Solvothermal synthesis of cobalt PCP pincer complexes from [Co2(CO)8].

Treatment of [Co2(CO)8] with the ipso-substituted P(C-X)PY ligands (X = Br, Cl; R = iPr, tBu) bearing Y = NH and CH2 linkers under solvothermal conditions affords the five-coordinate Co(I) and Co(III) complexes [CoI(PCPY-R)(CO)2] and [CoIII(PCPY-R)X2]. The later are paramagnetic exhibiting a solution magnetic moment in the range of 3.0-3.3 µB which is consistent with a d6 intermediate spin system corresponding to two unpaired electrons. In the case of P(C-X)PY ligands (X = Br, Cl; R = tBu; Y = NH) the formation of the square planar Co(II) complex [Co(PCPNH-tBu)X] was favored. This complex gives rise to a magnetic moment of 1.8 µB being consistent with a d7 low spin system corresponding to one unpaired electron. All complexes are characterized by means of spectroscopic techniques (NMR, IR), HR-MS. Representative complexes were also characterized by X-ray crystallography. Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-023-03123-x.

Cr(II) and Cr(III) NCN pincer complexes: synthesis, structure, and catalytic reactivity.

The synthesis, characterization, and reactivity of several new Cr(II) and Cr(III) complexes featuring an NCN pincer ligand with an arene backbone connected to amine donors NEt2 and NiPr2 via CH2-linkers is described. Reacting the in situ lithiated ligand precursor N(C-Br)NCH2-Et with [CrCl3(THF)3] resulted in the formation of the Cr(III) complex trans-[Cr(κ3NCN-NCNCH2-Et)(Cl)2(THF)]. Upon reaction of lithiated N(C-Br)NCH2-iPr with a suspension of anhydrous CrCl2, the Cr(II) complex [Cr(κ2NC-NCNCH2-iPr)2] is formed featuring two NCN ligands bound in κ2NC-fashion. In contrast, when lithiated N(C-Br)NCH2-iPr is reacted with a homogeneous solution of anhydrous CrX2 (X = Cl, Br), complexes [Cr(κ3NCN-NCNCH2-iPr)X] are obtained. Treatment of [Cr(κ3NCN-NCNCH2-iPr)Cl] with 1 equiv of PhCH2MgCl and LiCH2SiMe3 afforded the alkyl complexes [Cr(κ3NCN-NCNCH2-iPr)(CH2Ph)] and [Cr(κ3NCN-NCNCH2-iPr)(CH2SiMe3)]. All Cr(II) complexes exhibit effective magnetic moments in the range of 4.7-4.9 µB which is indicative for d4 high spin systems. If a solution of lithiated N(C-Br)NCH2-iPr is treated with CrCl2, followed by addition of an excess of Na[HB(Et)3], the dimeric complex [Cr(κ2NC-NCNCH2-iPr)(µ2-H)]2 is obtained bearing two bridging hydride ligands. [Cr(κ3NCN-NCNCH2-iPr)(CH2SiMe3)] turned out to be catalytically active for the hydrosilylation of ketones at room temperature with a catalyst loading of 1 mol%. X-ray structures of all complexes are presented. Supplementary Information: The online version contains supplementary material available at 10.1007/s00706-023-03128-6.

Multisite Qualification of an Automated Incubator and Colony Counter for Environmental and Bioburden Applications in Pharmaceutical Microbiology.

Traditional microbiological techniques have been used for well over a century as the basis for contamination testing of pharmaceutical products and processes. With more recent focus on faster product release and concerns around the integrity of the test data, new technologies have been implemented to detect and enumerate organisms faster and provide paperless processes to minimize data integrity issues. Manual colony counting technologies, where incubation is performed in a standard incubator, and the plate is manually transferred to the colony counter for a single read at the end of incubation, have been used for many years to reduce the potential for human error; however, they pose validation challenges due to poor counting accuracy. Colony counters that automatically perform both the incubation and enumeration functions (multiple enumeration calculations through the incubation phase) have recently been implemented for quality control (QC) laboratory analytical processes, supporting a cGMP environment. This article summarizes the findings of eight companies demonstrating the qualification of an automated colony counter technology to perform the majority of microbial tests required for QC, environmental monitoring, and bioburden for in-process, bulk drug substance, and water system testing. Comparable analytical performance and time to result data generated during individual studies at all companies allows the system to be qualified and implemented for cGMP processes while reducing data integrity risks.

