Precambrian Research 173 (2009) 19–26
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Micro-FTIR spectroscopic signatures of Bacterial lipids in Proterozoic microfossils Motoko Igisu a,b,c , Yuichiro Ueno d,e,∗ , Mie Shimojima e,f , Satoru Nakashima b , Stanley M. Awramik g , Hiroyuki Ohta e,h , Shigenori Maruyama a,e a
Department of Earth and Planetary Sciences, Tokyo Institute of Technology, O-okayama 2-12-1, Meguro-ku, Tokyo 152-8551, Japan Department of Earth and Space Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043, Japan c Department of Earth Science and Astronomy, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan d Global Edge Institute, Tokyo Institute of Technology, Post No. I2-21, Meguro-ku, Tokyo 152-8551, Japan e Research Center for the Evolving Earth and Planets, Tokyo Institute of Technology, 4259-B-14 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan f Department of Bioscience, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-14 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan g Department of Earth Science, University of California, Santa Barbara, CA 93106, USA h Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan b
a r t i c l e
i n f o
Article history: Received 31 December 2008 Received in revised form 1 March 2009 Accepted 16 March 2009 Keywords: Micro-FTIR Prokaryotic fossil Aliphatic CH moieties Lipid Bacteria Archaea
a b s t r a c t Precambrian microbial fossils show carbonaceous cellular structure, which often resemble in shape and size cyanobacteria and other prokaryotes. Morphological taxonomy of these minute, simple, and more or less degraded fossils is, however, often not enough to determine their precise phylogenetic positions. Here we report the results of micro-FTIR spectroscopic analyses of well-preserved microfossils in ∼850 Ma and ∼1900 Ma stromatolites, together with those of 8 species of extant prokaryotes and 5 of eukaryotes for comparison. These Proterozoic fossils have low CH3 /CH2 absorbance ratios (R3/2 < 0.5) of aliphatic CH moieties, suggesting selective preservation of long, straight, aliphatic carbon chains probably derived from bacterial membrane lipids. All the observed R3/2 values of coccoids, filaments and amorphous organic matter resemble lipid fractions of extant Bacteria including cyanobacteria, but not Archaea. The results indicate that Proterozoic microfossils belong to Bacteria, which is consistent with the cyanobacterial origin inferred from morphology. Moreover, the R3/2 value of fossilized cell would reflect chemical composition of its precursor membrane lipid, thus could be a useful new tracer for distinguishing Archaea, Bacteria and possibly Eucarya for fossilized and extant microorganisms. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Morphologically preserved microbial fossils provide direct evidence for the existence of life in the Precambrian (e.g. Schopf, 1992; Knoll, 2003). The microfossils show carbonaceous cellular structure, which often resemble cyanobacteria and other prokaryotes. But morphological analysis of these prokaryote-like fossils is often not adequate to define the biology of such fossils and to determine their precise phylogenetic positions. Even domainlevel classification of them has not been fully resolved. Modern micro-scale spectroscopic analysis is an alternative way to understand the chemical characteristics of individual microfossils (Ueno et al., 2001, 2006; Kudryavtsev et al., 2001; Schopf et al., 2002, 2005; Brasier et al., 2002, 2004; Pasteris and Wopenka, 2002, 2003; Arouri et al., 1999, 2000; Marshall et al., 2005; Igisu et al., 2006).
