Sulfur disproportionating microbial communities in a dynamic, microoxic-sulfidic karst system.

Biogeochemical sulfur cycling in sulfidic karst systems is largely driven by abiotic and biological sulfide oxidation, but the fate of elemental sulfur (S0 ) that accumulates in these systems is not well understood. The Frasassi Cave system (Italy) is intersected by a sulfidic aquifer that mixes with small quantities of oxygen-rich meteoric water, creating Proterozoic-like conditions and supporting a prolific ecosystem driven by sulfur-based chemolithoautotrophy. To better understand the cycling of S0 in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide-oxidizing mats dominated by Beggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related to Sulfurovum, Halothiobacillus, Thiofaba, Thiovirga, Thiobacillus, and Desulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated with Bacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae. Desulfocapsa and Sulfurovum populations accounted for 12%-26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S0 disproportionation in pure culture. Gibbs energy (∆Gr ) calculations revealed that S0 disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat-sediment interface showed that Beggiatoa mats consume dissolved sulfide and oxygen, but a net increase in acidity was only observed in the sediments below. Together, these findings suggest that disproportionation is an important sink for S0 generated by microbial sulfide oxidation in this oxygen-limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur-rich environments.

Well-hidden methanogenesis in deep, organic-rich sediments of Guaymas Basin.

Deep marine sediments (>1mbsf) harbor ~26% of microbial biomass and are the largest reservoir of methane on Earth. Yet, the deep subsurface biosphere and controls on its contribution to methane production remain underexplored. Here, we use a multidisciplinary approach to examine methanogenesis in sediments (down to 295 mbsf) from sites with varying degrees of thermal alteration (none, past, current) at Guaymas Basin (Gulf of California) for the first time. Traditional (13C/12C and D/H) and multiply substituted (13CH3D and 12CH2D2) methane isotope measurements reveal significant proportions of microbial methane at all sites, with the largest signal at the site with past alteration. With depth, relative microbial methane decreases at differing rates between sites. Gibbs energy calculations confirm methanogenesis is exergonic in Guaymas sediments, with methylotrophic pathways consistently yielding more energy than the canonical hydrogenotrophic and acetoclastic pathways. Yet, metagenomic sequencing and cultivation attempts indicate that methanogens are present in low abundance. We find only one methyl-coenzyme M (mcrA) sequence within the entire sequencing dataset. Also, we identify a wide diversity of methyltransferases (mtaB, mttB), but only a few sequences phylogenetically cluster with methylotrophic methanogens. Our results suggest that the microbial methane in the Guaymas subsurface was produced over geologic time by relatively small methanogen populations, which have been variably influenced by thermal sediment alteration. Higher resolution metagenomic sampling may clarify the modern methanogen community. This study highlights the importance of using a multidisciplinary approach to capture microbial influences in dynamic, deep subsurface settings like Guaymas Basin.

Sources and Fluxes of Organic Carbon and Energy to Microorganisms in Global Marine Sediments.

Marine sediments comprise one of the largest microbial habitats and organic carbon sinks on the planet. However, it is unclear how variations in sediment physicochemical properties impact microorganisms on a global scale. Here we investigate patterns in the distribution of microbial cells, organic carbon, and the amounts of power used by microorganisms in global sediments. Our results show that sediment on continental shelves and margins is predominantly anoxic and contains cells whose power utilization decreases with sediment depth and age. Sediment in abyssal zones contains microbes that use low amounts of power on a per cell basis, across large gradients in sediment depth and age. We find that trends in cell abundance, POC storage and degradation, and microbial power utilization are mainly structured by depositional setting and redox conditions, rather than sediment depth and age. We also reveal distinct trends in per-cell power regime across different depositional settings, from maxima of ∼10-16 W cell-1 in recently deposited shelf sediments to minima of <10-20 W cell-1 in deeper and ancient sediments. Overall, we demonstrate broad global-scale connections between the depositional setting and redox conditions of global sediment, and the amounts of organic carbon and activity of deep biosphere microorganisms.

Unique Geothermal Chemistry Shapes Microbial Communities on Mt. Erebus, Antarctica.