A Systematic Approach for the Evaluation, Validation, and Implementation of Automated Colony Counting Systems.

For several years, automated colony counting systems have been available with varying degrees of automation. Ever more sophisticated instruments are now increasingly used in microbiological laboratories of pharmaceutical quality control. In addition to the colony counting device, the instruments are now also equipped with robotic systems performing the entire handling of the petri dishes, e.g., automated internal transportation of petri dishes from the incubator chamber to the instrument's enumeration device and back. Moreover, the subjective evaluation of microbial enumeration tests by analysts is replaced with a more accurate and precise process. This leads to significant improvements to data integrity compliance. Automated colony counting systems also often enable cost reduction in the microbiological laboratory, e.g., by not requiring a contemporaneous verification by a second analyst. They also enable direct integration of count data into an existing laboratory information management system, reducing the hands-on time, costs per test and also preventing human errors caused by manual transcription. Altogether, these instruments will lead to improved monitoring and assurance of control of biopharmaceutical processes and manufacturing environments, as well as shortened cycle times in the supply chain. Regulators are encouraging the biopharmaceutical industry to adopt these innovative systems. For example, this year a BioPhorum member company received the first health authority approvals from EU, US, CH, Canada, Australia, and China for the use of automated colony counting systems for in-process bioburden testing and the release of drug substance lots, with an incubation time reduced by about 50%. Although these approvals are for release testing of drug substance lots, the instruments can also be used for environmental monitoring, testing of water samples, etc. This article describes a systematic 9-step approach to the evaluation, equipment qualification, and deployment of automated colony counting systems, which can be applied by biopharmaceutical companies wanting to take advantage of their numerous benefits.

Synthesis and Characterization of Cobalt NCN Pincer Complexes.

A series of cobalt complexes, stabilized by a monoanionic tridentate NCN pincer ligand, was synthetized and characterized. Preparation of the paramagnetic 15 VE complex [Co(NCNCH2-Et)Br] (1) was accomplished by transmetalation of Li[2,6-(Et2NCH2)2C6H3] with CoBr2 in THF. Treatment of this air-sensitive compound with NO gas resulted in the formation of the diamagnetic Co(III) species [Co(NCNCH2-Et)(NO)Br] (2) as confirmed by X-ray diffraction. This complex features a strongly bent NO ligand (Co-N-O∠135.0°). The νNO is observed at 1609 cm-1 which is typical for a bent metal-N-O arrangement. Coordinatively unsaturated 1 could further be treated with pyridine, isocyanides, phosphines and CO to form five-coordinate 17 VE complexes. Oxidation of 1 with CuBr2 led to the formation of the Co(III) complex [Co(NCNCH2-Et)Br2]. Treatment of [Co(NCNCH2-Et)Br2] with TlBF4 as halide scavenger in acetonitrile led to the formation of the cationic octahedral complex [Co(NCNCH2-Et)(MeCN)3](BF4)2. A combination of X-ray crystallography, IR-, NMR- and EPR-spectroscopy as well as DFT/CAS-SCF calculations were used to characterize all compounds.

Manganese and iron PCP pincer complexes - the influence of sterics on structure and reactivity.