∗ Corresponding author. Present address: Global Edge Institute, Tokyo Institute of Technology, Post No. I2-21, Meguro-ku, Tokyo 152-8551, Japan. Tel.: +81 3 5734 2618; fax: +81 3 5734 3536. E-mail address:
[email protected] (Y. Ueno). 0301-9268/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.precamres.2009.03.006
To date, micro-Raman spectroscopy is being used to demonstrate carbonaceous composition of putative organic-walled microfossils and to determine their degree of post-depositional alteration (Ueno et al., 2001, 2006; Kudryavtsev et al., 2001; Schopf et al., 2002, 2005; Brasier et al., 2002, 2004; Pasteris and Wopenka, 2002, 2003). Raman spectra of Precambrian microfossils, however, cannot provide useful information for their taxonomy because Raman bands generally reflect non-polar aromatic structure of carbonaceous matter, which is not taxonomically specific. On the other hand, infrared (IR) spectroscopy can detect many polar bonds, thus is potentially useful to detect taxon-specific chemical signature. IR spectroscopy has seldom been applied to various Precambrian microfossils (Arouri et al., 1999, 2000; Marshall et al., 2005; Igisu et al., 2006). Here, we report micro-FTIR spectroscopy of unequivocal prokaryotic fossils in ∼850 and ∼1900 million-years-old (Ma) stromatolitic cherts in order to obtain taxon-specific chemical signature. We found that these Proterozoic fossils show characteristic IR signature derived from aliphatic C–H bonding. In order to understand the origin of the aliphatic CH moieties observed in the prokaryotic fossils, we further applied this micro-FTIR method to
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M. Igisu et al. / Precambrian Research 173 (2009) 19–26
Fig. 1. Optical photomicrographs of analyzed Proterozoic microfossils in stromatolitic cherts. (A and B) Microfossils (A, Cephalophytarion; B, Glenobotrydion) from ∼850 Ma Bitter Springs Formation [from the previous study (Igisu et al., 2006)]. (C–F) Microfossils and amorphous organic matter from ∼1900 Ma Gunflint Formation. (C and D) Aggregated filamentous (Gunflintia) and coccoidal (Huroniospora) microfossils (Barghoorn and Tyler, 1965). (E and F) Amorphous organic matter. (C–E) Found within stromatolitic laminae mainly produced by concentrations of carbonaceous materials, while (F) is found in inner part of laminae where organic matter is less concentrated. Analyzed areas and their R3/2 values in micro-FTIR measurements are shown. Scale bars represent 20 m for (A and B) and 40 m for (C–F).
extant Bacteria, Archaea and Eucarya. Post-mortem degradation and selective preservation of specific compounds were evaluated by sequential chemical separation and heating experiments of extant prokaryote. Based on these results, we discuss domain-level taxonomy of Proterozoic microfossils. 2. Materials and methods 2.1. Samples Micro-Fourier transform infrared (FTIR) spectroscopy of individual prokaryotic fossils in stromatolitic cherts from the ∼850 Ma Bitter Springs Formation (Barghoorn and Schopf, 1965; Schopf, 1968; Schopf and Blacic, 1971) and the ∼1900 Ma Gunflint Formation (Barghoorn and Tyler, 1965; Awramik and Barghoorn, 1977)
were conducted as described previously (Igisu et al., 2006). Doubly polished petrographic thin sections of stromatolitic cherts were used for obtaining the IR spectra of the microfossils. These fossils are carbonaceous and are well-preserved in chert matrix (cryptocrystalline quartz) (Fig. 1). Based on size and their filamentous and coccoidal morphology they are all interpreted as cyanobacteria (Barghoorn and Schopf, 1965; Schopf, 1968; Schopf and Blacic, 1971; Barghoorn and Tyler, 1965; Awramik and Barghoorn, 1977). Extant prokaryotes were cultured at Tokyo Institute of Technology and JAMSTEC. Green algae (Volvox and Desmodesmus), diatom (Melosira) and dinoflagellate (Ceratium) were collected from the Lake Fukami-ike, Japan. They were washed carefully with water or sodium chloride aqueous solution to avoid infrared active medium. Whole cells, water-insoluble, water-soluble and lipid fractions were placed on 1 mm thick CaF2 disks, then dried, and measured by
M. Igisu et al. / Precambrian Research 173 (2009) 19–26
micro-FTIR spectrometer. Leaf segment of commercially available higher plant was also prepared for micro-FTIR measurement.
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to room temperature, the degraded samples were measured at the same position.
