Ketil Bernt Sørensen

Uncultured archaea in deep marine subsurface sediments: have we caught them all?

Deep marine subsurface sediments represent a novel archaeal biosphere with unknown physiology; the sedimentary subsurface harbors numerous novel phylogenetic lineages of archaea that are at present uncultured. Archaeal 16S rRNA analyses of deep subsurface sediments demonstrate their global occurrence and wide habitat range, including deep subsurface sediments, methane seeps and organic-rich coastal sediments. These subsurface archaeal lineages were discovered by PCR of extracted environmental DNA; their detection ultimately depends on the specificity of the archaeal PCR 16S rRNA primers. Surprisingly high mismatch frequencies for some archaeal PCR primers result in amplification bias against the corresponding archaeal lineages; this review presents some examples. Obviously, most archaeal 16S rRNA PCR primers were developed either before the discovery of these deep subsurface archaeal lineages, or without taking their sequence variants into account. PCR surveys with multiple primer combinations, revision and updates of primers whenever possible, and increasing use of PCR-independent methods in molecular microbial ecology will contribute to a more comprehensive view of subsurface archaeal communities.

Stratified Communities of Active Archaea in Deep Marine Subsurface Sediments

ABSTRACT Archaeal 16S rRNA was extracted from samples of deep marine subsurface sediments from Peru Margin site 1227, Ocean Drilling Program leg 201. The amounts of archaeal 16S rRNA in each extract were quantified by serial dilution and reverse transcription (RT)-PCR. The results indicated a 1,000-fold variation in rRNA content with depth in the sediment, with the highest concentrations found near the sediment surface and in the sulfate-methane transition zone (SMTZ). The phylogenetic composition of the active archaeal population revealed by cloning and sequencing of RT-PCR products changed with depth. Several phylotypes affiliated with marine benthic group B (MBGB) dominated clone libraries from the upper part of the SMTZ and were detected only in this layer. Members of the miscellaneous crenarchaeotal group (MCG) dominated clone libraries from the other layers. These results demonstrate that archaeal communities change in activity and community composition over short distances in geochemically distinct zones of deep subseafloor sediments and that these changes are traceable in the rRNA pool. It was shown for the first time that members of both the MCG and MBGB Archaea are more active in the SMTZ than in layers above and below. This indicates that they benefit either directly or indirectly from the anaerobic oxidation of methane. They also appear to be ecophysiologically flexible, as they have been retrieved from a wide range of marine sediments of various geochemical properties.

Community composition of a hypersaline endoevaporitic microbial mat

ABSTRACT A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4′,6′-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of α-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic γ- and δ-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.

Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment

ABSTRACT Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80°C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with Firmicutes (Bacteria) and Thermococcales (Archaea). Potential heterotrophic nitrate reducers (Deferribacterales) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of dsrAB genes revealed that archaeal SRP (Archaeoglobus) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (Desulfacinum and Desulfotomaculum). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [35S]sulfate incubations at 80°C. Sulfate reduction was found at 60°C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i) Archaeoglobus can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.

Salinity Responses of Benthic Microbial Communities in a Solar Saltern (Eilat, Israel)

ABSTRACT The salinity responses of cyanobacteria, anoxygenic phototrophs, sulfate reducers, and methanogens from the laminated endoevaporitic community in the solar salterns of Eilat, Israel, were studied in situ with oxygen microelectrodes and in the laboratory in slurries. The optimum salinity for the sulfate reduction rate in sediment slurries was between 100 and 120‰, and sulfate reduction was strongly inhibited at an in situ salinity of 215‰. Nevertheless, sulfate reduction was an important respiratory process in the crust, and reoxidation of formed sulfide accounted for a major part of the oxygen budget. Methanogens were well adapted to the in situ salinity but contributed little to the anaerobic mineralization in the crust. In slurries with a salinity of 180‰ or less, methanogens were inhibited by increased activity of sulfate-reducing bacteria. Unicellular and filamentous cyanobacteria metabolized at near-optimum rates at the in situ salinity, whereas the optimum salinity for anoxygenic phototrophs was between 100 and 120‰.

Antimicrobial properties of Dead Sea black mineral mud

Background  The unique, black, hypersaline mud mined from the Dead Sea shores is extensively used in mud packs, masks, and topical body and facial treatments in spas surrounding the lake, and in cosmetic preparations marketed worldwide, but little is known about its antimicrobiological properties.

Annual fluctuations in sulfur isotope fractionation in the water column of a euxinic marine basin

Abstract The sulfur cycle of Mariager Fjord was studied by following the pool of sulfide in the anoxic water and its isotopic composition during a period of 3 yr. Though most of the sulfide accumulating in the fjord was formed in the sediment, the isotopic composition of sulfide in the water was different from the isotopic composition of sulfide diffusing into the water from the sediment. The mean isotopic composition of the water column sulfide (δ34S) varied during the year between −13‰ and −21‰ with the most negative values reached during winter/early spring, while the sulfide diffusing into the water from the sediment had a mean isotope composition of −11.3‰. This annual pattern suggested that processes in the oxidative part of the sulfur cycle were responsible for the excess fractionation, and mass-balance considerations indicated that the excess fractionation of the sulfur isotopes could be accounted for by disproportionation of S0 or S2O32− in the water column, but not by water column sulfate reduction or sulfide oxidation alone. MPN counts demonstrated that a population of more than 3 × 104 cells mL−1 capable of growing by disproportionation of these two substrates was present in all depths of the fjord. The results presented in this communication demonstrate that the isotopic depletion of sulfide in anoxic systems may vary between periods of net sulfate reduction versus periods of net sulfide oxidation and indicate that disproportionation of sulfur compounds may be an important step in the sulfur cycle of euxinic basins.