The syntheses of various manganese and iron PCP pincer complexes via a solvothermal oxidative addition methodology is described. Upon reacting [Mn2(CO)10] with the ligands (P(C-Br)PCH2-iPr) (1a) and (P(C-Br)PO-iPr) (1b), Mn(I) PCP pincer complexes [Mn(PCPCH2-iPr)(CO)3] (2a) and [Mn(-PCPO-iPr)(CO)3] (2b) were obtained. Protonation of 2a with HBF4·Et2O led to the formation of [Mn(κ3P,CH,P-P(CH)PCH2-iPr)(CO)3]BF4 (3) featuring an η2-Caryl-H agostic bond. The solvothermal reaction of 1a with [Fe2(CO)9] afforded the Fe(II) PCP pincer complex [Fe(PCPCH2-iPr)(CO)2Br] (4). Treatment of 4 with Li[HBEt3] afforded the Fe(I) complex [Fe(PCPCH2-iPr)(CO)2] (5a). When using the sterically more demanding ligands (P(C-Br)PCH2-tBu) (1c) and (P(C-Br)PO-tBu)(1d) striking differences in reactivity were observed. While neither 1c nor 1d showed any reactivity towards [Mn2(CO)10], the reaction with [Fe2(CO)9] and [Fe(CO)5] led to the formation of the Fe(I) complexes [Fe(PCPCH2-tBu)(CO)2] (5b) and [Fe(PCPO-tBu)(CO)2] (5c). X-ray structures of representative complexes are provided.

Synthesis, Characterization, and Catalytic Reactivity of {CoNO}8 PCP Pincer Complexes.

The reaction of coordinatively unsaturated Co(II) PCP pincer complexes with nitric oxide leads to the formation of new, air-stable, diamagnetic mono nitrosyl compounds. The synthesis and characterization of five- and four-coordinate Co(III) and Co(I) nitrosyl pincer complexes based on three different ligand scaffolds is described. Passing NO through a solution of [Co(PCPNMe-iPr)Cl], [Co(PCPO-iPr)Cl] or [Co(PCPCH2-iPr)Br] led to the formation of the low-spin complex [Co(PCP-iPr)(NO)X] with a strongly bent NO ligand. Treatment of the latter species with (X = Cl, Br) AgBF4 led to chloride abstraction and formation of cationic square-planar Co(I) complexes of the type [Co(PCP-iPr)(NO)]+ featuring a linear NO group. This reaction could be viewed as a formal two electron reduction of the metal center by the NO radical from Co(III) to Co(I), if NO is counted as NO+. Hence, these systems can be described as {CoNO}8 according to the Enemark-Feltham convention. X-ray structures, spectroscopic and electrochemical data of all nitrosyl complexes are presented. Preliminary studies show that [Co(PCPNMe-iPr)(NO)]+ catalyzes efficiently the reductive hydroboration of nitriles with pinacolborane (HBpin) forming an intermediate {CoNO}8 hydride species.

Recognition of the unsuitability of DSM 12173 as the deposited type strain of Thermocrinis ruber Huber et al. 1999, recognition of DSM 23557 as an authentic sub-culture of strain OC 1/4, the nomenclatural type of Thermocrinis ruber Huber et al. 1999 and an emended description of Thermocrinis ruber Huber et al. 1999.

During the course of growing cell material for the extraction of genomic DNA for the Genomic Encyclopedia of Bacteria and Archaea, strain OC 1/4, the designated type strain of Thermocrinis ruber was cultivated at the Institute for Microbiology and Archaea Center of the University of Regensburg, Regensburg, Germany. Partial sequencing of the 16S rRNA gene indicated that the cell material initially cultivated and the strain held in the DSMZ as DSM 12173 did not correspond with that deposited as AJ005640 and was probably a strain of Thermocrinis albus. A subsequent search of the strain collection of the Institute for Microbiology and Archaea Center of the University of Regensburg held in liquid nitrogen indicated that a strain could be recovered from the liquid nitrogen stocks that corresponded with the properties originally given for strain OC 1/4. We report here on the characterization of this strain that has subsequently been deposited in the DSMZ as DSM 23557.

Lunar cycles and rainy seasons drive growth and reproduction in nummulitid foraminifera, important producers of carbonate buildups.

Representatives of the foraminifer Nummulites are important in Earth history for timing Cenozoic shallow-water carbonates. Taphonomic complexity explains the construction of carbonate buildups, but reproduction and life span of the constructing individuals are unknown. During the 15-month investigation period, asexually reproduced schizonts and gamonts showed equal proportions in the first half of this period, whereas gamonts predominated in the second half. Oscillations in cell growth are mainly caused by light intensities during chamber construction when minor differences in water depth increase the photosynthetic rate of endosymbiotic diatoms during neap tides. The continuous reproduction rate of N. venosus throughout the year is increased in subtropical calms by higher summer temperatures and the marginal input of inorganic nutrients during rainy seasons. The expected life span of both gamonts and schizonts are 18 months.

