Kai Mangelsdorf

Prokaryotic Populations and Activities in an Interbedded Coal Deposit, Including a Previously Deeply Buried Section (1.6–2.3 km) Above ∼ 150 Ma Basement Rock

A largely terrestrial, lignite/coal-bearing, 148 m core from the Waikato Basin, New Zealand, was studied, with a multidisciplinary approach, for subsurface microbiology. The top ≈76 m was Latest Miocene-Late Pleistocene (≈0.4–5.5 Ma) sediments, which overlay an unconformity and a previously deeply buried (1,600–2,300 m, × 55–75°C) ≈69 m section of moderately indurated, Late Eocene-Early Oligocene (≈32–35 Ma) deposits. Below this is weathered, Late Jurassic metasedimentary basement rock (145.5–157.0 Ma). Similar cell numbers (mean 1.2 × 106 cm −3 ), high viability (4–32%), intact phospholipids (biomarkers for living Bacteria) and activity (sulphate reduction, DNA replication) occurred heterogeneously throughout the core, including the weathered basement rock. Substantial numbers of viable anaerobic heterotrophic and lignite-utilizing bacteria (means 3.4 × 10 4 , 3.0 × 10 3 cm −3 ) were present throughout the core. This is similar to some deep terrestrial formations but contrasts with the generally exponentially decreasing prokaryotic populations in sub-seafloor sediments. For Bacteria, ≈76% of the 16S rRNA gene phylotypes were similar above (31.98 m) and below (133.55 m) the 76 m unconformity, which together with similar cell numbers indicates limited deep burial impact/palaeosterilization, or effective re-colonisation. Archaeal populations were not dominant being only detected with general primers at 31.98 m and those detected with methanogen functional primers were different above and below the ≈76 m unconformity. Both dominant bacterial (Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi) and archaeal (Miscellaneous Crenarchaeotic Group, Methanosarcinales and Methanobacteriales) sequences were similar to those previously detected in both marine and terrestrial subsurface environments, reflecting the changing depositional conditions of the formation. However, the presence of ANME sequences had not been previously found in the terrestrial subsurface. A large proportion of the bacterial 16S rDNA diversity was cultured (43% of commonest genera). Prokaryotic populations and activity changed with lithology and depth and substrates (formate, acetate, oxalate) may diffuse from high-carbon, lignite/coaly layers to support bacterial populations in adjacent sandy or clay-silt layers.

An interlaboratory study of TEX86 and BIT analysis of sediments, extracts, and standard mixtures

Two commonly used proxies based on the distribution of glycerol dialkyl glycerol tetraethers (GDGTs) are the TEX86 (TetraEther indeX of 86 carbon atoms) paleothermometer for sea surface temperature reconstructions and the BIT (Branched Isoprenoid Tetraether) index for reconstructing soil organic matter input to the ocean. An initial round-robin study of two sediment extracts, in which 15 laboratories participated, showed relatively consistent TEX86 values (reproducibility +/- 3-4 degrees C when translated to temperature) but a large spread in BIT measurements (reproducibility +/- 0.41 on a scale of 0-1). Here we report results of a second round-robin study with 35 laboratories in which three sediments, one sediment extract, and two mixtures of pure, isolated GDGTs were analyzed. The results for TEX86 and BIT index showed improvement compared to the previous round-robin study. The reproducibility, indicating interlaboratory variation, of TEX86 values ranged from 1.3 to 3.0 degrees C when translated to temperature. These results are similar to those of other temperature proxies used in paleoceanography. Comparison of the results obtained from one of the three sediments showed that TEX86 and BIT indices are not significantly affected by interlaboratory differences in sediment extraction techniques. BIT values of the sediments and extracts were at the extremes of the index with values close to 0 or 1, and showed good reproducibility (ranging from 0.013 to 0.042). However, the measured BIT values for the two GDGT mixtures, with known molar ratios of crenarchaeol and branched GDGTs, had intermediate BIT values and showed poor reproducibility and a large overestimation of the true (i.e., molar-based) BIT index. The latter is likely due to, among other factors, the higher mass spectrometric response of branched GDGTs compared to crenarchaeol, which also varies among mass spectrometers. Correction for this different mass spectrometric response showed a considerable improvement in the reproducibility of BIT index measurements among laboratories, as well as a substantially improved estimation of molar-based BIT values. This suggests that standard mixtures should be used in order to obtain consistent, and molar-based, BIT values.

