Marc Mußmann

Detoxification of sulphidic African shelf waters by blooming chemolithotrophs

Coastal waters support ∼90 per cent of global fisheries and are therefore an important food reserve for our planet. Eutrophication of these waters, due to human activity, leads to severe oxygen depletion and the episodic occurrence of hydrogen sulphide—toxic to multi-cellular life—with disastrous consequences for coastal ecosytems. Here we show that an area of ∼7,000 km2 of African shelf, covered by sulphidic water, was detoxified by blooming bacteria that oxidized the biologically harmful sulphide to environmentally harmless colloidal sulphur and sulphate. Combined chemical analyses, stoichiometric modelling, isotopic incubations, comparative 16S ribosomal RNA, functional gene sequence analyses and fluorescence in situ hybridization indicate that the detoxification proceeded by chemolithotrophic oxidation of sulphide with nitrate and was mainly catalysed by two discrete populations of γ- and ε-proteobacteria. Chemolithotrophic bacteria, accounting for ∼20 per cent of the bacterioplankton in sulphidic waters, created a buffer zone between the toxic sulphidic subsurface waters and the oxic surface waters, where fish and other nekton live. This is the first time that large-scale detoxification of sulphidic waters by chemolithotrophs has been observed in an open-ocean system. The data suggest that sulphide can be completely consumed by bacteria in the subsurface waters and, thus, can be overlooked by remote sensing or monitoring of shallow coastal waters. Consequently, sulphidic bottom waters on continental shelves may be more common than previously believed, and could therefore have an important but as yet neglected effect on benthic communities.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, 14CO2 fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of 13CO2, was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.

An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments

In situ identification of prokaryotic cells in subsurface sediments is hampered by the low cellular rRNA contents of the target organisms. Fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) has the potential to overcome this limitation, and was therefore optimized for a 40 cm deep sediment core sampled from a tidal sandy flat of the German Wadden Sea. Treatment with methanol and H(2)O(2) inactivated endogenous peroxidases and effectively reduced the background signal. Percentage of DAPI stained cells detected with the probe combination EUB(I-III), targeting nearly all the Bacteria, were comparable for CARD-FISH with a horseradish peroxidase (HRP)-labeled probe and FISH with a fluorescently monolabeled probe in the 2-3 cm depth interval (92% and 82%, respectively), but significantly higher with the HRP-labeled probe at 35-40 cm, the deepest layer sampled (63% with HRP vs. 26% with monolabeled probe). With CARD-FISH Alphaproteobacteria and the Desulfobulbaceae group of sulfate-reducing bacteria were detected only in the upper layers. In contrast, Desulfosarcinales, the Bacteroidetes group, Planctomycetes, Betaproteobacteria, and Gammaproteobacteria were found at all depths. Archaea were detectable with ARCH915-HRP after achromopeptidase treatment. Surprisingly, aggregates of Bacteria and Archaea were found, below 12 cm depth, that strongly resemble consortia involved in anoxic oxidation of methane that have previously been found in sediments near methane hydrate deposits. With the optimized CARD-FISH protocol, microbial populations could also be detected in deeper sediment horizons. Furthermore, the intensity of the CARD-FISH signals improved detection of rare organisms such as Archaea.

Insights into the genome of large sulfur bacteria revealed by analysis of single filaments.

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO2 fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments.

Reverse dissimilatory sulfite reductase as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes.

Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of dsrAB that encode the reversely operating sirohaem dissimilatory sulfite reductase (rDSR) and are present in many but not all published genomes of SOP. The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.

Ubiquitous Gammaproteobacteria dominate dark carbon fixation in coastal sediments

Marine sediments are the largest carbon sink on earth. Nearly half of dark carbon fixation in the oceans occurs in coastal sediments, but the microorganisms responsible are largely unknown. By integrating the 16S rRNA approach, single-cell genomics, metagenomics and transcriptomics with 14C-carbon assimilation experiments, we show that uncultured Gammaproteobacteria account for 70–86% of dark carbon fixation in coastal sediments. First, we surveyed the bacterial 16S rRNA gene diversity of 13 tidal and sublittoral sediments across Europe and Australia to identify ubiquitous core groups of Gammaproteobacteria mainly affiliating with sulfur-oxidizing bacteria. These also accounted for a substantial fraction of the microbial community in anoxic, 490-cm-deep subsurface sediments. We then quantified dark carbon fixation by scintillography of specific microbial populations extracted and flow-sorted from sediments that were short-term incubated with 14C-bicarbonate. We identified three distinct gammaproteobacterial clades covering diversity ranges on family to order level (the Acidiferrobacter, JTB255 and SSr clades) that made up >50% of dark carbon fixation in a tidal sediment. Consistent with these activity measurements, environmental transcripts of sulfur oxidation and carbon fixation genes mainly affiliated with those of sulfur-oxidizing Gammaproteobacteria. The co-localization of key genes of sulfur and hydrogen oxidation pathways and their expression in genomes of uncultured Gammaproteobacteria illustrates an unknown metabolic plasticity for sulfur oxidizers in marine sediments. Given their global distribution and high abundance, we propose that a stable assemblage of metabolically flexible Gammaproteobacteria drives important parts of marine carbon and sulfur cycles.

