1) Researchers analyzed microorganisms trapped in fluid inclusions in ancient halite samples to learn about past microbial communities and seawater chemistry. 2) They found 11% genetic similarity between microbes in modern brine and those trapped in halite millions of years ago, showing some microbes can survive long-term. 3) Analysis of fluid inclusions indicated seawater was calcium-rich in the early Carboniferous period, and the researchers hope to document shifts in chemistry over time.

1. Ancient Microorganism Communities in Fluid Inclusions in Halite
Geology Department: Nora Holt, Mike Timofeeff, Elliot Jagniecki, Yaicha Winters, Tim Lowenstein
Anthropology Department: Gabriel Wolfson, Koji Lum
Biology Department: Krithivasan Sankaranarayanan
Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 13902
Microorganisms have been known to survive in the fluid inclusions of
ancient halite samples for tens of thousands of years (Schubert et al. 2010).
Through finding halite deposits that contain microorganisms and culturing
these samples, it is possible to obtain a better picture of which organisms
thrived at the time the halite was formed. The brine that the
microorganisms inhabit can also be analyzed to determine the chemistry of
ancient seawaters, which is instrumental in understanding how seawater
chemistry has changed in the past. Once the chemistry of seawaters is
determined, it is possible to draw connections between the marine
environment of the time and how it affected the organisms, in particular
shell builders, that inhabited the oceans. These connections are useful not
only in better understanding the histories of our oceans but in predicting
how the oceans will change in the future and what effect that will have on
our marine ecosystems.
Biology
We successfully retrieved partial archaeal 16s rRNA fragments [V1-V3 region]
from modern brines collected from Saline Valley [04]. Using BLAST, we were able
to confirm that all sequences retrieved belong to the family Halobacteriaceae.
Organisms in this family survive in highly saline environments. We then used
phylogenetic clustering (similarity >95%) of the sequences to generate genus
level diversity profiles. We found that of the ~130 operational taxonomic units
(OTUs), 11% of the sequences were shared between brine and crystal. Negative
controls used to monitor the sterility of reagents and the environment did not
yield any sequences.
Geology
The analysis of fluid inclusions in halite samples from the Mississippian
(318-359 million years ago) and the Pennsylvanian (299-318 million years
ago) has supported our hypothesis that early Carboniferous seawater was
calcium rich. We continue to analyze samples from the late Pennsylvanian
in order to document the shift in seawater chemistry we believe to have
happened during the Carboniferous period. The sulfur isotope and
bromide analyses of these samples have proven that the fluid inclusions we
are analyzing are ancient seawater and not from a freshwater basin.
The results show an 11% match between the community in the brine and in
fluid inclusions in halite. We are investigating why the overlap is not higher.
These preliminary results are beginning to show the microorganism
communities in Saline Valley. This work will help our understanding of
which halobacterium species are best equipped to handle extreme
environments and which ones are best equipped for long term survival.
The results of the analysis of our samples using scanning electron
microscopy have confirmed our hypothesis that seawater was calcium rich
during the early Carboniferous period. Through the continued analysis of
fluid inclusions from Nova Scotia and the Pennsylvanian Paradox Basin of
Utah, we hope to document a shift in seawater chemistry during the late
Carboniferous. Future work involves analysis of additional halite samples
with primary fluid inclusions.
OTUs only
represented in
brine
48%
OTUs only
represented in
crystal
41%
OTUs
represented
in both
11%
OTUs Represented in Brine and Crystals
A: Algae in a brine from Saline
Valley
B: Microorganisms in fluid
inclusions in halite from Death
Valley
C: Microorganisms in a fluid
inclusion from Searles Lake
D&E: Pennsylvanian halite
samples with bottom growth
crystals which include primary
fluid inclusions.
Citations: Sandberg, Altschul SF, et al. (1990) Basic local alignment search tool. J Mol Biol 215: 403-410.
Dereeper et al. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist Nucleic Acids Research. 36 W465-9.
Lliros M, et. al. (2008) High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study. FEMS
Microbial Ecol 66: 331–342.
McGenity TR, et al. (2000) Origins of halophilic microorganisms in ancient salt deposits. Environ Microbiol 2: 243–250.
Risacher & Clement (2001). Elsevier Scient Ltd.
Sandberg, P.A. (1983). “An oscillating trend in Phanerozoic non-skeletal carbonate mineralogy”. Nature305 (5929): 19-22.
Sankaranarayanan K, et al. (2011) Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction. PLoS ONE 6(6):
e20683.
Schubert BA, et al. (2010a) Halophilic Archaea cultured from ancient halite, Death Valley, California. Environ Microbiol 12: 440–454.
Yanan Yu, et al. (2006)FastGroupII: A web-based bioinformatics platform for analyses of large 16S rDNA libraries, BMC Bioinformatics,7:57.
Biology
Identify microbial communities
•Obtain halite samples
•Surface sterilize and dissolve salt
•PCR (amplify DNA)
•Insert amplified DNA into E. Coli
•Sequence DNA
•Analyze sequences
Geology
Obtaining good
samples
•Collect samples
•Cut thin sections
•Microscopy of thin
sections
Determining sea water
chemistry
•Cleave halite crystal
•Place in scanning
electron microscope and
focus electron beam on
fluid inclusion
•Analyze results
This diagram shows what percentage of OTUs we found represented in each medium
C
Oscillations in the Mg/Ca ratio of seawater (Demicco personal communication). This diagram illustrates the approximate age of our samples (indicated
by the black box). Our samples show that Carboniferous seawater had [Ca]>[SO4] and Mg/Ca ratios <2.
The major-ion composition of seawater has varied with time primarily
based on changes in the discharge of mid-ocean ridge emissions, and the
change in concentration of various ions and salts determines which shell-
building species will be able to survive and therefore affects the entire
marine ecosystem. As the concentration of ions such as Mg2+ and Ca2+
fluctuates with time, so do many other aspects of marine life. Halite (NaCl)
is the most common mineral that forms from the deposition of marine
evaporites on the seafloor, and the growth of halite crystals traps small
amounts of seawater within the crystal lattice. These pockets of water,
called fluid inclusions, remain unchanged through time; preserving
communities of microscopic organisms that are still alive today and serving
as evidence of seawater chemistry at the time the crystal was formed. We
can analyze these fluid inclusions to identify the organisms trapped within
the crystals and determine the chemistry of the water. From our analyses
we can decipher how the communities changed over short and long
periods of time in response to changes in seawater chemistry. The viability
of these microorganisms is pushing the limits of our knowledge of long-
term survival.
Abstract
Introduction
Methods
A B C
D
E
Results
Discussion
Two evaporation curves of Carboniferous seawater were obtained through the analysis of our fluid inclusions using a geochemistry computer program
(Risacher 2001). Points on both diagrams indicate the ionic compositions of our samples obtained by scanning electron microscopy. These diagrams
show that our samples fall along expected evaporation paths and therefore are accurate indicators of the major ion composition chemistry of seawater
during the time they were formed.
Phylogenetic tree of Saline Valley (04) brine and crystal samples. Branches with shared sequences are highlighted.
0.05
1000
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[Mg]mmol/(kgH2O)
[Cl] mmol/(kg H2O)
Mississippian and Early Pennsylvanian [Mg] v [Cl]
Paradox
Nova Scotia
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[Na]mmol/(kgH2O)
[Cl] mmol/(kg H2O)
Mississippian and Early Pennsylvanian [Na] v [Cl]
Paradox
Nova Scotia