Organic matter preservation on the Pakistan continental margin as revealed by biomarker geochemistry

In order to assess whether the oxygen-minimum zone (OMZ) in the Arabian sea has an eAect on the preservation and composition of organic matter in surface sediments we investigated samples from three diAerent transects on the Pakistan continental margin across the OMZ. In addition to determining the total amount of organic carbon (TOC), we analyzed the extractable lipids by gas chromatography, combined gas chromatography/mass spectrometry, and compound-specific stable carbon isotope measurements. The extractable lipids are dominated by marine organic matter as indicated by the abundance of lipids typical of marine biota and by the bulk and molecular isotopic composition. Sediments from within the OMZ are enriched in organic carbon and in several extractable lipids (i.e. phytol, n-alcohols, total sterols, n-C35 alkane) relative to stations above and below this zone. Other lipid concentrations, such as those of total n-fatty acids and total n-alkanes fail to show any relation to the OMZ. Only a weak correlation of TOC with mineral surface area was found in sediments deposited within the OMZ. In contrast, sediments from outside the OMZ do not show any relationship between TOC and surface area. Among the extractable lipids, only the n-alkane concentration is highly correlated with surface area in sediments from the Hab and Makran transects. In sediments from outside the OMZ, the phytol and sterol concentrations are also weakly correlated with mineral surface area. The depositional environment of the Indus Fan oAers the best conditions for an enhanced preservation of organic matter. The OMZ, together with the undisturbed sedimentation at moderate rates, seems to be mainly responsible for the high TOC values in this area. Overall, the type of organic matter and its lability toward oxic degradation, the mineral surface area, the mineral composition, and possibly the secondary productivity by (sedimentary) bacteria also appear to have an influence on organic matter accumulation and composition. # 2000 Elsevier Science Ltd. All rights reserved.

Cold water coral mounds revealed

The discovery of mounds and reefs hosting cold-water coral ecosystems along the northeastern Atlantic continental margins has propelled a vigorous effort over the past decade to study the distribution of the mounds, surface sediments, the ecosystems they host, and their environments [Hovland et al., 1994; Freiwald and Roberts, 2005].This effort has involved swath bathymetry, remotely operated vehicle deployments, shallow coring, and seismic surveys. Global coverage is difficult to gauge, but studies indicate that cold-water corals may cover as large an area as the better known warm-water corals that form shallow reefs (284,300 square kilometers) [Freiwald et al., 2005]. Cold-water corals occur in a variety of forms and settings, from small isolated colonies or patch reefs to giant mound structures such as those found west of Ireland.

Methanosarcina soligelidi sp. nov., a desiccation- and freeze-thaw-resistant methanogenic archaeon from a Siberian permafrost-affected soil.

A methanogenic archaeon, strain SMA-21(T), was isolated from a permafrost-affected soil by serial dilution in liquid medium. The cells were non-motile, stained Gram-negative and grew as irregular cocci with a diameter of 1.3-2.5 µm. Optimal growth was observed at 28 °C, pH 7.8 and 0.02 M NaCl. The strain grew on H2/CO2, methanol and acetate, but not on formate, ethanol, 2-butanol, 2-propanol, monomethylamine, dimethylamine, trimethylamine or dimethyl sulfide. Major membrane lipids of strain SMA-21(T) were archaeol phosphatidylglycerol, archaeol phosphatidylethanolamine and the corresponding hydroxyarchaeol compounds. The G+C content of the genomic DNA was 40.9 mol%. The 16S rRNA gene sequence was closely related to those of Methanosarcina mazei DSM 2053(T) (similarity 99.9 %) and Methanosarcina horonobensis HB-1(T) (similarity 98.7 %). On basis of the level of DNA-DNA hybridization (22.1 %) between strain SMA-21(T) and Methanosarcina mazei DSM 2053(T) as well as of phenotypic and genotypic differences, strain SMA-21(T) was assigned to a novel species of the genus Methanosarcina, for which the name Methanosarcina soligelidi sp. nov. is proposed. The type strain is SMA-21(T) (=DSM 26065(T) [corrected] = JCM 18468).