Drivers of bacterial colonization patterns in stream biofilms

Dispersal and colonization are important for the assembly and biodiversity of microbial communities. While emigration as the initial step of dispersal has become increasingly understood in model bacterial biofilms, the drivers of dispersal and colonization in complex biofilms remain elusive. We grew complex biofilms in microcosms from natural surface water in laminar and turbulent flow, and investigated dispersal and colonization patterns of fluorescently labeled cells and microbeads in nascent and mature biofilms. Settling occurred in nonrandom spatial patterns governed by the interplay of local flow patterns and biofilm topography. Settling was higher in treatments with nascent biofilms, with fewer cells remaining in the water column than in treatments with mature biofilms. The flow regime had no effect on settling velocity, even though in mature biofilms the formation of streamers under turbulent flow enhanced particle trapping compared with the laminar flow treatment. Hence, small-scale variations in the flow pattern seemed to be more important than the overall flow regime. Furthermore, spatial analysis of the colonizer patterns suggests that bacteria have moved in the biofilm after settling. Our results show that colonization of biofilms in a model stream environment is a heterogeneous process differently affected by biological and physical factors.

Structure of microbial communities and hydrocarbon-dependent sulfate reduction in the anoxic layer of a polluted microbial mat.

The bacterial communities in the anoxic layer of a heavily polluted microbial mat and their growth on hydrocarbons under sulfate-reducing conditions were investigated. Microbial communities were dominated by members of Alphaproteobacteria (27% of the total rRNA), Planctomycetes (21.1%) and sulfate-reducing bacteria (SRB: 17.5%). 16S rRNA cloning revealed sequences beloning to the same bacterial groups with SRB affiliated to the genera Desulfobulbus, Desulfocapsa, Desulfomicrobium, Desulfobacterium and Desulfosarcina/Desulfococcus. The derived enrichment cultures on crude oil, hexadecane and toluene were dominated by SRB. While most SRB sequences of the toluene and hexadecane cultures were related to the sequence of Desulfotignum toluolica, the crude oil enrichment showed a more diverse bacterial community with sequences from the genera Desulfotignum, Desulfobacter, Desulfatibacillus, Desulfosalina, and Desulfococcus. We conclude that the anoxic layer of the studied mats contains a diverse community of anaerobic bacteria, dominated by SRB, some of which are able to grow on hydrocarbons.

Microbial consumption of zero-valence sulfur in marine benthic habitats.

Zero-valence sulfur (S°) is a central intermediate in the marine sulfur cycle and forms conspicuous accumulations at sediment surfaces, hydrothermal vents and in oxygen minimum zones. Diverse microorganisms can utilize S°, but those consuming S° in the environment are largely unknown. We identified possible key players in S° turnover on native or introduced S° in benthic coastal and deep-sea habitats using the 16S ribosomal RNA approach, (in situ) growth experiments and activity measurements. In all habitats, the epsilonproteobacterial Sulfurimonas/Sulfurovum group accounted for a substantial fraction of the microbial community. Deltaproteobacterial Desulfobulbaceae and Desulfuromonadales were also frequently detected, indicating S° disproportionation and S° respiration under anoxic conditions. Sulfate production from S° particles colonized in situ with Sulfurimonas/Sulfurovum suggested that this group oxidized S°. We also show that the type strain Sulfurimonas denitrificans is able to access cyclooctasulfur (S₈), a metabolic feature not yet demonstrated for sulfur oxidizers. The ability to oxidize S°, in particular S8 , likely facilitates niche partitioning among sulfur oxidizers in habitats with intense microbial sulfur cycling such as sulfidic sediment surfaces. Our results underscore the previously overlooked but central role of Sulfurimonas/Sulfurovum group for conversion of free S° at the seafloor surface.