Bente Aagaard Lomstein

Spirosoma spitsbergense sp. nov. and Spirosoma luteum sp. nov., isolated from a high Arctic permafrost soil, and emended description of the genus Spirosoma.

Two pigmented, Gram-negative, non-motile, pleomorphic rod-shaped bacteria (strains SPM-9(T) and SPM-10(T)) were isolated from a permafrost soil collected from the Adventdalen valley, Spitsbergen, northern Norway. A third isolate (strain M5-H2) was recovered from the same soil sample after the sample had been exposed to simulated Martian environmental conditions. The three strains were characterized taxonomically by using a polyphasic approach. Phylogenetic, chemotaxonomic, physiological and morphological analyses demonstrated that the three isolates were most closely related to members of the genus Spirosoma. 16S rRNA gene sequence data indicated that the three isolates could be divided into two clusters: (i) strain SPM-9(T) and (ii) strains SPM-10(T) and M5-H2. This grouping was confirmed by DNA-DNA hybridization experiments. Strains SPM-9(T) and SPM-10(T) exhibited 92 % 16S rRNA gene sequence similarity to both Spirosoma linguale LMG 10896(T) and Spirosoma rigui WPCB 118(T). The major fatty acids present in all three isolates were summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16 : 1)omega7c; 43.0-48.2 % of the total), C(16 : 1)omega5c (19.1-21.3 %), C(16 : 0) (6.7-7.3 %), iso-C(17 : 0) 3-OH (4.7-6.0 %) and iso-C(15 : 0) (2.6-5.7 %). On the basis of their phenotypic and genotypic characteristics, the new strains are assigned to two novel species of the genus Spirosoma, for which the names Spirosoma spitsbergense sp. nov. and Spirosoma luteum sp. nov. are proposed. The type strain of Spirosoma spitsbergense is SPM-9(T) (=NCIMB 14407(T)=DSM 19989(T)) and the type strain of Spirosoma luteum is SPM-10(T) (=NCIMB 14406(T)=DSM 19990(T)). An emended description of the genus Spirosoma is also proposed.

A Facility for Long-Term Mars Simulation Experiments: The Mars Environmental Simulation Chamber (MESCH)

We describe the design, construction, and pilot operation of a Mars simulation facility comprised of a cryogenic environmental chamber, an atmospheric gas analyzer, and a xenon/mercury discharge source for UV generation. The Mars Environmental Simulation Chamber (MESCH) consists of a double-walled cylindrical chamber. The double wall provides a cooling mantle through which liquid N(2) can be circulated. A load-lock system that consists of a small pressure-exchange chamber, which can be evacuated, allows for the exchange of samples without changing the chamber environment. Fitted within the MESCH is a carousel, which holds up to 10 steel sample tubes. Rotation of the carousel is controlled by an external motor. Each sample in the carousel can be placed at any desired position. Environmental data, such as temperature, pressure, and UV exposure time, are computer logged and used in automated feedback mechanisms, enabling a wide variety of experiments that include time series. Tests of the simulation facility have successfully demonstrated its ability to produce temperature cycles and maintain low temperature (down to -140 degrees C), low atmospheric pressure (5-10 mbar), and a gas composition like that of Mars during long-term experiments.

Formate, acetate, and propionate as substrates for sulfate reduction in sub-arctic sediments of Southwest Greenland