Techniques for Contamination Assessment During Drilling for Terrestrial Subsurface Sediments

Details about the procedures for drilling a ca. 150 m long drill core in a terrestrial setting under contamination controlled conditions are presented. Different to previous studies we only used commercially available drilling equipment to reduce the cost of operation significantly. The goals were (1) to minimize, (2) to monitor and, if possible, to quantify the contamination of the recovered sediments, and (3) to identify the different sources of contamination. Both the potential contamination of the sample material by surface microorganisms and non-indigenous material was assessed. To estimate the infiltration of drill mud into the core, fluorescent microspheres, having about half the size as microorganisms, were added to the mud. The drilling technique used was mud rotary drilling. With the exception of the very beginning of the drilling operations, the drill mud was devoid of any allochthonous hydrocarbons potentially derived from the drilling equipment or drill additives, and its biomarker composition reflected the varying organo-facies that were penetrated. Due to the lack of allochthonous hydrocarbons in the drill mud, its infiltration into the sediment cannot be traced by organic geochemical biomarker analysis. Microspheres proved to be a sensitive tool for the assessment of infiltration of drill mud into the core. The concentration of microspheres in the drill mud decreased continuously during the drilling, most probably caused by seepage of mud through leaks and attachment of spheres to the surface scum in the mud pit. Microscopic enumeration of the microspheres showed great variability in the depth of penetration of mud into the core, apparently unaffected of lithology. The sampling of the core material in the laboratory was carried out inside an anaerobic chamber. Several techniques for subsampling were used, according to sediment properties. The overall results indicate that, if strict contamination control protocols are employed, it is possible to recover uncontaminated samples at reasonable cost with commercially available drilling equipment.

Arthrobacter livingstonensis sp. nov. and Arthrobacter cryotolerans sp. nov., salt-tolerant and psychrotolerant species from Antarctic soil

Two novel cold-tolerant, Gram-stain-positive, motile, facultatively anaerobic bacterial strains, LI2(T) and LI3(T), were isolated from moss-covered soil from Livingston Island, Antarctica, near the Bulgarian station St Kliment Ohridski. A rod-coccus cycle was observed for both strains. 16S rRNA gene sequence analysis revealed an affiliation to the genus Arthrobacter, with the highest similarity to Arthrobacter stackebrandtii and Arthrobacter psychrochitiniphilus for strain LI2(T) (97.8 and 97.7 % similarity to the respective type strains) and to Arthrobacter kerguelensis and Arthrobacter psychrophenolicus for strain LI3(T) (97.4 and 97.3 % similarity to the respective type strains). The growth temperature range was -6 to 28 °C for LI2(T) and -6 to 24 °C for LI3(T), with an optimum at 16 °C for both strains. Growth occurred at 0-10 % (w/v) NaCl, with optimum growth at 0-1 % (w/v) for LI2(T) and 0.5-3 % (w/v) for LI3(T). The pH range for growth was pH 4-9.5 with an optimum of pH 8 for LI2(T) and pH 6.5 for LI3(T). The predominant fatty acids were anteiso-C(15 : 0), C(18 : 0) and anteiso-C(17 : 0) for LI2(T) and anteiso-C(15 : 0) and C(18 : 0) for LI3(T). Physiological and biochemical tests clearly differentiated strain LI2(T) from A. stackebrandtii and A. psychrochitiniphilus and strain LI3(T) from A. kerguelensis and A. psychrophenolicus. Therefore, two novel species within the genus Arthrobacter are proposed: Arthrobacter livingstonensis sp. nov. (type strain LI2(T)  = DSM 22825(T)  = NCCB 100314(T)) and Arthrobacter cryotolerans sp. nov. (type strain LI3(T)  = DSM 22826(T)  = NCCB 100315(T)).

Methanosarcina spelaei sp. nov., a methanogenic archaeon isolated from a floating biofilm of a subsurface sulphurous lake.