Nitrogen dynamics and removal in a horizontal flow biofilm reactor for wastewater treatment

A horizontal flow biofilm reactor (HFBR) designed for the treatment of synthetic wastewater (SWW) was studied to examine the spatial distribution and dynamics of nitrogen transformation processes. Detailed analyses of bulk water and biomass samples, giving substrate and proportions of ammonia oxidising bacteria (AOB) and nitrite oxidising bacteria (NOB) gradients in the HFBR, were carried out using chemical analyses, sensor rate measurements and molecular techniques. Based on these results, proposals for the design of HFBR systems are presented. The HFBR comprised a stack of 60 polystyrene sheets with 10-mm deep frustums. SWW was intermittently dosed at two points, Sheets 1 and 38, in a 2 to 1 volume ratio respectively. Removals of 85.7% COD, 97.4% 5-day biochemical oxygen demand (BOD(5)) and 61.7% TN were recorded during the study. In the nitrification zones of the HFBR, which were separated by a step-feed zone, little variation in nitrification activity was found, despite decreasing in situ ammonia concentrations. The results further indicate significant simultaneous nitrification and denitrification (SND) activity in the nitrifying zones of the HFBR. Sensor measurements showed a linear increase in potential nitrification rates at temperatures between 7 and 16 degrees C, and similar rates of nitrification were measured at concentrations between 1 and 20mg NH(4)-N/l. These results can be used to optimise HFBR reactor design. The HFBR technology could provide an alternative, low maintenance, economically efficient system for carbon and nitrogen removal for low flow wastewater discharges.

Succession of Deferribacteres and Epsilonproteobacteria through a nitrate-treated high-temperature oil production facility

Members of Epsilonproteobacteria and Deferribacteres have been implied in nitrate-induced souring control in high-temperature oil production facilities. Here we report on their diversity and abundance in the injection and production part of a nitrate-treated, off-shore oil facility (Halfdan, Denmark) and aimed to assess their potential in souring control. Nitrate addition to deoxygenated seawater shifted the low-biomass seawater community dominated by Gammaproteobacteria closely affiliated with the genus Colwellia to a high-biomass community with significantly higher species richness. Epsilonproteobacteria accounted for less than 1% of the total bacterial community in the nitrate-amended injection water and were most likely outcompeted by putative nitrate-reducing, methylotrophic Gammaproteobacteria of the genus Methylophaga. Reservoir passage and recovery of the oil resulted in a significant change in the bacterial community. Members of the thermophilic Deferribacteres were the second major fraction of the bacterial community in the production water (~30% of the total bacterial community). They were not found in the injection water and were therefore assumed to be indigenous to the reservoir. Additional diversity analysis and targeted quantification of periplasmic nitrate reductase (napA) genes indicated that most resident Deferribacteres possessed the functional potential to contribute to nitrate reduction in the system. In sum, the dominance of nitrate-reducing Deferribacteres and the low relative abundance of Epsilonproteobacteria throughout the production facility suggested that the Deferribacteres play a major role in nitrate-induced souring control at high temperatures.

Distributions and activities of ammonia oxidizing bacteria and polyphosphate accumulating organisms in a pumped-flow biofilm reactor

The spatial distributions and activities of ammonia oxidizing bacteria (AOB) and polyphosphate accumulating organisms (PAOs) were investigated for a novel laboratory-scale sequencing batch pumped-flow biofilm reactor (PFBR) system that was operated for carbon, nitrogen and phosphorus removal. The PFBR comprised of two 16.5l tanks (Reactors 1 and 2), each with a biofilm module of 2m(2) surface area. To facilitate the growth of AOB and PAOs in the reactor biofilms, the influent wastewater was held in Reactor 1 under stagnant un-aerated conditions for 6 h after feeding, and was then pumped over and back between Reactors 1 and 2 for 12 h, creating aerobic conditions in the two reactors during this period; as a consequence, the biofilm in Reactor 2 was in an aerobic environment for almost all the 18.2 h operating cycle. A combination of micro-sensor measurements, molecular techniques, batch experiments and reactor studies were carried out to analyse the performance of the PFBR system. After 100 days operation at a filtered chemical oxygen demand (COD(f)) loading rate of 3.46 g/m(2) per day, the removal efficiencies were 95% COD(f), 87% TN(f) and 74% TP(f). While the PFBR microbial community structure and function were found to be highly diversified with substantial AOB and PAO populations, about 70% of the phosphorus release potential and almost 100% of the nitrification potential were located in Reactors 1 and 2, respectively. Co-enrichment of AOB and PAOs was realized in the Reactor 2 biofilm, where molecular analyses revealed unexpected microbial distributions at micro-scale, with population peaks of AOB in a 100-250 microm deep sub-surface zone and of PAOs in the 0-150 microm surface zone. The micro-distribution of AOB coincided with the position of the nitrification peak identified during micro-sensor analyses. The study demonstrates that enrichment of PAOs can be realized in a constant or near constant aerobic biofilm environment. Furthermore, the findings suggest that when successful co-enrichment of AOB and PAOs occur in biofilm environments, such as in the PFBR system, they do so at different zone depths in the biofilm.