Michael N. Timofeeff

Secular variation in seawater chemistry and the origin of calcium chloride basinal brines

CaCl 2 basinal brines, which are present in most Phanerozoic sedimentary basins, inherited their chemistries and salinities from evaporated paleoseawaters when the world oceans were Ca rich and SO 4 poor (CaCl 2 seas). CaCl 2 seas coincided with periods of rapid seafloor spreading, high influxes of mid-ocean-ridge brines rich in CaCl 2 , and elevated sea levels, conditions that favored accumulation of marine CaCl 2 brines in marginal and interior continental basins. Typical basinal brines in Silurian-Devonian formations of the interior Illinois basin, United States, show the same compositional trends as those of progressively evaporated CaCl 2 -rich Silurian seawater. Chemical deviations can be accounted for quantitatively by brine-rock reactions during burial (dolomitization, dolomite and K-feldspar cement). This explanation for the origin of CaCl 2 basinal brines contrasts with others that assume constancy of seawater chemistry and involve more complex brine-rock interactions.

Microbial communities in fluid inclusions and long-term survival in halite

Fluid inclusions in modern and ancient buried halite from Death Valley and Saline Valley, California, USA, contain an ecosystem of “salt-loving” (halophilic) prokaryotes and eukaryotes, some of which are alive. Prokaryotes may survive inside fluid inclusions for tens of thousands of years using carbon and other metabolites supplied by the trapped microbial community, most notably the single-celled alga Dunaliella, an important primary producer in hypersaline systems. Deeper understanding of the long-term survival of prokaryotes in fluid inclusions will complement studies that further explore microbial life on Earth and elsewhere in the solar system, where materials that potentially harbor microorganisms are millions and even billions of years old.

Microscopic Identification of Prokaryotes in Modern and Ancient Halite, Saline Valley and Death Valley, California

Primary fluid inclusions in halite crystallized in Saline Valley, California, in 1980, 2004-2005, and 2007, contain rod- and coccoid-shaped microparticles the same size and morphology as archaea and bacteria living in modern brines. Primary fluid inclusions from a well-dated (0-100,000 years), 90 m long salt core from Badwater Basin, Death Valley, California, also contain microparticles, here interpreted as halophilic and halotolerant prokaryotes. Prokaryotes are distinguished from crystals on the basis of morphology, optical properties (birefringence), and uniformity of size. Electron micrographs of microparticles from filtered modern brine (Saline Valley), dissolved modern halite crystals (Saline Valley), and dissolved ancient halite crystals (Death Valley) support in situ microscopic observations that prokaryotes are present in fluid inclusions in ancient halite. In the Death Valley salt core, prokaryotes in fluid inclusions occur almost exclusively in halite precipitated in perennial saline lakes 10,000 to 35,000 years ago. This suggests that trapping and preservation of prokaryotes in fluid inclusions is influenced by the surface environment in which the halite originally precipitated. In all cases, prokaryotes in fluid inclusions in halite from the Death Valley salt core are miniaturized (<1 microm diameter cocci, <2.5 microm long, very rare rod shapes), which supports interpretations that the prokaryotes are indigenous to the halite and starvation survival may be the normal response of some prokaryotes to entrapment in fluid inclusions for millennia. These results reinforce the view that fluid inclusions in halite and possibly other evaporites are important repositories of microbial life and should be carefully examined in the search for ancient microorganisms on Earth, Mars, and elsewhere in the Solar System.

Halophilic Archaea cultured from ancient halite, Death Valley, California

Halophilic Archaea cultured from ancient fluid inclusions in a 90-m-long (0- to 100,000-year-old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34,000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface-sterilized halite crystals exclusively in one section of the core (13.0-17.8 m; 22,000-34,000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1-86.7 m (10,000-100,000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra-laboratory reproducibility. Inter-laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.

ESEM-EDS: an improved technique for major element chemical analysis of fluid inclusions

Abstract Quantitative chemical analyses of fluid inclusions in sedimentary and diagenetic minerals can greatly improve our understanding of the chemistry of ancient surface and near surface waters. The environmental scanning electron microscope (ESEM) with an attached X-ray energy dispersive system (EDS) is capable of producing rapid and accurate major element chemical analyses of individual fluid inclusions in crystals of halite greater than about 30 μm in diameter. The ESEM-EDS technique uses the same basic principles as the X-ray microanalysis of frozen fluid inclusions reported by Ayora and Fontarnau [Ayora, C., Fontarnau, R., 1990. X-ray microanalysis of frozen fluid inclusion. Chemical Geology, 89, pp. 135–148.] but operates in a low-vacuum environment, which allows direct observation of the fluid inclusions to be analyzed without the need for a conductive coating. Modifications of the technique of Ayora and Fontarnau [Ayora, C., Fontarnau, R., 1990. X-ray microanalysis of frozen fluid inclusion. Chemical Geology, 89, pp. 135–148.] also include a newly designed sample holder, modified methods for the preparation of standard brine solutions to obtain “glass-like” homogeneous freezing behavior by addition of ethylene glycol, and a technique to obtain flat smooth surfaces on standard brine solutions. The ability to observe samples before analysis led to the discovery of heterogeneous freezing behavior of some standard solutions and fluid inclusions, which adversely influenced measurement reproducibility. The ESEM-EDS technique yielded quantitative results for Ca, Mg, K, SO 4 , and Cl at concentrations above about 0.1 wt.%; for Na, quantitative analyses were achieved at concentrations above 0.5 wt.%. Accuracies for the major elements in aqueous standards were better than 7% and precisions (relative standard deviations) ranged from 2% to 7%. Results on fluid inclusions in laboratory-grown halite gave accuracies of 6% to 10% for Mg, Ca, and K and precisions from 3% to 16%.