Mt. Erebus, Antarctica, is the world's southernmost active volcano and is unique in its isolation from other major active volcanic systems and its distinctive geothermal systems. Using 16S rRNA gene amplicon sequencing and physicochemical analyses, we compared samples collected at two contrasting high-temperature (50°C-65°C) sites on Mt. Erebus: Tramway Ridge, a weather-protected high biomass site, and Western Crater, an extremely exposed low biomass site. Samples were collected along three thermal gradients, one from Western Crater and two within Tramway Ridge, which allowed an examination of the heterogeneity present at Tramway Ridge. We found distinct soil compositions between the two sites, and to a lesser extent within Tramway Ridge, correlated with disparate microbial communities. Notably, pH, not temperature, showed the strongest correlation with these differences. The abundance profiles of several microbial groups were different between the two sites; class Nitrososphaeria amplicon sequence variants (ASVs) dominated the community profiles at Tramway Ridge, whereas Acidobacteriotal ASVs were only found at Western Crater. A co-occurrence network, paired with physicochemical analyses, allowed for finer scale analysis of parameters correlated with differential abundance profiles, with various parameters (total carbon, total nitrogen, soil moisture, soil conductivity, sulfur, phosphorous, and iron) showing significant correlations. ASVs assigned to Chloroflexi classes Ktedonobacteria and Chloroflexia were detected at both sites. Based on the known metabolic capabilities of previously studied members of these groups, we predict that chemolithotrophy is a common strategy in this system. These analyses highlight the importance of conducting broader-scale metagenomics and cultivation efforts at Mt. Erebus to better understand this unique environment.

Novel nitrite reductase domain structure suggests a chimeric denitrification repertoire in the phylum Chloroflexi.

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported NOR subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

Unique mobile elements and scalable gene flow at the prokaryote-eukaryote boundary revealed by circularized Asgard archaea genomes.

Eukaryotic genomes are known to have garnered innovations from both archaeal and bacterial domains but the sequence of events that led to the complex gene repertoire of eukaryotes is largely unresolved. Here, through the enrichment of hydrothermal vent microorganisms, we recovered two circularized genomes of Heimdallarchaeum species that belong to an Asgard archaea clade phylogenetically closest to eukaryotes. These genomes reveal diverse mobile elements, including an integrative viral genome that bidirectionally replicates in a circular form and aloposons, transposons that encode the 5,000 amino acid-sized proteins Otus and Ephialtes. Heimdallaechaeal mobile elements have garnered various genes from bacteria and bacteriophages, likely playing a role in shuffling functions across domains. The number of archaea- and bacteria-related genes follow strikingly different scaling laws in Asgard archaea, exhibiting a genome size-dependent ratio and a functional division resembling the bacteria- and archaea-derived gene repertoire across eukaryotes. Bacterial gene import has thus likely been a continuous process unaltered by eukaryogenesis and scaled up through genome expansion. Our data further highlight the importance of viewing eukaryogenesis in a pan-Asgard context, which led to the proposal of a conceptual framework, that is, the Heimdall nucleation-decentralized innovation-hierarchical import model that accounts for the emergence of eukaryotic complexity.

Structure and metabolic potential of the prokaryotic communities from the hydrothermal system of Paleochori Bay, Milos, Greece.

Introduction: Shallow hydrothermal systems share many characteristics with their deep-sea counterparts, but their accessibility facilitates their study. One of the most studied shallow hydrothermal vent fields lies at Paleochori Bay off the coast of Milos in the Aegean Sea (Greece). It has been studied through extensive mapping and its physical and chemical processes have been characterized over the past decades. However, a thorough description of the microbial communities inhabiting the bay is still missing. Methods: We present the first in-depth characterization of the prokaryotic communities of Paleochori Bay by sampling eight different seafloor types that are distributed along the entire gradient of hydrothermal influence. We used deep sequencing of the 16S rRNA marker gene and complemented the analysis with qPCR quantification of the 16S rRNA gene and several functional genes to gain insights into the metabolic potential of the communities. Results: We found that the microbiome of the bay is strongly influenced by the hydrothermal venting, with a succession of various groups dominating the sediments from the coldest to the warmest zones. Prokaryotic diversity and abundance decrease with increasing temperature, and thermophilic archaea overtake the community. Discussion: Relevant geochemical cycles of the Bay are discussed. This study expands our limited understanding of subsurface microbial communities in acidic shallow-sea hydrothermal systems and the contribution of their microbial activity to biogeochemical cycling.

The Energetic Potential for Undiscovered Manganese Metabolisms in Nature.

