ALBERT

Description: The electrocopolymerization of 3,4-ethylenedioxythiophene (EDOT) with the branched thiophene building block 2,2′:3′,2″-terthiophene (3T) is presented as a versatile route to functional polymer films. Comparisons to blend systems of the respective homopolymers PEDOT and P3T by in situ spectroelectrochemistry and Raman spectroscopy prove the successful copolymer formation and the access to tailored redox properties and energy levels. The use of EDOT-N 3 as co-monomer furthermore allows modifications of the films by polymer analogous reactions. Here, we exemplarily describe the post-functionalization with ionic moieties by 1,3-dipolar cycloaddition (“click”-chemistry) which allows to tune the surface polarity of the copolymer films from water contact angles of 140° down to 40°. Beilstein J. Org. Chem. 2015, 11, 335–347. doi:10.3762/bjoc.11.39

Description: The electrocopolymerization of 3,4-ethylenedioxythiophene (EDOT) with the branched thiophene building block 2,2′:3′,2″-terthiophene (3T) is presented as a versatile route to functional polymer films. Comparisons to blend systems of the respective homopolymers PEDOT and P3T by in situ spectroelectrochemistry and Raman spectroscopy prove the successful copolymer formation and the access to tailored redox properties and energy levels. The use of EDOT-N3 as co-monomer furthermore allows modifications of the films by polymer analogous reactions. Here, we exemplarily describe the post-functionalization with ionic moieties by 1,3-dipolar cycloaddition (“click”-chemistry) which allows to tune the surface polarity of the copolymer films from water contact angles of 140° down to 40°.

Description: 16S rRNA and metabolic gene abundances as well as gene reconstructions were generated using a novel, modified version of the phyloFlash pipeline (https://github.com/HRGV/phyloFlash) called funcFlash for metabolic genes. In brief, the generated reads were mapped with BBMap at minimal global nucleotide identity of 70% against curated nucleotide databases: The SILVA SSURef v119 database [75], a published dsrAB gene database (http://www.microbial-ecology.net/db_download/dsr_v3.zip, [83]) and two newly generated pmoA and mcrA gene databases that are publicly available at PANGAEA, see [61]. The mapped read pairs were counted when at least one read had a positive mapping. Full length (〉70% of the target length) genes were assembled with SPAdes [84] or reconstructed with EMIRGE [85]. The mapping of reconstructed metabolic genes to curated databases using funcFlash can be used to distinguish, whether a gene of interest is affiliated with organisms performing the reductive or oxidative pathway. In our case this new analysis allowed us to distinguish between dsrAB genes from sulfate reducers and from sulfur oxidizers, as well as between mcrA genes from methanogens and from methanotrophs.

Description: Organic-rich, brackish water bodies are common along coastlines and important for the biogeochemical cycling of carbon, nitrogen, sulfur, and phosphorus. These ecosystems are dynamic and frequently disturbed by weather, tides, erosion, and human activities. Here, we investigated a shallow, brackish lagoon (Trunk River, Woods Hole, MA) that contains layers of decaying organic matter, which releases hydrogen sulfide upon physical disturbance. To study the microbial habitat and community response to perturbations, we carried out replicated in situ experiments, analyzing the physicochemistry and microbial community succession. At each site, yellow blooms of microorganisms formed within three days after disturbance. The water column changed substantially, establishing steep gradients of temperature, oxygen, sulfide, and salinity. The diverse microbial community at early timepoints was replaced by a community largely dominated by a clonal population of green sulfur bacteria (GSB) Prosthecochloris vibrioformis. Despite its dominance, this population coexisted with less abundant GSBs affiliating with Chlorobaculum. This population represents a new Chlorobaculum species, as indicated by phylogenetic and phylogenomic placement, ANI values, and CRISPR-Cas genes. Interestingly, despite their dominance the GSB coexisted with purple sulfur bacteria (Halochromatium sp. and Allochromatium sp.), anoxygenic phototrophic Chloroflexi (Chloroploca sp.) and other phototrophs. A high relative sequence abundance of Microviridae viruses was found in the metagenome, indicating their activity in the bloom. After two weeks the bloom subsided and the ecosystem slowly returned towards a diverse state and ecosystem functions, indicating its resilience after disturbance. This work provides insights into the assembly, succession, and coexistence of oxygenic and anoxygenic phototrophs in a common coastal ecosystem. The transient bloom was spatially structured analogous to a phototrophic microbial mat with distinct ecological niches for multiple clades of Cyanobacteria, purple and green sulfur bacteria. We suggest that a cryptic sulfur cycle in the water column between sulfur-oxidizing phototrophs, sulfate reducers, and sulfur oxidizers enhanced the development of the bloom. The bloom was likely driven by new species in the order Chlorobiales and possibly impacted by viruses of the family Microviridae.