2.2. Cell culture and fraction 2.5. Micro-Raman measurements Extant cyanobacteria (Synechocystis sp. PCC 6803) were grown in BG11 medium as described in Hagio et al. (2000). For microFTIR spectroscopic observations, 7-day-old cyanobacteria cells (OD750 approximately 1.0) were collected by centrifugation at 1390 × g for 10 min and washed by water to avoid a liquid growth medium (BG11) whose IR signatures prevent IR observation of organic signatures from cyanobacteria. This procedure was repeated three times. Then, cyanobacteria were sonicated, and followed by brief centrifugation to eliminate unbroken cell contamination. Then cell-free extracts were ultracentrifuged twice at 120,000 × g for 1 h (Beckman Optra TL) with the same volume of water. After the analysis of washed whole cells of Synechocystis sp., the sample was separated into a water-insoluble membrane fraction and a water-soluble fraction by sonication and centrifugation. The membrane fraction is mainly composed of lipids, peptidoglycan (glycoprotein), proteins, and pigments combined with proteins. The water-soluble fraction mainly contains proteins, pigments, and nucleic acid. Furthermore, we extracted lipids from the membrane fraction by using chloroform–methanol as a solvent according to Bligh and Dyer (1959). Similar preparation procedure was also applied to other prokaryote samples. 2.3. Micro-FTIR measurements In situ IR measurements of Proterozoic microfossils and 8 species of extant prokaryotes including cyanobacteria (Synechocystis), and 5 species of eukaryotes were conducted using an FTIR microspectrometer equipped with MCT detector (JASCO FTIR620+IRT30). A reference background spectrum (T0 ) was first measured at a place away from sample, and then transmission IR spectrum of sample (T) was measured. The IR spectrum is described as IR absorbance (abs = −log10 T/T0 ) as a function of the wavenumber (cm−1 ). Spot size of the analysis ranged from 25 m × 25 m to 300 m × 300 m depending on the choice of rectangular aperture. This micro-FTIR technique, with such a spatial resolution of 25 m, allows for the in situ analysis of microfossils in petrographic thin section. One hundred to one thousand scans were accumulated at 4 cm−1 spectral resolution in a range from 4000 cm−1 to 1000 cm−1 according to the analyzed spot size and IR signal intensities. All the IR spectral data were analyzed with Spectra Manager (JASCO). 2.4. Thermal degradation experiments of extant prokaryotes In order to examine thermal changes of R3/2 values in whole cells and separated fractions, heating experiments were also conducted. A heating stage (Linkam LK600) was modified and used for the thermal degradation experimental. About 2 l of samples were placed on an IR inactive CaF2 disk (1 mm thickness). Then, the samples except for lipid fraction were heated several minutes at 70 ◦ C under atmospheric condition to evaporate water. Lipid fraction was left at room temperature under atmospheric condition for dehydration. Then, each sample on a CaF2 disk (1 mm thickness) was mounted on the heating stage. The samples were isothermally heated for 5 h at 250–350 ± 1 ◦ C under dysoxic (99.99% Ar gas) or atmospheric condition. Although the temperature conditions during thermal degradation might be below 200 ◦ C, the degradations of organic functional groups below 200 ◦ C were extremely slow for monitoring in reasonable time periods. Therefore, acceleration experiments were conducted at higher temperatures. After cooling
Laser Raman micro-spectrometer (JASCO NRS-2000) was used for characterizing degree of degradation for prokaryotic fossils and experimental run products. The samples were exposed to an Ar laser (514.5 nm) for 5 s twice at a laser power of 20 mW to obtain Raman spectra in a range from 1750 cm−1 to 1100 cm−1 at 1 cm−1 resolution. A 100× objective lens (NA = 0.84) was used so the spatial resolution of the Raman analysis was 1–2 m. Using obtained Raman spectra, Raman Index of Preservation (RIP) (Schopf et al., 2005) was calculated for evaluating degradation degree of organic matter. RIP is defined as follows (Schopf et al., 2005):
1300 RIP =
1100
I(v)dv
1300
I(v)dv
1370
(1)
where I() is intensity at Raman shift (cm−1 ). Calibration of the RIP value was conducted for adapting the scale used by Schopf et al. (2005). Integral intensity of 1370–1300 cm−1 region is mainly due to graphitization-evidencing disordered (D) band, while that of 1300–1100 cm−1 region is mainly due to D band shoulder (Schopf et al., 2005). Therefore, more degraded organic matter gives smaller RIP value. All the Raman spectral data were also analyzed with Spectra Manager (JASCO).
