Mahoney Lake represents an extreme meromictic model system and is a valuable site for examining the organisms and processes that sustain photic zone euxinia (PZE). A single population of purple sulfur bacteria (PSB) living in a dense phototrophic plate in the chemocline is responsible for most of the primary production in Mahoney Lake. Here, we present metagenomic data from this phototrophic plate--including the genome of the major PSB, as obtained from both a highly enriched culture and from the metagenomic data--as well as evidence for multiple other taxa that contribute to the oxidative sulfur cycle and to sulfate reduction. The planktonic PSB is a member of the Chromatiaceae, here renamed Thiohalocapsa sp. strain ML1. It produces the carotenoid okenone, yet its closest relatives are benthic PSB isolates, a finding that may complicate the use of okenone (okenane) as a biomarker for ancient PZE. Favorable thermodynamics for non-phototrophic sulfide oxidation and sulfate reduction reactions also occur in the plate, and a suite of organisms capable of oxidizing and reducing sulfur is apparent in the metagenome. Fluctuating supplies of both reduced carbon and reduced sulfur to the chemocline may partly account for the diversity of both autotrophic and heterotrophic species. Collectively, the data demonstrate the physiological potential for maintaining complex sulfur and carbon cycles in an anoxic water column, driven by the input of exogenous organic matter. This is consistent with suggestions that high levels of oxygenic primary production maintain episodes of PZE in Earth's history and that such communities should support a diversity of sulfur cycle reactions.

Mahoney Lake in British Columbia is an extreme meromictic system with unusually high levels of sulfate and sulfide present in the water column. As is common in strongly stratified lakes, Mahoney Lake hosts a dense, sulfide-oxidizing phototrophic microbial community where light reaches the chemocline. Below this “plate,” the euxinic hypolimnion is anoxic, eutrophic, saline, and rich in sulfide, polysulfides, elemental sulfur, and other sulfur intermediates. While much is known regarding microbial communities in sunlit portions of euxinic systems, the composition and genetic potential of organisms living at aphotic depths have rarely been studied. Metagenomic sequencing of samples from the hypolimnion and the underlying sediments of Mahoney Lake indicate that multiple taxa contribute to sulfate reduction below the chemocline and that the hypolimnion and sediments each support distinct populations of sulfate reducing bacteria (SRB) that differ from the SRB populations observed in the chemocline. After assembling and binning the metagenomic datasets, we recovered near-complete genomes of dominant populations including two Deltaproteobacteria. One of the deltaproteobacterial genomes encoded a 16S rRNA sequence that was most closely related to the sulfur-disproportionating genus Dissulfuribacter and the other encoded a 16S rRNA sequence that was most closely related to the fatty acid- and aromatic acid-degrading genus Syntrophus. We also recovered two near-complete genomes of Firmicutes species. Analysis of concatenated ribosomal protein trees suggests these genomes are most closely related to extremely alkaliphilic genera Alkaliphilus and Dethiobacter. Our metagenomic data indicate that these Firmicutes contribute to carbon cycling below the chemocline. Lastly, we recovered a nearly complete genome from the sediment metagenome which represents a new genus within the FCB (Fibrobacteres, Chlorobi, Bacteroidetes) superphylum. Consistent with the geochemical data, we found little or no evidence for organisms capable of sulfide oxidation in the aphotic zone below the chemocline. Instead, comparison of functional genes below the chemocline are consistent with recovery of multiple populations capable of reducing oxidized sulfur. Our data support previous observations that at least some of the sulfide necessary to support the dense population of phototrophs in the chemocline is supplied from sulfate reduction in the hypolimnion and sediments. These studies provide key insights regarding the taxonomic and functional diversity within a euxinic environment and highlight the complexity of biogeochemical carbon and sulfur cycling necessary to maintain euxinia.

