Domain-level Identification of Single Prokaryotic Cells by Optical Photothermal Infrared Spectroscopy.

Infrared spectroscopy is used for the chemical characterization of prokaryotes. However, its application has been limited to cell aggregates and lipid extracts because of the relatively low spatial resolution of diffraction. We herein report optical photothermal infrared (O-PTIR) spectroscopy of prokaryotes for a domain-level diagnosis at the single-cell level. The technique provided infrared spectra of individual bacterial as well as archaeal cells, and the resulting aliphatic CH3/CH2 intensity ratios showed domain-specific signatures, which may reflect distinctive cellular lipid compositions; however, there was interference by other cellular components. These results suggest the potential of O-PTIR for a domain-level diagnosis of single prokaryotic cells in natural environments.

Spectroscopic and Biophysical Methods to Determine Differential Salt-Uptake by Primitive Membraneless Polyester Microdroplets.

α-Hydroxy acids are prebiotic monomers that undergo dehydration synthesis to form polyester gels, which assemble into membraneless microdroplets upon aqueous rehydration. These microdroplets are proposed as protocells that can segregate and compartmentalize primitive molecules/reactions. Different primitive aqueous environments with a variety of salts could have hosted chemistries that formed polyester microdroplets. These salts could be essential cofactors of compartmentalized prebiotic reactions or even directly affect protocell structure. However, fully understanding polyester-salt interactions remains elusive, partially due to technical challenges of quantitative measurements in condensed phases. Here, spectroscopic and biophysical methods are applied to analyze salt uptake by polyester microdroplets. Inductively coupled plasma mass spectrometry is applied to measure the cation concentration within polyester microdroplets after addition of chloride salts. Combined with methods to determine the effects of salt uptake on droplet turbidity, size, surface potential and internal water distribution, it was observed that polyester microdroplets can selectively partition salt cations, leading to differential microdroplet coalescence due to ionic screening effects reducing electrostatic repulsion forces between microdroplets. Through applying existing techniques to novel analyses related to primitive compartment chemistry and biophysics, this study suggests that even minor differences in analyte uptake can lead to significant protocellular structural change.

Geochemistry and the Origin of Life: From Extraterrestrial Processes, Chemical Evolution on Earth, Fossilized Life's Records, to Natures of the Extant Life.

In 2001, the first author (S.N.) led the publication of a book entitled "Geochemistry and the origin of life" in collaboration with Dr. Andre Brack aiming to figure out geo- and astro-chemical processes essential for the emergence of life. Since then, a great number of research progress has been achieved in the relevant topics from our group and others, ranging from the extraterrestrial inputs of life's building blocks, the chemical evolution on Earth with the aid of mineral catalysts, to the fossilized records of ancient microorganisms. Here, in addition to summarizing these findings for the origin and early evolution of life, we propose a new hypothesis for the generation and co-evolution of photosynthesis with the redox and photochemical conditions on the Earth's surface. Besides these bottom-up approaches, we introduce an experimental study on the role of water molecules in the life's function, focusing on the transition from live, dormant, and dead states through dehydration/hydration. Further spectroscopic studies on the hydrogen bonding behaviors of water molecules in living cells will provide important clues to solve the complex nature of life.

Changes of aliphatic C-H bonds in cyanobacteria during experimental thermal maturation in the presence or absence of silica as evaluated by FTIR microspectroscopy.

Aliphatic C-H bonds are one of the major organic signatures detected in Proterozoic organic microfossils, and their origin is a topic of interest. To investigate the influence of the presence of silica on the thermal alteration of aliphatic C-H bonds in prokaryotic cells during diagenesis, cyanobacteria Synechocystis sp. PCC6803 were heated at temperatures of 250-450°C. Changes in the infrared (IR) signals were monitored by micro-Fourier transform infrared (FTIR) spectroscopy. Micro-FTIR shows that absorbances at 2,925 cm-1 band (aliphatic CH2 ) and 2,960 cm-1 band (aliphatic CH3 ) decrease during heating, indicating loss of the C-H bonds, which was delayed by the presence of silica. A theoretical approach using solid-state kinetics indicates that the most probable process for the aliphatic C-H decrease is three-dimensional diffusion of alteration products under both non-embedded and silica-embedded conditions. The extrapolation of the experimental results obtained at 250-450°C to lower temperatures implies that the rate constant for CH3 (kCH3 ) is similar to or lower than that for CH2 (kCH2 ; i.e., CH3 decreases at a similar rate or more slowly than CH2 ). The peak height ratio of 2,960 cm-1 band (CH3 )/2,925 cm-1 band (CH2 ; R3/2 values) either increased or remained constant during the heating. These results reveal that the presence of silica does affect the decreasing rate of the aliphatic C-H bonds in cyanobacteria during thermal maturation, but that it does not significantly decrease the R3/2 values. Meanwhile, studies of microfossils suggest that the R3/2 values of Proterozoic prokaryotic fossils from the Bitter Springs Group and Gunflint Formation have decreased during fossilization, which is inconsistent with the prediction from our experimental results that R3/2 values did not decrease after silicification. Some process other than thermal degradation, possibly preservation of specific classes of biomolecules with low R3/2 values, might have occurred during fossilization.