Volatile fatty acids (VFAs) are key intermediates in the anaerobic mineralization of organic matter in marine sediments. We studied the role of VFAs in the carbon and energy turnover in the sulfate reduction zone of sediments from the sub-arctic Godthåbsfjord (SW Greenland) and the adjacent continental shelf in the NE Labrador Sea. VFA porewater concentrations were measured by a new two-dimensional ion chromatography-mass spectrometry method that enabled the direct analysis of VFAs without sample pretreatment. VFA concentrations were low and surprisingly constant (4–6 μmol L−1 for formate and acetate, and 0.5 μmol L−1 for propionate) throughout the sulfate reduction zone. Hence, VFAs are turned over while maintaining a stable concentration that is suggested to be under a strong microbial control. Estimated mean diffusion times of acetate between neighboring cells were <1 s, whereas VFA turnover times increased from several hours at the sediment surface to several years at the bottom of the sulfate reduction zone. Thus, diffusion was not limiting the VFA turnover. Despite constant VFA concentrations, the Gibbs energies (ΔGr) of VFA-dependent sulfate reduction decreased downcore, from −28 to −16 kJ (mol formate)−1, −68 to −31 kJ (mol acetate)−1, and −124 to −65 kJ (mol propionate)−1. Thus, ΔGr is apparently not determining the in-situ VFA concentrations directly. However, at the bottom of the sulfate zone of the shelf station, acetoclastic sulfate reduction might operate at its energetic limit at ~ −30 kJ (mol acetate)−1. It is not clear what controls VFA concentrations in the porewater but cell physiological constraints such as energetic costs of VFA activation or uptake could be important. We suggest that such constraints control the substrate turnover and result in a minimum ΔGr that depends on cell physiology and is different for individual substrates.

Activity and stability of a complex bacterial soil community under simulated Martian conditions

A simulation experiment with a complex bacterial soil community in a Mars simulation chamber was performed to determine the effect of Martian conditions on community activity, stability and survival. At three different depths in the soil core short-term effects of Martian conditions with and without ultraviolet (UV) exposure corresponding to 8 Martian Sol were compared. Community metabolic activities and functional diversity, measured as glucose respiration and versatility in substrate utilization, respectively, decreased after UV exposure, whereas they remained unaffected by Martian conditions without UV exposure. In contrast, the numbers of culturable bacteria and the genetic diversity were unaffected by the simulated Martian conditions both with and without UV exposure. The genetic diversity of the soil community and of the colonies grown on agar plates were evaluated by denaturant gradient gel electrophoresis (DGGE) on DNA extracts. Desiccation of the soil prior to experimentation affected the functional diversity by decreasing the versatility in substrate utilization. The natural dominance of endospores and Gram-positive bacteria in the investigated Mars-analogue soil may explain the limited effect of the Mars incubations on the survival and community structure. Our results suggest that UV radiation and desiccation are major selecting factors on bacterial functional diversity in terrestrial bacterial communities incubated under simulated Martian conditions. Furthermore, these results suggest that forward contamination of Mars is a matter of great concern in future space missions.

Demequina lutea sp. nov., isolated from a high Arctic permafrost soil.

Two Gram-stain-positive, pigmented, non-motile, non-spore-forming, pleomorphic, rod-shaped bacteria (strains SV45(T) and SV47), isolated from a permafrost soil collected from the Adventdalen valley, Spitsbergen, northern Norway, have been characterized taxonomically using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two permafrost isolates formed a distinct phyletic line within the suborder Micrococcineae of the order Actinomycetales. DNA-DNA hybridization analyses indicate that strains SV45(T) and SV47 are closely related (60-69 % relatedness) and belong to the same species, although they show slightly different colony pigmentation. The closest phylogenetic neighbour was Demequina aestuarii JC2054(T), with 96 % 16S rRNA gene sequence similarity. Optimum growth of SV45(T) and SV47 occurred aerobically in the absence of NaCl, but both isolates tolerated up to 2 % NaCl (w/v) in the growth medium. Growth under anaerobic conditions was slow and weak. The peptidoglycan of both isolates was of the A4beta type with l-ornithine as the diamino acid and serine as a component of the interpeptide bridge with either d-aspartate (SV45(T)) or d-glutamate (SV47) as the N-terminal amino acid. The major fatty acids present in both isolates were C(15 : 0) (3.2-8.6 %), iso-C(16 : 0) (5.0-8.9 %), anteiso-C(15 : 0) (59.4-61.5 %), anteiso-C(17 : 0) (4.1-8.8 %) and anteiso-C(15 : 1) (4.4-6.4 %). Isoprenoid quinones were present at exceptionally low levels in both isolates, and only demethylmenaquinone DMK-9(H(4)) could be identified with any degree of confidence. Phylogenetic analysis and differences in physiological and biochemical characteristics between the strains and Demequina aestuarii JC2054(T) indicate that these isolates belong to a novel species within the genus Demequina, for which the name Demequina lutea sp. nov. is proposed. The type strain is SV45(T) (=LMG 24795(T) =DSM 19970(T)).