A novel methanogenic archaeon, strain MC-15(T), was isolated from a floating biofilm on a sulphurous subsurface lake in Movile Cave (Mangalia, Romania). Cells were non-motile sarcina-like cocci with a diameter of 2-4 µm, occurring in aggregates. The strain was able to grow autotrophically on H2/CO2. Additionally, acetate, methanol, monomethylamine, dimethylamine and trimethylamine were utilized, but not formate or dimethyl sulfide. Trypticase peptone and yeast extract were not required for growth. Optimal growth was observed at 33 °C, pH 6.5 and a salt concentration of 0.05 M NaCl. The predominant membrane lipids of MC-15(T) were archaeol and hydroxyarchaeol phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol as well as hydroxyarchaeol phosphatidylserine and archaeol glycosaminyl phosphatidylinositol. The closely related species, Methanosarcina vacuolata and Methanosarcina horonobensis, had a similar composition of major membrane lipids to strain MC-15(T). The 16S rRNA gene sequence of strain MC-15(T) was similar to those of Methanosarcina vacuolata DSM 1232(T) (sequence similarity 99.3%), Methanosarcina horonobensis HB-1(T) (98.8%), Methanosarcina barkeri DSM 800(T) (98.7%) and Methanosarcina siciliae T4/M(T) (98.4%). DNA-DNA hybridization revealed 43.3% relatedness between strain MC-15(T) and Methanosarcina vacuolata DSM 1232(T). The G+C content of the genomic DNA was 39.0 mol%. Based on physiological, phenotypic and genotypic differences, strain MC-15(T) represents a novel species of the genus Methanosarcina, for which the name Methanosarcina spelaei sp. nov. is proposed. The type strain is MC-15(T) ( = DSM 26047(T) = JCM 18469(T)).

Methanobacterium movilense sp. nov., a hydrogenotrophic, secondary-alcohol-utilizing methanogen from the anoxic sediment of a subsurface lake

A novel strain of methanogenic archaea, designated MC-20(T), was isolated from the anoxic sediment of a subsurface lake in Movile Cave, Mangalia, Romania. Cells were non-motile, Gram-stain-negative rods 3.5-4.0 µm in length and 0.6-0.7 µm in width, and occurred either singly or in short chains. Strain MC-20(T) was able to utilize H2/CO2, formate, 2-propanol and 2-butanol as substrate, but not acetate, methanol, ethanol, dimethyl sulfide, monomethylamine, dimethylamine or trimethylamine. Neither trypticase peptone nor yeast extract was required for growth. The major membrane lipids of strain MC-20(T) were archaeol phosphatidylethanolamine and diglycosyl archaeol, while archaeol phosphatidylinositol and glycosyl archaeol were present only in minor amounts. Optimal growth was observed at 33 °C, pH 7.4 and 0.08 M NaCl. Based on phylogenetic analysis of 16S rRNA gene sequences, strain MC-20(T) was closely affiliated with Methanobacterium oryzae FPi(T) (similarity 97.1%) and Methanobacterium lacus 17A1(T) (97.0%). The G+C content of the genomic DNA was 33.0 mol%. Based on phenotypic and genotypic differences, strain MC-20(T) was assigned to a novel species of the genus Methanobacterium for which the name Methanobacterium movilense sp. nov. is proposed. The type strain is MC-20(T) ( = DSM 26032(T) = JCM 18470(T)).

Cryobacterium arcticum sp. nov., a psychrotolerant bacterium from an Arctic soil

A psychrotolerant, Gram-stain-positive, yellow-pigmented, aerobic rod, designated SK1(T), was isolated from a soil sample collected from Store Koldewey, north-east Greenland. Cells were catalase- and methyl red-positive, produced H(2)S and produced acid from glucose, mannitol and salicin. Strain SK1(T) was able to grow between -6 and 28 °C, with an optimum at 20 °C. The isolate contained 2,4-diaminobutyrate, glycine, alanine and glutamic acid in the cell wall and the major menaquinones were MK-10 and MK-11. Identified polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C(15 : 0) (53.5 %), anteiso-C(17 : 0) (17.0 %) and C(18 : 0) (12.1 %). The genomic DNA G+C content was 67.8 mol%. Strain SK1(T) showed the highest 16S rRNA gene sequence similarity with Cryobacterium psychrotolerans 0549(T) (97.6 %) and Cryobacterium roopkundense RuGl7(T) (96.8 %). Considering morphological, physiological, biochemical and chemotaxonomic characters and phylogenetic analysis, strain SK1(T) represents a novel species in the genus Cryobacterium, for which the name Cryobacterium arcticum sp. nov. is proposed. The type strain is SK1(T) ( = DSM 22823(T)  = NCCB 100316(T)).