How do prokaryotes survive in fluid inclusions in halite for 30 k.y.

Long-term survival of microorganisms has been demonstrated by prokaryotes cultured from ancient halite, but previous results are controversial. Three genera of non-spore-forming halophilic Archaea were cultured from 22–34 k.y. old subsurface halite from Death Valley, California. Primary, brine-filled inclusions in this halite contained prokaryotic organisms in miniaturized starvation-survival forms and dead cells of the algal genus Dunaliella . The energy needed for protracted survival of halophilic Archaea , including repair of damaged DNA, may have been provided by glycerol and other carbon molecules leaked from Dunaliella cells. These results provide further evidence that fluid inclusions in halite are a favorable refuge for long-term survival of microorganisms, and indicate that the original depositional environment influences the distribution and viability of prokaryotes.

Evaluating seawater chemistry from fluid inclusions in halite: examples from modern marine and nonmarine environments

Fluid inclusions from marine halites have long been studied to determine the chemical composition of ancient seawater. Chemical analyses of the major ions in fluid inclusions in halites from the solar saltwork of Great Inagua Island, Bahamas, and from the supratidal sabkha, Baja California, Mexico, show that modern marine halites faithfully record the chemical signature of seawater. The major ions in Great Inagua and Baja California fluid inclusions display distinctive linear trends when plotted against one another (ie., Na+, K+, and SO42− vs. Mg2+ and Cl−), which track the evaporation path of seawater as it evolved during the crystallization of halite. These evaporation paths defined for the major ions by fluid inclusions in halite overlap findings of computer simulations of the evaporation of modern seawater by the Harvie, Moller, and Weare (HMW) computer program. The close match between the HMW seawater evaporation paths and the Great Inagua fluid inclusion data is not surprising considering the carefully controlled inflow, evaporation, and discharge of seawater at the Great Inagua saltwork. The major ion chemistry of fluid inclusions from the Baja California halites matches the HMW seawater evaporation paths in most respects, but one Baja fluid inclusion has lower concentrations of Mg2+ than evaporated seawater. Nonmarine inflows and syndepositional recycling of preexisting salts in the Baja California supratidal setting were not large enough to override the chemical signature of evaporating seawater as the primary control on the Baja fluid inclusion compositions. Fluid inclusions in halites from the nonmarine Qaidam Basin, Qinghai Province, western China, have a distinctly different major ion chemical signature than does “global” seawater. The fluid inclusion chemistries from the Qaidam Basin halites do not lie on the evaporation pathways defined by modern seawater and can clearly be differentiated from fluid inclusions containing evaporated seawater. If fluid inclusions in halites from modern natural settings contain unmistakable samples of evaporated seawater, then evaluation of the chemistry of ancient seawater by chemical analysis of fluid inclusions in ancient marine halites by means of the same approach should be valid.

Dunaliella Cells in Fluid Inclusions in Halite: Significance for Long-term Survival of Prokaryotes

A 90-m-long (100,000 year old) salt core from Death Valley, California, contains cells of the algal genus Dunaliella co-trapped with prokaryote cells in fluid inclusions in halite. It is hypothesized that Dunaliella cells provided glycerol, the carbon source needed by halophilic Archaea for survival over periods of tens of thousands of years. Support for this hypothesis includes: observations that intracellular materials leaked from Dunaliella cells into fluid inclusions; the distribution of Dunaliella cells in the Death Valley core, which matches the distribution of culturable prokaryotic cells; and halophilic Archaea cultured from the Death Valley core grew in media containing glycerol as the only carbon source.

Haloarchaeal diversity in 23, 121 and 419 MYA salts

DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium. The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.

Identification of Carotenoids in Ancient Salt from Death Valley, Saline Valley, and Searles Lake, California, Using Laser Raman Spectroscopy

Carotenoids are common components of many photosynthetic organisms and are well known from the red waters of hypersaline ecosystems where they are produced by halophilic algae and prokaryotes. They are also of great interest as biomarkers in extraterrestrial samples. Few laser Raman spectroscopy studies have examined ancient field samples, where pigments and microscopic life are less defined. Here, we have identified carotenoids in ancient halite brine inclusions, 9 ka to 1.44 Ma in age, from borehole cores taken from Death Valley, Saline Valley, and Searles Lake, California, for the first time with laser Raman spectroscopy. Carotenoids occurred in fluid inclusions as colorless to red-brown amorphous and crystalline masses associated with spheroidal algal cells similar in appearance to the common halophilic alga Dunaliella. Spectra from carotenoid standards, including β-carotene, lycopene, and lutein, were compared to microscopically targeted carotenoids in fluid inclusions. Carotenoids produced characteristic bands in the Raman spectrum, 1000-1020 cm⁻¹ (v₃), 1150-1170 cm⁻¹ (v₂), and 1500-1550 cm⁻¹ (v₁), when exposed to visible laser excitation. Laser Raman analyses confirmed the presence of carotenoids with these characteristic peaks in ancient halite. A number of band sets were repeated at various depths (ages), which suggests the stability of this class of organic molecules. Carotenoids appear well preserved in ancient salt, which supports other observations, for example, preserved DNA and live cells, that fluid inclusions in buried halite deposits preserve intact halophilic microbial ecosystems. This work demonstrates the value of laser Raman spectroscopy and carotenoids in extraterrestrial exploration for remnants of microbial life.