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10−5–11×10−5 yr−1. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.

Identity, abundance and reactivation kinetics of thermophilic fermentative endospores in cold marine sediment and seawater

Cold marine sediments harbor endospores of fermentative and sulfate-reducing, thermophilic bacteria. These dormant populations of endospores are believed to accumulate in the seabed via passive dispersal by ocean currents followed by sedimentation from the water column. However, the magnitude of this process is poorly understood because the endospores present in seawater were so far not identified, and only the abundance of thermophilic sulfate-reducing endospores in the seabed has been quantified. We investigated the distribution of thermophilic fermentative endospores (TFEs) in water column and sediment of Aarhus Bay, Denmark, to test the role of suspended dispersal and determine the rate of endospore deposition and the endospore abundance in the sediment. We furthermore aimed to determine the time course of reactivation of the germinating TFEs. TFEs were induced to germinate and grow by incubating pasteurized sediment and water samples anaerobically at 50°C. We observed a sudden release of the endospore component dipicolinic acid immediately upon incubation suggesting fast endospore reactivation in response to heating. Volatile fatty acids (VFAs) and H2 began to accumulate exponentially after 3.5 h of incubation showing that reactivation was followed by a short phase of outgrowth before germinated cells began to divide. Thermophilic fermenters were mainly present in the sediment as endospores because the rate of VFA accumulation was identical in pasteurized and non-pasteurized samples. Germinating TFEs were identified taxonomically by reverse transcription, PCR amplification and sequencing of 16S rRNA. The water column and sediment shared the same phylotypes, thereby confirming the potential for seawater dispersal. The abundance of TFEs was estimated by most probable number enumeration, rates of VFA production, and released amounts of dipicolinic acid during germination. The surface sediment contained ∼105–106 inducible TFEs cm-3. TFEs thus outnumber thermophilic sulfate-reducing endospores by an order of magnitude. The abundance of cultivable TFEs decreased exponentially with sediment depth with a half-life of 350 years. We estimate that 6 × 109 anaerobic thermophilic endospores are deposited on the seafloor per m2 per year in Aarhus Bay, and that these thermophiles represent >10% of the total endospore community in the surface sediment.

Contrasting community composition of endospores and vegetative Firmicutes in a marine sediment suggests both endogenous and exogenous sources of endospore accumulation: Identity and origin of endospores in the seafloor

Bacterial endospores are highly abundant in marine sediments, but their taxonomic identity and ecology is largely unknown. We selectively extracted DNA from endospores and vegetative cells and sequenced 16S rRNA genes to characterize the composition of the endospore and vegetative Firmicutes communities in the sediment and water column of Aarhus Bay (Denmark). The endospore community in the sediment was dominated by the families Bacillaceae, Lachnospiraceae, Clostridiaceae and Ruminoccocaceae. These families were also represented in the vegetative community in the sediment and the endospore community in the water column. OTUs of high relative abundance in the endospore community were also represented in the vegetative Firmicutes community. Other OTUs were exclusively found in the endospore communities. This suggests that endospores accumulate in marine sediments due to passive deposition from the water column and sporulation of vegetative cells in the sediment. Some OTUs were detected in the endospore community of the water column and the vegetative community the sediment indicating that endospores deposited from the water column may germinate upon burial/deposition in the sediment. We provide novel insight into the composition of endospore communities in marine sediments and highlight their role in microbial dispersal and as a seed bank in subsurface sediments.