Early trace of life from 3.95 Ga sedimentary rocks in Labrador, Canada.

The vestiges of life in Eoarchean rocks have the potential to elucidate the origin of life. However, gathering evidence from many terrains is not always possible, and biogenic graphite has thus far been found only in the 3.7-3.8 Ga (gigayears ago) Isua supracrustal belt. Here we present the total organic carbon contents and carbon isotope values of graphite (δ13Corg) and carbonate (δ13Ccarb) in the oldest metasedimentary rocks from northern Labrador. Some pelitic rocks have low δ13Corg values of -28.2, comparable to the lowest value in younger rocks. The consistency between crystallization temperatures of the graphite and metamorphic temperature of the host rocks establishes that the graphite does not originate from later contamination. A clear correlation between the δ13Corg values and metamorphic grade indicates that variations in the δ13Corg values are due to metamorphism, and that the pre-metamorphic value was lower than the minimum value. We concluded that the large fractionation between the δ13Ccarb and δ13Corg values, up to 25‰, indicates the oldest evidence of organisms greater than 3.95 Ga. The discovery of the biogenic graphite enables geochemical study of the biogenic materials themselves, and will provide insight into early life not only on Earth but also on other planets.

Domain-level identification and quantification of relative prokaryotic cell abundance in microbial communities by Micro-FTIR spectroscopy.

Domain-level identification of microbial cells or cell-like structures is crucial for investigating natural microbial communities and their ecological significance. By using micro-Fourier transform infrared (micro-FTIR) spectroscopy, we established a technical basis for the domain-level diagnosis and quantification of prokaryotic cell abundance in natural microbial communities. Various prokaryotic cultures (12 species of bacteria and 10 of archaea) were examined using micro-FTIR spectroscopic analysis. The aliphatic CH3 /CH2 absorbance ratios (R3/2 ) showed domain-specific signatures, possibly reflecting distinctive cellular lipid compositions. The signatures were preserved even after chemical cell fixation (formaldehyde) and nucleic acid staining (DAPI) processes - techniques that are essential in studying microbial ecology. The micro-FTIR technique was successfully applied for quantification of the bacteria/archaea abundance ratio in an active microbial mat community in a subsurface hot aquifer stream. We conclude that the micro-FTIR R3/2 measurement is both fast and effective for domain-level diagnosis and quantification of first-order prokaryotic community structures.

In situ infrared microspectroscopy of approximately 850 million-year-old prokaryotic fossils.

In situ infrared (IR) and Raman microspectroscopy have been conducted on Neoproterozoic, organic-walled microfossils (prokaryotic fossils) in doubly polished, petrographic thin sections in order to detect their spectral signatures. The microfossils are very well preserved and occur in black chert from the approximately 850 million-year-old Bitter Springs Formation, Northern Territory, Australia. Raman microspectroscopy on two species of microfossils, one a filament and the other a coccoid, shows disordered peaks (D peak, 1340 cm(-1)) and graphite peaks (G peak, 1600 cm(-1)), indicating that they consist of disordered carbonaceous materials. IR micro-mapping results of the filament reveal that the distributions of peak heights at 2920 cm(-1) (aliphatic CH(2)), 1585 cm(-1) (aromatic C-C), and 1370 cm(-1) (aliphatic CH(3)) match the shape of the filamentous microfossil. These results suggest that IR microspectroscopy can be used for in situ characterization of organic polar signatures that morphologically indicate microfossils embedded in chert by using doubly-polished rock (petrographic) thin section samples. Further, these methods can be applied to controversial microfossil-like structures to test their biogenic nature.