Morphometric analysis of Eocene nummulitids in western and central Cuba: taxonomy, biostratigraphy and evolutionary trends.

Megalospheric specimens of Nummulitidae from eight localities in western and central Cuba were morphometrically investigated using test characters described by 11 growth-independent and growth-invariant attributes that provide a complete geometric reconstruction of nummulitid equatorial morphology. The species Nummulites striatoreticulatus, Palaeonummulites trinitatensis, Operculinoides floridensis and O. soldadensis were classified by an agglomerative cluster analysis. Discriminant analysis yielded significant morphological separators between the species such as the backbend angle, marginal radius increase, perimeter ratio and first chamber length. The transition of tightness to laxity of the spiral was an important morphological separator at the generic level, representing a clear general trend coupled with the change in palaeodepth. Based on further discriminant analysis, an increase in proloculus size was detected in Nummulites striatoreticulatus from the middle Eocene to early late Eocene, supporting this important evolutionary pattern in many lineages of Nummulites. Operculinid forms showed an opposite and more weakly pronounced time-dependent trend in the size decrease of the proloculus. In the Cuban localities, Nummulites striatoreticulatus occurs from the Lutetian to Priabonian, while Palaeonummulites trinitatensis is restricted to the Bartonian to Priabonian. The moderately to loosely coiled operculinid taxa O. floridensis and O. soldadensis have longer stratigraphical ranges from the middle Eocene to probably the early Oligocene. Operculinoides floridensis and O. soldadensis show a broader variability in marginal radius increase, and thus probably occupied wider niches than N. striatoreticulatus. The latter seems to be restricted to the shelf edge and to the shallowest parts of the upper slope. A possible phylogenetic connection between Heterostegina and Operculinoides is suggested by the closest equatorial morphology of Heterostegina sp. indet. to tightly coiled forms of Operculinoides floridensis. Discriminant analysis documents the strongest similarities in perimeter ratio, backbend angle, initial marginal radius and proloculus mean diameter.

Growth estimation of the larger foraminifer Heterostegina depressa by means of population dynamics.

In Heterostegina depressa, the flagship species of laboratory investigations of larger benthic foraminifera (LBF) since the 70's, the timing of reproduction, longevity and natural chamber building rates are still understudied. A recently developed method, the natural laboratory (sensu Hohenegger), has been applied on H. depressa populations from Sesoko Jima, NW Okinawa, Japan. An averaged chamber building rate and longevity of H. depressa were calculated based on 17 monthly samplings at fixed stations. All samples were collected at 20 and 50 m water depths using SCUBA. Live populations were dried and investigated by microCT. The monthly frequency distributions of chamber numbers and test diameters have been decomposed in normally distributed components. For each month, mean and standard deviations of the components were used to calculate the maximum chamber number and maximum test diameter. Based on these values, the natural chamber building rate (CBR) or diameter increase rate (DIR) could be estimated using the Michaelis-Menten function. CBR and DIR were inverted to estimate the 'birthdate' of all investigated individuals. Based on frequencies of these 'birthdates', main reproduction events could be detected and compared to the reproduction timing of other subtropical and tropical LBF taxa. Furthermore, peaks in reproduction could be linked to monsoon wet seasons (="rainy seasons") and winter rains.

Growth, chamber building rate and reproduction time of Palaeonummulites venosus (Foraminifera) under natural conditions.

We investigated the symbiont-bearing benthic foraminifer Palaeonummulites venosus to determine the chamber building rate (CBR), test diameter increase rate (DIR), reproduction time and longevity using the 'natural laboratory' approach. This is based on the decomposition of monthly obtained frequency distributions of chamber number and test diameter into normally distributed components. Test measurements were taken using MicroCT. The shift of the mean and standard deviation of component parameters during the 15-month investigation period was used to calculate Michaelis-Menten functions applied to estimate the averaged CBR and DIR under natural conditions. The individual dates of birth were estimated using the inverse averaged CBR and the inverse DIR fitted by the individual chamber number or the individual test diameter at the sampling date. Distributions of frequencies and densities (i.e., frequency divided by sediment weight) based on both CBR and DIR revealed continuous reproduction throughout the year with two peaks, a stronger one in June determined as the onset of the summer generation (generation 1) and a weaker one in November determined as the onset of the winter generation (generation 2). This reproduction scheme explains the presence of small and large specimens in the same sample. Longevity, calculated as the maximum difference in days between the individual's birth date and the sampling date, is approximately 1.5 yr, an estimation obtained by using both CBR and DIR.