3. Results and discussions 3.1. Proterozoic microfossils Typical infrared transmission spectra of Proterozoic microfossils in stromatolitic chert are shown in Fig. 2. Aggregated filamentous and coccoidal microfossils as well as amorphous organic matter in ∼1900 Ma Gunflint Formation shows IR absorption bands of aliphatic CH moieties (∼2960, ∼2925, ∼2875 and ∼2850 cm−1 ) in the range of >2500 cm−1 (Figs. 1C–F and 2C,D). The bands around 2960 cm−1 and ∼2925 cm−1 are derived from asymmetric stretching of end-methyl aliphatic CH3 and methylene-chain CH2 , respectively (Bellamy, 1954). Weaker bands from symmetric stretching of aliphatic CH3 and CH2 are also seen around 2875 cm−1 and 2850 cm−1 , respectively. In addition, smaller IR band due to aliphatic CH at ∼2890 cm−1 may possibly exist between asymmetric CH2 and symmetric CH3 bands as reported in the previous studies (Lin and Ritz, 1993; Marshall et al., 2005). However, we could not obtain unequivocal evidence of the presence of aliphatic CH because of ambiguity of deconvolution method in this study. Similar results were obtained in our previous study of cyanobacteria-like filamentous and coccoidal fossils in ∼850 Ma Bitter Springs Formation chert (Figs. 1A, B and 2A, B; Igisu et al., 2006). These results indicate all the Proterozoic microfossils still possess hydrocarbon moieties, in spites of their highly aromatized carbon structure shown as ∼1600 cm−1 (graphite) and ∼1350 cm−1 (disordered) bands by their Raman spectra (Fig. 4A and B). Stronger band around 3400 cm−1 is originated from molecular H2 O (Fig. 2). This band is typically observed in chert and probably reflects H2 O present at grain boundary of cryptocrystalline quartz (Nakashima et al., 1995; Ito and Nakashima, 2002; Igisu et al., 2006). In the lower wavenumber regions less than 2500 cm−1 , many intense bands of the matrix quartz masked the small signal from organic matter (Fig. 2). Hence, we focus further discussions on IR bands originated from aliphatic CH moieties.
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Fig. 2. Typical transmission IR absorbance spectra of fossilized prokaryotes. (A) Stacked IR spectra of a coccoidal (Glenobotrydion: C) and filamentous (Cephalophytarion: F) fossils, and quartz matrix (Qz) from ∼850 Ma Bitter Springs Formation shown in arbitrary absorbance unit for comparison. IR bands at 2960 cm−1 , 2925 cm−1 and 2850 cm−1 are due to asymmetric aliphatic CH3 (end-methyl), asymmetric and symmetric aliphatic CH2 (methylene-chain), respectively. Broadband around 3400 cm−1 is due to molecular water probably within the grain boundary of micro-quartz. Saturated bands (1400–1000 cm−1 ) and seven bands (1995, 1870, 1793, 1684, 1610, 1525, and 1492 cm−1 ) are due to Si–O bonds of quartz. (B) Enlarged view of IR spectra of (A) in range of 2800–3000 cm−1 . An example of baseline correction for elucidation of peak height is described as dotted line. (C) Stacked IR spectra of ∼1900 Ma Gunflint Formation stromatolitic black chert (aggregated filaments and coccoids: F + C, amorphous organic matter: OM, quartz matrix: Qz). Peak assignments are the same as (A). (D) Enlarged view of IR spectra of (C) in range of 2800–3000 cm−1 .
3.2. R3/2 value In order to evaluate these spectral characteristics, we introduce the aliphatic CH3 /CH2 absorbance ratio (R3/2 ): R3/2 =
[as CH3 ] [as CH2 ]
(2)
where [as CH3 ] and [as CH2 ] represent peak heights of asymmetric stretching bands for aliphatic CH3 (end-methyl; ∼2960 cm−1 ) and CH2 (methylene-chain; ∼2925 cm−1 ), respectively. The R3/2 values of the Bitter Springs Formation fossils are 0.33 ± 0.09 (n = 4) for filaments and 0.32 ± 0.10 (n = 3) for coccoids (Fig. 1). Gunflint Formation fossils are 0.45 ± 0.04 (n = 5) for aggregated filament and coccoid, and 0.43 ± 0.11 (n = 10) for amorphous organic matter (Fig. 1). The R3/2 value generally reflects the degree of branching and chain length of the aliphatic hydrocarbon moiety (Lin and Ritz, 1993; Marshall et al., 2005). The low R3/2 values (