Throughout the Proterozoic δ(13)C values for preserved n-alkyl lipids are more positive than for syngenetic kerogen. This pattern is the inverse of biosynthetic expectations. It has been suggested that this isotopic inversion results from selective preservation of lipids from (13)C-enriched heterotrophic populations, while the bulk of kerogen derives from primary producers. Here, we formulate a degradation model to calculate the (13)C content of sedimentary total organic carbon and lipid. The model addresses two scenarios. The first scenario explores preferential preservation of heterotrophic lipid, thereby quantifying the existing hypothesis. In the second, we suggest that an inverse signature could be the result of prokaryotic phytoplankton contributing the majority of the total ecosystem biomass. Photosynthetic prokaryotes bearing a relative (13)C enrichment would contribute much of the resulting preserved lipids, while primary eukaryotic biomass would dominate the total organic carbon. We find that our hypothesis of a mixed primary producer community generates inverse isotopic patterns while placing far fewer requirements on specific degradation conditions. It also provides a possible explanation as to why there are large variations in the (13)C content of the isoprenoid lipids pristane and phytane relative to n-alkyl lipid, while the difference between n-alkyl lipid and kerogen is more constant. Our results suggest that the disappearance of the inverse (13)C signature in the late Ediacaran is a natural consequence of the fundamental shift to oceans in which export production has a higher ratio of eukaryotic biomass.

Protein stable isotope fingerprinting (P-SIF) is a method to measure the carbon isotope ratios of whole proteins separated from complex mixtures, including cultures and environmental samples. The goal of P-SIF is to expose the links between taxonomic identity and metabolic function in microbial ecosystems. To accomplish this, two dimensions of chromatography are used in sequence to resolve a sample containing ca. 5-10 mg of mixed proteins into 960 fractions. Each fraction then is split in two aliquots: The first is digested with trypsin for peptide sequencing, while the second has its ratio of (13)C/(12)C (value of δ(13)C) measured in triplicate using an isotope-ratio mass spectrometer interfaced with a spooling wire microcombustion device. Data from cultured species show that bacteria have a narrow distribution of protein δ(13)C values within individual taxa (±0.7-1.2‰, 1σ). This is moderately larger than the mean precision of the triplicate isotope measurements (±0.5‰, 1σ) and may reflect heterogeneous distribution of (13)C among the amino acids. When cells from different species are mixed together prior to protein extraction and separation, the results can predict accurately (to within ±1σ) the δ(13)C values of the original taxa. The number of data points required for this endmember prediction is ≥20/taxon, yielding a theoretical resolution of ca. 10 taxonomic units/sample. Such resolution should be useful to determine the overall trophic breadth of mixed microbial ecosystems. Although we utilize P-SIF to measure natural isotope ratios, it also could be combined with experiments that incorporate stable isotope labeling.

Planktonic sulfur bacteria growing in zones of photic zone euxinia (PZE) are important primary producers in stratified, sulfur-rich environments. The potential for export and burial of microbial biomass from anoxic photic zones remains relatively understudied, despite being of fundamental importance to interpreting the geologic record of bulk total organic carbon (TOC) and individual lipid biomarkers. Here we report the relative concentrations and carbon isotope ratios of lipid biomarkers from the water column and sediments of meromictic Mahoney Lake. The data show that organic matter in the central basin sediments is indistinguishable from material at the lake shoreline in both its lipid and carbon isotopic compositions. However, this material is not consistent with either the lipid profile or carbon isotope composition of biomass obtained directly from the region of PZE. Due to the strong density stratification and the intensive carbon and sulfur recycling pathways in the water column, there appears to be minimal direct export of the sulfur-oxidizing planktonic community to depth. The results instead suggest that basinal sediments are sourced via the littoral environment, a system that integrates an indigenous shoreline microbial community, the degraded remains of laterally rafted biomass from the PZE community, and detrital remains of terrigenous higher plants. Material from the lake margins appears to travel downslope, traverse the strong density gradient, and become deposited in the deep basin; its final composition may be largely heterotrophic in origin. This suggests an important role for clastic and/or authigenic minerals in aiding the burial of terrigenous and mat-derived organic matter in euxinic systems. Downslope or mineral-aided transport of anoxygenic, photoautotrophic microbial mats may have been a significant sedimentation process in early Earth history.

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