Additional file 1: Figure S1. of Amino acid compositions in heated carbonaceous chondrites and their compound-specific nitrogen isotopic ratios

GC/C/IRMS chromatogram of the 12 M HCl-hydrolyzed hot-water extracts of the Piv/iPr amino acid esters in Y-980115. The subgraph (a) shows the magnified region with the alanine and glycine peaks. The single-ion traces GC/MS chromatograms of (b) Y-980115 and (c) amino acid standard mixture of the same region as shown in (a) indicate that no coeluting compounds were present that could have interfered the IRMS analysis of Y-980115 and that only alanine and glycine are the main nitrogen-bearing compounds in the region

Compound-Specific Nitrogen Isotope Analysis of d-Alanine, l-Alanine, and Valine: Application of Diastereomer Separation to δ¹⁵N and Microbial Peptidoglycan Studies

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (<i>R</i>)-(−)-2-butanol or (<i>S</i>)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of d- and l-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; <i></i><i>Enterococcus faecalis</i>, <i></i><i>Staphylococcus aureus</i>, <i></i><i>Staphylococcus staphylolyticus</i>, <i></i><i>Lactobacillus acidophilus</i>,<i></i> <i>Bacillus subtilis</i>, <i></i><i>Micrococcus luteus</i>, and <i>Streptomyces</i> sp.), natural whole bacterial cells (<i></i><i>Bacillus subtilis</i> var. <i>natto</i>), (pseudo)-peptidoglycan from archaea (<i>Methanobacterium</i> sp.), and cell wall from eukaryota (<i>Saccharomyces cerevisiae</i>). We observed statistically significant differences in nitrogen isotopic compositions; e.g., δ¹⁵N (‰ vs air) in <i>Staphylococcus staphylolyticus</i> for d-alanine (19.2 ± 0.5‰, <i>n</i> = 4) and l-alanine (21.3 ± 0.8‰, <i>n</i> = 4) and in <i>Bacillus subtilis</i> for d-alanine (6.2 ± 0.2‰, <i>n</i> = 3) and l-alanine (8.2 ± 0.4‰, <i>n</i> = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in ¹⁵N-depleted d-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers

Trophic discrimination factors.

<p>Linear regression analysis of trophic discrimination factors (Δ¹⁵N) deriving from A) CSIA analysis, and B) bulk ¹⁵N analysis. Each point represents the trophic discrimination factor of an individual organism. Discrimination factors depicted at trophic levels 2, 3, and 4 represent the isotopic shifts from levels 1 to 2, 2 to 3, and 3 to 4, respectively.</p

Trophic position estimates, controlled-feeding trials.

<p>Trophic position estimates (mean ± 1σ) from the controlled-feeding trials. A) Plots of observed trophic positions vs. their corresponding known trophic positions. Black circles indicate TP_glu-phe estimates, and gray triangles indicate TP_bulk estimates. The dotted line (<i>y</i> = <i>x</i>) represents perfect agreement between observed and known trophic positions. B) Degree of departure between the observed and known trophic positions (mean ± 1σ).</p

Trophic position estimates, apple orchard.

<p>Trophic position estimates (mean ± 1σ) from an apple orchard food chain (pictured): apple leaves, apple aphid, hover fly, parasitoid, and hyperparasitoid. Observed trophic positions are plotted against expected trophic positions. Black circles and gray triangles respectively indicate TP_glu-phe and TP_bulk estimates. The dotted line (<i>y</i> = <i>x</i>) represents perfect agreement between observed and expected trophic positions.</p

Quantitative Analysis of Coenzyme F430 in Environmental Samples: A New Diagnostic Tool for Methanogenesis and Anaerobic Methane Oxidation

Coenzyme F430 is a nickel hydrocorphinoid and is the prosthetic group of methyl–coenzyme M reductase that catalyzes the last step of the methanogenic reaction sequence and its reversed reaction for anaerobic methane oxidation by ANME. As such, function-specific compound analysis has the potential to reveal the microbial distribution and activity associated with methane production and consumption in natural environments and, in particular, in deep subsurface sediments where microbiological and geochemical techniques are restricted. Herein, we report the development of a technique for high-sensitivity analysis of F430 in environmental samples, including paddy soils, marine sediments, microbial mats, and an anaerobic groundwater. The lower detection limit of F430 analysis by liquid chromatography/mass spectrometry is 0.1 femto mol, which corresponds to 6 × 10² to 1 × 10⁴ cells of methanogens. F430 concentrations in these natural environmental samples range from 63 × 10^–6 to 44 nmol g^–1 and are consistent with the methanogenic archaeal biomass estimated by microbiological analyses

Time series of <i>rbcL</i> sequence composition in <i>Plakobranchus ocellatus</i>.

<p>(A) Relative abundance of respective <i>rbcL</i> in individuals of <i>Plakobranchus ocellatus</i> collected monthly in 2005 and 2007. Source algae of kleptoplasts are shown by abbreviations of the clades (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042024#pone-0042024-t002" target="_blank">Table 2</a>). (B) Tukey's boxplots of the relative abundance of <i>rbcL</i> sequences. Data points of fewer than three individuals of <i>P. ocellatus</i> are shown as diamonds. The brackets with “*” indicate a significant difference of the percentage value in the permuted Brunner-Munzel test. The value beside the symbol “*” indicates p-value.</p

Number of clones obtained from kleptoplast <i>rbcL</i> clone sequences.

<p>Number of clones obtained in kleptoplast <i>rbcL</i> clone sequencing from each of 7 <i>Plakobranchus ocellatus</i> individuals. The source algae were identified from the phylogenetic analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042024#pone-0042024-g001" target="_blank">Figure 1</a>).</p

Phylogenetic tree based on <i>rbcL</i> sequences (continued).

<p>Phylogenetic tree based on <i>rbcL</i> sequences (continued).</p