Microorganisms are found in nearly every surface and near-surface environment, where they gain energy by catalyzing reactions among a wide variety of chemical compounds. The discovery of new catabolic strategies and microbial habitats can therefore be guided by determining which redox reactions can supply energy under environmentally-relevant conditions. In this study, we have explored the thermodynamic potential of redox reactions involving manganese, one of the most abundant transition metals in the Earth's crust. In particular, we have assessed the Gibbs energies of comproportionation and disproportionation reactions involving Mn2+ and several Mn-bearing oxide and oxyhydroxide minerals containing Mn in the +II, +III, and +IV oxidation states as a function of temperature (0-100°C) and pH (1-13). In addition, we also calculated the energetic potential of Mn2+ oxidation coupled to O2, NO2 -, NO3 -, and FeOOH. Results show that these reactions-none of which, except O2 + Mn2+, are known catabolisms-can provide energy to microorganisms, particularly at higher pH values and temperatures. Comproportionation between Mn2+ and pyrolusite, for example, can yield 10 s of kJ (mol Mn)-1. Disproportionation of Mn3+ can yield more than 100 kJ (mol Mn)-1 at conditions relevant to natural settings such as sediments, ferromanganese nodules and crusts, bioreactors and suboxic portions of the water column. Of the Mn2+ oxidation reactions, the one with nitrite as the electron acceptor is most energy yielding under most combinations of pH and temperature. We posit that several Mn redox reactions represent heretofore unknown microbial metabolisms.

Geothermobacter hydrogeniphilus sp. nov., a mesophilic, iron(III)-reducing bacterium from seafloor/subseafloor environments in the Pacific Ocean, and emended description of the genus Geothermobacter.

A novel mesophilic, anaerobic, mixotrophic bacterium, with designated strains EPR-MT and HR-1, was isolated from a semi-extinct hydrothermal vent at the East Pacific Rise and from an Fe-mat at Lo'ihi Seamount, respectively. The cells were Gram-negative, pleomorphic rods of about 2.0 µm in length and 0.5 µm in width. Strain EPR-MT grew between 25 and 45 °C (optimum, 37.5-40 °C), 10 and 50 g l-1 NaCl (optimum, 15-20 g l-1) and pH 5.5 and 8.6 (optimum, pH 6.4). Strain HR-1 grew between 20 and 45 °C (optimum, 37.5-40 °C), 10 and 50 g l-1 NaCl (optimum, 15-25 g l-1) and pH 5.5 and 8.6 (optimum, pH 6.4). Shortest generation times with H2 as the primary electron donor, CO2 as the carbon source and ferric citrate as terminal electron acceptor were 6.7 and 5.5 h for EPR-MT and HR-1, respectively. Fe(OH)3, MnO2, AsO4 3-, SO4 2-, SeO4 2-, S2O3 2-, S0 and NO3 - were also used as terminal electron acceptors. Acetate, yeast extract, formate, lactate, tryptone and Casamino acids also served as both electron donors and carbon sources. G+C content of the genomic DNA was 59.4 mol% for strain EPR-MT and 59.2 mol% for strain HR-1. Phylogenetic and phylogenomic analyses indicated that both strains were closely related to each other and to Geothermobacter ehrlichii, within the class δ-Proteobacteria (now within the class Desulfuromonadia). Based on phylogenetic and phylogenomic analyses in addition to physiological and biochemical characteristics, both strains were found to represent a novel species within the genus Geothermobacter, for which the name Geothermobacter hydrogeniphilus sp. nov. is proposed. Geothermobacter hydrogeniphilus is represented by type strain EPR-MT (=JCM 32109T=KCTC 15831T=ATCC TSD-173T) and strain HR-1 (=JCM 32110=KCTC 15832).

Electrochemical evidence for in situ microbial activity at the Deep Mine Microbial Observatory (DeMMO), South Dakota, USA.

The subsurface is Earth's largest reservoir of biomass. Micro-organisms are the dominant lifeforms in this habitat, but the nature of their in situ activities remains largely unresolved. At the Deep Mine Microbial Observatory (DeMMO) located in the Sanford Underground Research Facility (SURF) in Lead, South Dakota (USA), we performed in situ electrochemical incubations designed to assess the potential for deep groundwater microbial communities to utilize extracellular electron transfer to support microbial respiration. DeMMO 4 was chosen for its stable geochemistry and microbial community. Graphite and indium tin oxide electrodes poised at -200 mV versus SHE were incubated along with open circuit controls and various minerals in a parallel flow reactor that split access to fluids across different treatments. From the patterns of net current over time (fluctuating between anodic and cathodic currents over the course of a few days to weeks) and the catalytic features measured using periodic cyclic voltammetry, evidence of both oxidative and reductive microbe-electrode interactions was observed. The predominant catalytic activity ranged from -210 to -120 mV. The observed temporal variability in electrochemical activity was unexpected given the documented stability in major geochemical parameters. This suggests that the accessed fluids are more heterogeneous in electrochemically active microbial populations than previously predicted from the stable community composition. As previously reported, the fracture fluid and surface-attached microbial communities at SURF differed significantly. However, only minimal differences in community composition were observed between poised potential electrodes, open circuit electrodes, and mineral incubations. These data support that in this environment the ability to attach to surfaces is a stronger driver of microbial community structure than the type or reactivity of the surface. We demonstrate that insight into specific activities can be gained from electrochemical methods, specifically chronoamperometry coupled with routine cyclic voltammetry, which provide a sensitive approach to evaluate microbial activities in situ.

Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer.

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter, Methyloversatilis, and Pseudomonas. Subsequent experimental deployments included a methanogen-dominated stage (Methanobacteriales and Methanomicrobiales) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.

Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface.

A novel, obligately anaerobic bacterium (strain SURF-ANA1T) was isolated from deep continental subsurface fluids at a depth of 1500 m below surface in the former Homestake Gold Mine (now Sanford Underground Research Facility, in Lead, South Dakota, USA). Cells of strain SURF-ANA1T were Gram-negative, helical, non-spore-forming and were 0.25-0.55×5.0-75.0 µm with a wavelength of 0.5-0.62 µm. Strain SURF-ANA1T grew at 15-50 °C (optimally at 40 °C), at pH 4.8-9.0 (pH 7.2) and in 1.0-40.0 g l-1 NaCl (10 g l-1 NaCl). The strain grew chemoheterotrophically with hydrogen or mono-, di- and polysaccharides as electron donors. The major cellular fatty acids in order of decreasing abundance (comprising >5% of total) were 10-methyl C16:0, iso-C15:0, C18:2 and C18:0 dimethyl acetal (DMA) and C20:0 methylene-nonadecanoic acid. Phylogenetic analysis based on the 16S rRNA gene sequence of strain SURF-ANA1T indicated a closest relationship with the recently characterized Rectinema cohabitans (99%). Despite high sequence identity, because of its distinct physiology, morphology and fatty acid profile, strain SURF-ANA1T is considered to represent a novel species within the genus Rectinema, for which the name Rectinema subterraneum sp. nov. is proposed. To our knowledge, this is the first report of an isolate within the phylum Spirochaetes from the deep (>100 m) terrestrial subsurface. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genomic sequences of strain SURF-ANA1T are KU359248 and GCF 009768935.1, respectively. The type strain of Rectinema subterraneum is SURF-ANA1T (=ATCC TSD-67=JCM 32656).

Bioenergetic characterization of a shallow-sea hydrothermal vent system: Milos Island, Greece.

Shallow-sea hydrothermal systems, like their deep-sea and terrestrial counterparts, can serve as relatively accessible portals into the microbial ecology of subsurface environments. In this study, we determined the chemical composition of 47 sediment porewater samples along a transect from a diffuse shallow-sea hydrothermal vent to a non-thermal background area in Paleochori Bay, Milos Island, Greece. These geochemical data were combined with thermodynamic calculations to quantify potential sources of energy that may support in situ chemolithotrophy. The Gibbs energies (ΔGr) of 730 redox reactions involving 23 inorganic H-, O-, C-, N-, S-, Fe-, Mn-, and As-bearing compounds were calculated. Of these reactions, 379 were exergonic at one or more sampling locations. The greatest energy yields were from anaerobic CO oxidation with NO2- (-136 to -162 kJ/mol e-), followed by reactions in which the electron acceptor/donor pairs were O2/CO, NO3-/CO, and NO2-/H2S. When expressed as energy densities (where the concentration of the limiting reactant is taken into account), a different set of redox reactions are the most exergonic: in sediments affected by hydrothermal input, sulfide oxidation with a range of electron acceptors or nitrite reduction with different electron donors provide 85~245 J per kg of sediment, whereas in sediments less affected or unaffected by hydrothermal input, various S0 oxidation reactions and aerobic respiration reactions with several different electron donors are most energy-yielding (80~95 J per kg of sediment). A model that considers seawater mixing with hydrothermal fluids revealed that there is up to ~50 times more energy available for microorganisms that can use S0 or H2S as electron donors and NO2- or O2 as electron acceptors compared to other reactions. In addition to revealing likely metabolic pathways in the near-surface and subsurface mixing zones, thermodynamic calculations like these can help guide novel microbial cultivation efforts to isolate new species.

Another chemolithotrophic metabolism missing in nature: sulfur comproportionation.