Description: CARD-FISH was performed as previously described in Ruff et al., (2013; doi:10.1371/journal.pone.0072627) with the following modifications. 4-6 µl of 25-fold diluted sediment were used for filtration. Archaeal cell walls were permeabilized with 0.1M HCl for 2 min to detect ANME-3 cells, or Proteinase K solution (15 µg ml-1 (Merck, Darmstadt, Germany) in 0.05 M EDTA (pH 8), 0.1 M Tris-HCl (pH 8), 0.5 M NaCl) for 2-4 min at room temperature for all other archaea. Bacterial cell walls were permeabilized with lysozyme solution (1000kU/ml) for 60 min at 37°. Cells were stained with DAPI (1µg/ml), embedded in mounting medium and counted in 40-60 independent microscopic fields using an Axiophot II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Description: DNA extraction was carried out as described on the MICROBIS project pages (http://icomm.mbl.edu/microbis ) using a commercially available extraction kit. We amplified the hypervariable regions V4-V6 of archaeal and bacterial 16S rRNA genes using PCR and several sets of forward and reverse primers (http://vamps.mbl.edu/resources/primers.php). Massively parallel tag sequencing of the PCR products was carried out on a 454 Life Sciences GS FLX sequencer at Marine Biological Laboratory, Woods Hole, MA, following the same experimental conditions for all samples. Sequence reads were submitted to a rigorous quality control procedure based on mothur v30 (doi:10.1128/AEM.01541-09) including denoising of the flow grams using an algorithm based on PyroNoise (doi:10.1038/nmeth.1361), removal of PCR errors and a chimera check using uchime (doi:10.1093/bioinformatics/btr381). The reads were taxonomically assigned according to the SILVA taxonomy (SSURef v119, 07-2014; doi:10.1093/nar/gks1219) implemented in mothur and clustered at 98% ribosomal RNA gene V4-V6 sequence identity. V4-V6 amplicon sequence abundance tables were standardized to account for unequal sampling effort using 1000 (Archaea) and 2300 (Bacteria) randomly chosen sequences without replacement using mothur and then used to calculate inverse Simpson diversity indices and Chao1 richness (doi:10.2307/4615964). Bray-Curtis dissimilarities (doi:10.2307/1942268) between all samples were calculated and used for 2-dimensional non metric multidimensional scaling (NMDS) ordinations with 20 random starts (doi:10.1007/BF02289694). Stress values below 0.2 indicated that the multidimensional dataset was well represented by the 2D ordination. NMDS ordinations were compared and tested using Procrustes correlation analysis (doi:10.1007/BF02291478). All analyses were carried out with the R statistical environment and the packages vegan (available at: http://cran.r-project.org/package=vegan), labdsv (available at: http://cran.r-project.org/package=labdsv), as well as with custom R scripts. Operational taxonomic units at 98% sequence identity (OTU0.03) that occurred only once in the whole dataset were termed absolute single sequence OTUs (SSOabs; doi:10.1038/ismej.2011.132). OTU0.03 sequences that occurred only once in at least one sample, but may occur more often in other samples were termed relative single sequence OTUs (SSOrel). SSOrel are particularly interesting for community ecology, since they comprise rare organisms that might become abundant when conditions change.16S rRNA amplicons and metagenomic reads have been stored in the sequence read archive under SRA project accession number SRP042162.

Description: Emission of the greenhouse gas methane from the seabed is globally controlled by marine aerobic and anaerobic methanotrophs gaining energy via methane oxidation. However, the processes involved in the assembly and dynamics of methanotrophic populations in complex natural microbial communities remain unclear. Here we investigated the development of a methanotrophic microbiome following subsurface mud eruptions at Håkon Mosby mud volcano (1250 m water depth). We analyzed surface and subsurface sediment samples across HMMV mud flows from most recently discharged subsurface muds towards old consolidated muds as well as one reference site located approximately 0.5 km outside of the HMMV. Surface sediment samples (0-20 cm) were recovered either by TV-guided Multicorer or by push cores. Subsurface sediments of all zones (〉2 m below sea floor) were obtained in by gravity corer. After recovery, sediments were immediately subsampled in a refrigerated container (0°C) and further processed for biogeochemical analyses or preserved at -20°C for later DNA analyses. Our study show that freshly erupted muds hosted deep-subsurface communities, which were dominated by Bathyarchaeota, Atribacteria and Chloroflexi. Methanotrophy was initially limited to a thin surface layer of Methylococcales populations consuming methane aerobically. With increasing distance to the eruptive center, anaerobic methanotrophic archaea, sulfate-reducing Desulfobacterales and thiotrophic Beggiatoaceae developed, and their respective metabolic capabilities dominated the biogeochemical functions of the community. Microbial richness, evenness, and cell numbers of the entire microbial community increased up to tenfold within a few years downstream of the mud flow from the eruptive center. The increasing diversity was accompanied by an up to fourfold increase in sequence abundance of relevant metabolic genes of the anaerobic methanotrophic and thiotrophic guilds. The communities fundamentally changed in their structure and functions as reflected in the metagenome turnover with distance from the eruptive center, and this was reflected in the biogeochemical zonation across the mud volcano caldera. The observed functional succession provides a framework for the response time and recovery of complex methanotrophic communities after disturbances of the deep-sea bed.