Iron reduction in subseafloor sulfate-depleted and methane-rich marine sediments is currently a subject of interest in subsurface geomicrobiology. While iron reduction and microorganisms involved have been well studied in marine surface sediments, little is known about microorganisms responsible for iron reduction in deep methanic sediments. Here, we used quantitative PCR-based 16S rRNA gene copy numbers and pyrosequencing-based relative abundances of bacteria and archaea to investigate covariance between distinct microbial populations and specific geochemical profiles in the top 5 m of sediment cores from the Helgoland mud area, North Sea. We found that gene copy numbers of bacteria and archaea were specifically higher around the peak of dissolved iron in the methanic zone (250–350 cm). The higher copy numbers at these depths were also reflected by the relative sequence abundances of members of the candidate division JS1, methanogenic and Methanohalobium/ANME-3 related archaea. The distribution of these populations was strongly correlated to the profile of pore-water Fe2+ while that of Desulfobacteraceae corresponded to the pore-water sulfate profile. Furthermore, specific JS1 populations also strongly co-varied with the distribution of Methanosaetaceae in the methanic zone. Our data suggest that the interplay among JS1 bacteria, methanogenic archaea and Methanohalobium/ANME-3-related archaea may be important for iron reduction and methane cycling in deep methanic sediments of the Helgoland mud area and perhaps in other methane-rich depositional environments.

The role of microorganisms in the cycling of sedimentary organic carbon is a crucial one. To better understand relationships between molecular composition of a potentially bioavailable fraction of organic matter and microbial populations, bacterial and archaeal communities were characterized using pyrosequencing-based 16S rRNA gene analysis in surface (top 30 cm) and subsurface/deeper sediments (30–530 cm) of the Helgoland mud area, North Sea. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) was used to characterize a potentially bioavailable organic matter fraction (hot-water extractable organic matter, WE-OM). Algal polymer-associated microbial populations such as members of the Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia were dominant in surface sediments while members of the Chloroflexi (Dehalococcoidales and candidate order GIF9) and Miscellaneous Crenarchaeota Groups (MCG), both of which are linked to degradation of more recalcitrant, aromatic compounds and detrital proteins, were dominant in subsurface sediments. Microbial populations dominant in subsurface sediments (Chloroflexi, members of MCG, and Thermoplasmata) showed strong correlations to total organic carbon (TOC) content. Changes of WE-OM with sediment depth reveal molecular transformations from oxygen-rich [high oxygen to carbon (O/C), low hydrogen to carbon (H/C) ratios] aromatic compounds and highly unsaturated compounds toward compounds with lower O/C and higher H/C ratios. The observed molecular changes were most pronounced in organic compounds containing only CHO atoms. Our data thus, highlights classes of sedimentary organic compounds that may serve as microbial energy sources in methanic marine subsurface environments.

Carbon flow from benthic diatoms to heterotrophic bacterial was traced in an intertidal sediment for 5 consecutive days. 13 C-labeled bicarbonate was sprayed onto the sediment surface during low tide and 13 C-label incorporation in major carbon pools, intermediate metabolites, and biomarkers were monitored. Phospholipidderived fatty acid (PLFA) and ribosomal ribonucleic acid (rRNA) were used to identify the responsible members of the microbial community at class and family phylogenetic resolution. Diatoms were the predominant primary producers, and Gammaproteobacteria, Bacteroidetes, and Deltaproteobacteria (21%, 8%, and 7% of 16S rRNAderived clone library) were major heterotrophic bacterial groups. Both 13 C-PLFA and 13 C-rRNA data suggest a fast transfer of label from diatoms (60 nmol 13 C g 21 dry weight [dry wt]) to bacteria (7 nmol 13 C g 21 dry wt) during the first 24 h, which was probably due to the exudation of low-molecular-weight organic compounds by diatoms that could be directly utilized by bacteria. After this initial fast transfer, labeling of bacteria proceeded at a slower rate to 13 nmol 13 C g 21 dry wt on the third day of the experiment, which coincided with the degradation of carbohydrates in water-extractable extracellular polymeric substances (EPS) initially produced by the diatoms. Water-extractable EPS (primarily as glucose) was a major intermediate and its turnover explained 75% of the total carbohydrate processing in the sediment. Labeling in bacteria tracked labeling in the diatoms, suggesting a closely coupled system. The heterotrophic bacterial groups benefited equally from the organic matter released by the diatoms, suggesting limited specialization in this microbial food web.

We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine 13 C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotopelabeled [13 C]glucose, [ 13 C]propionate, or [ 13 C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that 13 C labeling can be much lower, to the extent that ␦ 13 C ratios can be studied even at their natural abundances.

Stable isotope probing of magnetic-bead-captured rRNA (Mag-SIP) indicated clear differences in in situ organic substrate utilization by major microbial groups between the more oxidized (0 to 2 cm) and sulfate-reducing (2 to 5 cm) horizons of marine intertidal sediment. We also showed that cyanobacteria and diatoms may survive by glucose utilization under dark anoxic conditions. T he microbial community in marine sediments is highly diverse and consists mainly of microorganisms related only distantly to described isolates, whose functions are therefore difficult to predict (1-3). A recent study indicated a higher functional redundancy in the oxidized top layer of marine sediments, where higher disturbance rates and higher availability of substrates may lead to the formation of a community of fast-growing generalists (4). In contrast, a consortium of microbes consisting of specialized fermenting and sulfate-reducing bacteria is thought to be involved in organic matter degradation under sulfate-reducing conditions (5). However, intermediate metabolites are generally at low concentrations due to their high turnover rates, making in situ identification of the microbial groups utilizing them difficult. We utilized a recently developed stable isotope-probing method based on magnetic bead capturing of specific 16S rRNA and subsequent sensitive 13 C analysis of the captured material (Mag-SIP) (6, 7) to show major differences in substrate utilization by predominant microbial groups between the oxidized top layer and sulfate-reducing deeper layer of an intertidal marine sediment.In this study, sediment cores (internal diameter, 5.2 cm) were collected at an intertidal flat in the for amino acids and 0.9 mol 13 C cm Ϫ3 for the other three substrates. These substrates represent both major constituents of the organic matter pool (carbohydrates and amino acids) and the main fermentation products (acetate and propionate) in marine sediments. Cores were incubated (24 h, 14°C) and sectioned in surface layers (0 to 2 cm) and deeper layers (2 to 5 cm), which corresponded to a clear color change of the sediment from brown-yellow to dark gray. A nested set of probes targeting approximately 80% of the rRNA sequences recovered from the two sediment layers was used with the Mag-SIP protocol (7). The testing of the probes for total bacterial rRNA (EUB338), Deltaproteobacteria rRNA (DELTA495a), and Desulfobacteraceae rRNA (Dbact653) was previously described by Miyatake et al. (7). Probe CYA361 was used to target cyanobacterium and chloroplast rRNA (20% formamide [8]). We designed a new specific probe and matching helper probes that target the 16S rRNA of most Beta-and Gammaproteobacteria (specific probe BG553 sequence, CGC CCA GTA ATT CCG ATT [60% formamide]; helper probe BG553_up_help sequence, AAC CGC CTR CGN RCG CTT TA; helper probe BG553_down_help sequence, AAC GCT YGC ACC CTM CTG ATT). In this study, the BG553 probe is basically Gammaproteobacteria specific, as we did not detect Betaproteobacteriarelated sequences in any of the clone ...