Chemotrophic microorganisms gain energy for cellular functions by catalyzing oxidation-reduction (redox) reactions that are out of equilibrium. Calculations of the Gibbs energy ( ΔG r ) can identify whether a reaction is thermodynamically favourable and quantify the accompanying energy yield at the temperature, pressure and chemical composition in the system of interest. Based on carefully calculated values of ΔG r , we predict a novel microbial metabolism - sulfur comproportionation (3H2 S + SO 4 2 - + 2H+ ⇌ 4S0 + 4H2 O). We show that at elevated concentrations of sulfide and sulfate in acidic environments over a broad temperature range, this putative metabolism can be exergonic ( ΔG r <0), yielding ~30-50 kJ mol-1 . We suggest that this may be sufficient energy to support a chemolithotrophic metabolism currently missing from the literature. Other versions of this metabolism, comproportionation to thiosulfate (H2 S + SO 4 2 - ⇌ S 2 O 3 2 - + H2 O) and to sulfite (H2 S + 3 SO 4 2 - ⇌ 4 SO 3 2 - + 2H+ ), are only moderately exergonic or endergonic even at ideal geochemical conditions. Natural and impacted environments, including sulfidic karst systems, shallow-sea hydrothermal vents, sites of acid mine drainage, and acid-sulfate crater lakes, may be ideal hunting grounds for finding microbial sulfur comproportionators.

Genome Sequence of Mariprofundus sp. Strain EBB-1, a Novel Marine Autotroph Isolated from an Iron-Sulfur Mineral.

Mariprofundus sp. strain EBB-1 was isolated from a pyrrhotite biofilm incubated in seawater from East Boothbay (ME, USA). Strain EBB-1 is an autotrophic member of the class Zetaproteobacteria with the ability to form iron oxide biominerals. Here, we present the 2.88-Mb genome sequence of EBB-1, which contains 2,656 putative protein-coding sequences.

Minireview: demystifying microbial reaction energetics.

The biology literature is rife with misleading information on how to quantify catabolic reaction energetics. The principal misconception is that the sign and value of the standard Gibbs energy ( Δ G r 0 ) define the direction and energy yield of a reaction; they do not. Δ G r 0 is one part of the actual Gibbs energy of a reaction (ΔGr ), with a second part accounting for deviations from the standard composition. It is also frequently assumed that Δ G r 0 applies only to 25 °C and 1 bar; it does not. Δ G r 0 is a function of temperature and pressure. Here, we review how to determine ΔGr as a function of temperature, pressure and chemical composition for microbial catabolic reactions, including a discussion of the effects of ionic strength on ΔGr and highlighting the large effects when multi-valent ions are part of the reaction. We also calculate ΔGr for five example catabolisms at specific environmental conditions: aerobic respiration of glucose in freshwater, anaerobic respiration of acetate in marine sediment, hydrogenotrophic methanogenesis in a laboratory batch reactor, anaerobic ammonia oxidation in a wastewater reactor and aerobic pyrite oxidation in acid mine drainage. These examples serve as templates to determine the energy yields of other catabolic reactions at environmentally relevant conditions.

The Cell and the Sum of Its Parts: Patterns of Complexity in Biosignatures as Revealed by Deep UV Raman Spectroscopy.

The next NASA-led Mars mission (Mars 2020) will carry a suite of instrumentation dedicated to investigating Martian history and the in situ detection of potential biosignatures. SHERLOC, a deep UV Raman/Fluorescence spectrometer has the ability to detect and map the distribution of many organic compounds, including the aromatic molecules that are fundamental building blocks of life on Earth, at concentrations down to 1 ppm. The mere presence of organic compounds is not a biosignature: there is widespread distribution of reduced organic molecules in the Solar System. Life utilizes a select few of these molecules creating conspicuous enrichments of specific molecules that deviate from the distribution expected from purely abiotic processes. The detection of far from equilibrium concentrations of a specific subset of organic molecules, such as those uniquely enriched by biological processes, would comprise a universal biosignature independent of specific terrestrial biochemistry. The detectability and suitability of a small subset of organic molecules to adequately describe a living system is explored using the bacterium Escherichia coli as a model organism. The DUV Raman spectra of E. coli cells are dominated by the vibrational modes of the nucleobases adenine, guanine, cytosine, and thymine, and the aromatic amino acids tyrosine, tryptophan, and phenylalanine. We demonstrate that not only does the deep ultraviolet (DUV) Raman spectrum of E. coli reflect a distinct concentration of specific organic molecules, but that a sufficient molecular complexity is required to deconvolute the cellular spectrum. Furthermore, a linear combination of the DUV resonant compounds is insufficient to fully describe the cellular spectrum. The residual in the cellular spectrum indicates that DUV Raman spectroscopy enables differentiating between the presence of biomolecules and the complex uniquely biological organization and arrangements of these molecules in living systems. This study demonstrates the ability of DUV Raman spectroscopy to interrogate a complex biological system represented in a living cell, and differentiate between organic detection and a series of Raman features that derive from the molecular complexity inherent to life constituting a biosignature.