Growth of Heterostegina depressa under natural and laboratory conditions.

The use of micro-computed tomography (µCT) provides a unique opportunity to look inside the shells of larger benthic foraminifera to investigate their structure by measuring linear and volumetric parameters. For this study, gamonts/schizonts and agamonts of the species Heterostegina depressa d'Orbigny were examined by µCT; each single chamber's volume was digitally measured. This approach enables cell growth to be recognised in terms of chamber volume sequence, which progressively increases until reproduction occurs. This sequence represents the ontogeny of the foraminiferal cell and has been used here to investigate controlling factors potentially affecting the process of chamber formation. This is manifested as instantaneous or periodic deviations of the realised chamber volumes derived from modelled growth functions. The results obtained on naturally grown specimens show oscillations in chamber volumes which can be modelled by sums of sinusoidal functions. A set of functions with similar periods in all investigated specimens points to lunar and tidal cycles. To determine whether such cyclic signals are genuine and not the effects of a theoretical model, the same analysis was conducted on specimens held in a closed laboratory facility, as they should not be affected by natural environmental effects. Surprisingly, similar cyclicities were observed in such samples. However, a solely genetic origin of these cycles couldn't be verified either. Therefore, detailed analysis on the phase equality of these growth oscillations have been done. This approach is pivotal for proving that the oscillatory patterns discovered in LBF are indeed genuine signals, and on how chamber growth might be influenced by tidal currents or lunar months.

Hydrobacter penzbergensis gen. nov., sp. nov., isolated from purified water.

A Gram-negative, oxidase- and catalase-positive bacterium, designated strain EM 4(T), which varied in shape from rod-shaped to curved or helical with frequently observed bulb-shaped protuberances, was isolated from purified water. 16S rRNA gene sequence analysis indicated that the novel strain belongs to the family Chitinophagaceae within the phylum Bacteroidetes; the closest relative among bacterial species with validly published names was determined to be Sediminibacterium salmoneum NBRC 103935(T), with 93.4 % sequence identity. The main fatty acids of strain EM 4(T) were iso-C15 : 0, iso-C15 : 1 and iso-C17 : 0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, aminolipids, aminophospholipids and unknown lipids; the quinone system consisted of menaquinone MK-7. 16S rRNA gene sequence analysis and the polar lipid and fatty acid profiles suggest that the strain represents a novel genus and species, for which the name Hydrobacter penzbergensis gen. nov., sp. nov. is proposed. The type strain of Hydrobacter penzbergensis is strain EM 4(T) ( = DSM 25353(T) = CCUG 62278(T)).

Crystal structure of N,N'-bis-(diiso-propyl-phosphan-yl)-4-methyl-pyridine-2,6-di-amine.

In the mol-ecule of the title compound, C18H35N3P2, the methyl-pyridine-2,6-di-amine moiety is almost planar, with a maximum deviation of 0.0129 (9) Šfor one of the amine N atoms. Whereas one of the P atoms is co-planar with this mean plane [deviation = 0.0158 (10) Å], the other P atom is considerably displaced out of the mean plane by 0.5882 (10) Å. In the crystal, no directional intermolecular interactions beyond van der Waals contacts could be identified.

Description of Undibacterium oligocarboniphilum sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum.

