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Marine sediments as excellent climate archives, contain among other biomolecules substantial amounts of extracellular DNA. Through mineral binding, some of the DNA remains protected from degradation which aids its preservation. While this pool of DNA represents genomic ecosystem fingerprints spanning over millions of years, the capability of current DNA extraction methods in recovering mineral‐bound DNA remains poorly understood. We evaluated current sedimentary DNA extraction approaches and their ability to recover short DNA fragments from artificially created DNA‐mineral complexes involving pure clay minerals or quartz, as well as from different types of natural marine sediments. We separately investigated lysis (DNA release) and isolation steps (purification of DNA) comparing five different lysis buffers across two commonly used DNA isolation approaches: silica magnetic beads and liquid‐phase organic extraction and purification. The choice of lysis buffer significantly impacted the amount of recovered mineral‐bound DNA and facilitated selective desorption of DNA fragments. High molarity EDTA and phosphate lysis buffers recovered on average an order of magnitude more DNA from clay minerals than other tested buffers, while both isolation approaches recovered comparable amounts of DNA. In marine sediments, however, liquid‐phase organic extraction caused inhibitory effects in subsequent downstream applications (e.g., PCR), across all assessed DNA extracts, while silica magnetic beads induced inhibition only in half of the tested DNA extracts. Thus, the isolation approach, together with the lysis buffer, played a decisive role in successful library preparation with lysis buffer choice ultimately impacting final library fragment distribution. With this study, we underscore the critical importance of lysis buffer selection to maximize the recovery of mineral‐bound DNA and show its profound impact on recovered fragment lengths in sedimentary DNA extractions, a crucial factor alongside existing isolation approaches in facilitating high‐quality DNA extracts for downstream analysis related to ancient environmental DNA research. Choice of lysis buffer and DNA isolation approach impacts yield and fragment composition in aeDNA extracts from marine sediments. Created with BioRender.com.

In coastal marine sediments, oxygen availability varies greatly, and anoxic conditions can develop quickly over low spatial resolution. Although benthic fungi are important players in the marine carbon cycle, little is known about their adaptation to fluctuating availability of oxygen as terminal electron acceptor. Here, we study which part of a mycobenthic community from oxic coastal sediments can thrive under temporarily anoxic conditions. We test whether phylogeny or certain fungal traits promote plasticity in respect to changes in oxygen availability. Therefore, we incubated mycobenthos under oxic and anoxic conditions, performed ITS2 Illumina tag-sequencing and an additional meta-analysis on a literature survey. Half of all OTUs showed a plasticity towards changing oxygen availability and exhibited different strategies towards anoxic conditions, with rapid response within hours or a delayed one after several days. The strategy of dimorphism and facultative yeasts were significantly linked to OTU occurrence in anoxic conditions, while phylogeny and other traits had less effect. Our results suggest that different fungal niches are formed over the duration of prolonged anoxic conditions. The taxon-specific proliferation seems to be regulated by the fine-tuning of various traits and factors. It is essential to take these results into account when conducting conceptual work on the functionality of the marine benthos.

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes) , Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe 2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.

Asgard is a recently discovered archaeal superphylum, closely linked to the emergence of eukaryotes. Among Asgard archaea, Lokiarchaeota are abundant in marine sediments, but their in situ activities are largely unknown except for Candidatus ‘Prometheoarchaeum syntrophicum’. Here, we tracked the activity of Lokiarchaeota in incubations with Helgoland mud area sediments (North Sea) by stable isotope probing (SIP) with organic polymers, 13 C-labelled inorganic carbon, fermentation intermediates and proteins. Within the active archaea, we detected members of the Lokiarchaeota class Loki-3, which appeared to mixotrophically participate in the degradation of lignin and humic acids while assimilating CO 2 , or heterotrophically used lactate. In contrast, members of the Lokiarchaeota class Loki-2 utilized protein and inorganic carbon, and degraded bacterial biomass formed in incubations. Metagenomic analysis revealed pathways for lactate degradation, and involvement in aromatic compound degradation in Loki-3, while the less globally distributed Loki-2 instead rely on protein degradation. We conclude that Lokiarchaeotal subgroups vary in their metabolic capabilities despite overlaps in their genomic equipment, and suggest that these subgroups occupy different ecologic niches in marine sediments.

In this study, we investigated how co-occurrence patters of ammonia and nitrite oxidizers, which drive autotrophic nitrification, are influenced by tree species composition as well as soil pH in different forest soils. We expected that a decline of ammonia oxidizers in coniferous forests, as a result of excreted nitrification inhibitors and at acidic sites with low availability of ammonia, would reduce the abundance of nitrite-oxidizing bacteria (NOB). To detect shifts in co-occurrence patterns, the abundance of key players was measured at 50 forest plots with coniferous respectively deciduous vegetation and different soil pH levels in the region Schwäbische Alb (Germany). We found ammonia-oxidizing archaea (AOA) and Nitrospira -like NOB (NS) to be dominating in numbers over their counterparts across all forest types. AOA co-occurred mostly with NS, while bacterial ammonia oxidizers (AOB) were correlated with Nitrobacter -like NOB (NB). Co-occurrence patterns changed from tight significant relationships of all ammonia and nitrite oxidizers in deciduous forests to a significant relationship of AOB and NB in coniferous forests, where AOA abundance was reduced. Surprisingly, no co-occurrence structures between ammonia and nitrite oxidizers could be determined at acidic sites, although abundances were correlated to the respective nitrogen pools. This raises the question whether interactions with heterotrophic nitrifiers may occur, which needs to be addressed in future studies.

