BE-4
551
Halomonas sp. HS207
Halomonas sp. GX9A2-1
Halomonas denitrificans strain D7030
798
Halomonas sp. SB J85
Halomoas sp. HB-N
694
632
973
659
BE-2
1000
Haloferax volcanii DS2
Halobacterium volcanii
Haloferax alexandrinus
412
Haloferax sp. H53
Haloferax sp. CY-4W
770
BE-5
529
Haloferax viridis
927
Haloferax prahovense
659
1500
1000
500
1 2 3 4