A Gram-negative, oxidase- and catalase-positive, flagellated, rod-shaped bacterium, designated strain EM 1(T), was isolated from purified water. 16S rRNA gene sequence analysis indicated that the novel strain belonged to the family Oxalobacteraceae within the class Betaproteobacteria; the closest phylogenetic relative was Undibacterium pigrum DSM 19792(T) (96.7 % gene sequence similarity). The new isolate could be distinguished from the type strain of U. pigrum DSM 19792(T) (=CCUG 49009(T)=CIP 109318(T)) and from strain CCUG 49012(T), which has been described as a second genomovar of this species, on the basis of genotypic data and several phenotypic properties. An S-layer was present in the cell envelope in U. pigrum DSM 19792(T), but was absent in strains EM 1(T) and CCUG 49012(T). Test conditions were established that enabled strain CCUG 49012(T) to be distinguished from U. pigrum DSM 19792(T). As found for U. pigrum, the main fatty acids of strains EM 1(T) and CCUG 49012(T) were summed feature 3 (including unsaturated C(16 : 1)ω7c), straight-chain C(16 : 0) and unsaturated C(18 : 1)ω7c (low percentage in strain CCUG 49012(T)), and C(10 : 0) 3-OH was the sole hydroxylated fatty acid. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The polyamine profile was mainly composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. In contrast to U. pigrum and strain CCUG 49012(T), where ubiquinone Q8 was reported as the sole quinone component, the quinone system of strain EM 1(T) consisted of ubiquinone Q-8 (64 %) and Q-7 (36 %). The 16S rRNA gene sequence similarity, the polar lipid profile and the absence of C(12)-hydroxylated fatty acids all indicated that strain EM 1(T) was affiliated with the genus Undibacterium. 16S rRNA gene sequence similarity values lower than 97.0 % and several differentiating phenotypic traits demonstrated that strain EM 1(T) represents a novel species for which the name Undibacterium oligocarboniphilum sp. nov. is proposed (type strain EM 1(T)=DSM 21777(T)=CCUG 57265(T)). In addition, based on previously published results and this study, a separate species, Undibacterium parvum sp. nov., is proposed with strain CCUG 49012(T) (=DSM 23061(T)=CIP 109317(T)) as the type strain.

A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay.

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 microm and 900 microm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario.

Salinisphaera shabanensis gen. nov., sp. nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea.

A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea. A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium. Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH. In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium. Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis. No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88%. The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed.

Prokaryotic phylogenetic diversity and corresponding geochemical data of the brine-seawater interface of the Shaban Deep, Red Sea.

The interface between the hypersaline brine and the overlying sea-water (brine-seawater interface) of the Shaban Deep, northern Red Sea was investigated for the presence of microorganisms using the 16S rRNA gene as a molecular marker. Samples of the south and east basin (depth: 1331 m and 1332 m respectively) were selected to ascertain the microbial diversity of this extreme and, so far, unexplored environment. Phylogenetic analysis revealed novel lineages within the Bacteria, the Crenarchaeota and the Euryarchaeota. Novel representatives of the KB1 sequence group (Eder et al., 1999 Arch Microbiol 172: 213-218) were detected indicating a widespread distribution of the corresponding Bacteria in Deep Sea brine pools. Our results contribute to the understanding of the hitherto unknown microbial diversity at the chemical gradient of the Shaban Deep, and suggest the presence of novel Bacteria and Archaea thriving under extreme environmental conditions.

New isolates and physiological properties of the Aquificales and description of Thermocrinis albus sp. nov.

The ecology of the Aquificales was studied using a combination of phylogenetic and cultivation approaches. Enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the United States (Yellowstone National Park), Russia (Kamchatka), Italy, Germany, Djibouti, Iceland, and Africa (Lake Tanganyika). Isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. Comparisons of their 16S rRNA gene sequences showed that most of the isolates were new representatives of the major lineages of the Aquificaceae, represented by the genera Aquifex, Thermocrinis, Hydrogenobaculum, and Hydrogenobacter. Isolate HI 11/12, which was obtained from whitish streamers in the Hveragerthi area of Iceland, represents a separate branch within the Aquificaceae. The organism grew at salinities up to 0.7% NaCl and at temperatures up to 89 degrees C. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed whitish streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, sulfur, or thiosulfate as the electron donor under microaerophilic conditions. It represents a second species within the order Thermocrinis, which we name Thermocrinis albus HI 11/12 (DSM 14484, JCM 11386).