Background The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy—the utilization of organic carbon as energy source but inorganic carbon as sole carbon source—has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8). Results To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13 C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13 C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13 C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility. Conclusion We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. A6zKEwmPSHRJuwdsc_HgHb Video Abstract

Background Rare species, especially of the marine sedimentary biosphere, have long been overlooked owing to the complexity of sediment microbial communities, their sporadic temporal and patchy spatial abundance, and challenges in cultivating environmental microorganisms. In this study, we combined enrichments, targeted metagenomic sequencing, and extensive data mining to uncover uncultivated members of the archaeal rare biosphere in marine sediments. Results In protein-amended enrichments, we detected the ecologically and metabolically uncharacterized class Candidatus Penumbrarchaeia within the phylum Thermoplasmatota. By screening more than 8000 metagenomic runs and 11,479 published genome assemblies, we expanded the phylogeny of Ca . Penumbrarchaeia by 3 novel orders. All six identified families of this class show low abundance in environmental samples characteristic of rare biosphere members. Members of the class Ca . Penumbrarchaeia were predicted to be involved in organic matter degradation in anoxic, carbon-rich habitats. All Ca . Penumbrarchaeia families contain high numbers of taxon-specific orthologous genes, highlighting their environmental adaptations and habitat specificity. Besides, members of this group exhibit the highest proportion of unknown genes within the entire phylum Thermoplasmatota, suggesting a high degree of functional novelty in this class. Conclusions In this study, we emphasize the necessity of targeted, data-integrative approaches to deepen our understanding of the rare biosphere and uncover the functions and metabolic potential hidden within these understudied taxa. BAgTGZ4wyFcCX_DqugzsU7 Video Abstract

Permanently cold marine sediments are heavily influenced by increased input of iron as a result of accelerated glacial melt, weathering, and erosion. The impact of such environmental changes on microbial communities in coastal sediments is poorly understood. We investigated geochemical parameters that shape microbial community compositions in anoxic surface sediments of four geochemically differing sites (Annenkov Trough, Church Trough, Cumberland Bay, Drygalski Trough) around South Georgia, Southern Ocean. Sulfate reduction prevails in Church Trough and iron reduction at the other sites, correlating with differing local microbial communities. Within the order Desulfuromonadales , the family Sva1033, not previously recognized for being capable of dissimilatory iron reduction, was detected at rather high relative abundances (up to 5%) while other members of Desulfuromonadales were less abundant (<0.6%). We propose that Sva1033 is capable of performing dissimilatory iron reduction in sediment incubations based on RNA stable isotope probing. Sulfate reducers, who maintain a high relative abundance of up to 30% of bacterial 16S rRNA genes at the iron reduction sites, were also active during iron reduction in the incubations. Thus, concurrent sulfate reduction is possibly masked by cryptic sulfur cycling, i.e., reoxidation or precipitation of produced sulfide at a small or undetectable pool size. Our results show the importance of iron and sulfate reduction, indicated by ferrous iron and sulfide, as processes that shape microbial communities and provide evidence for one of Sva1033’s metabolic capabilities in permanently cold marine sediments.

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13 C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60–86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens , up to 12% of methane was produced from CO 2 , indicating that CO 2 -dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO 2 -dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO 2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Metagenomic analysis has facilitated prediction of a variety of carbon utilization potentials by uncultivated archaea including degradation of protein, which is a wide-spread carbon polymer in marine sediments. However, the activity of detrital catabolic protein degradation is mostly unknown for the vast majority of archaea. Here, we show actively executed protein catabolism in three archaeal phyla (uncultivated Thermoplasmata, SG8-5; Bathyarchaeota subgroup 15; Lokiarchaeota subgroup 2c) by RNA- and lipid-stable isotope probing in incubations with different marine sediments. However, highly abundant potential protein degraders Thermoprofundales (MBG-D) and Lokiarchaeota subgroup 3 were not incorporating 13 C-label from protein during incubations. Nonetheless, we found that the pathway for protein utilization was present in metagenome associated genomes (MAGs) of active and inactive archaea. This finding was supported by screening extracellular peptidases in 180 archaeal MAGs, which appeared to be widespread but not correlated to organisms actively executing this process in our incubations. Thus, our results have important implications: (i) multiple low-abundant archaeal groups are actually catabolic protein degraders; (ii) the functional role of widespread extracellular peptidases is not an optimal tool to identify protein catabolism, and (iii) catabolic degradation of sedimentary protein is not a common feature of the abundant archaeal community in temperate and permanently cold marine sediments.