Polar lipids and pigments as biomarkers for the study of the microbial community structure of solar salterns

Whole community lipids and pigments have been examined over a 3–5-year period in commercial salterns located in the United States, Israel, and Spain. There were significant differences in the types of lipids and pigments within the California saltern system during the 5-year period. These patterns differed seasonally despite examination of ponds with approximately the same salinities. The solar saltern in Eilat, Israel had fewer lipids on the thin-layer chromatography plates and confirmed previous analyses. The biota in the crystallizer pond in Alicante, Spain, resembled the microbial community in Israel. In the crystallizers at all three locations, phosphatidyl glycerol, methyl-phosphatidyl glycerophosphate, phosphatidyl glycerosulfate, and the sulfated diglycosyldiether lipid were identified regardless of season. This was not true for pans with salinities below 25% where no distinctive pattern was observed. Thus, we hypothesize that the more eutrophic inlet waters of the California saltern and the cooler temperatures, which result in longer retention times of water in the different pans, allow for the more diverse microbial community to develop. This is reflected then in more complex lipid and pigment patterns. However, the oligotrophic inlet waters to the Eilat saltern coupled with a drier and warmer climate result in a shorter retention time of water in the pans and a less diverse microbial community as evidenced by fewer extractable lipids and pigments.     

Investigation of physiological limits and conditions for robust bioprocessing of an extreme halophilic archaeon using external cell retention system

High biological activity and volumetric productivity are considered as prerequisites for efficient bioprocesses, extreme halophilic Archaea have, however, lower growth rates, for which reason halophilic Archaea are so far not used in industrial bioprocesses. To overcome this physiological limit and to achieve increased volumetric productivity, the produced biomass must be retained in a bioreactor, for example equipped with an external cell retention system. In this study, the characterization and parameterization of a bioreactor setup with cell retention was carried out with an extreme halophilic archaeon. Bioprocess quantification was used to demonstrate the process controllability. Focussing on maximizing the volumetric productivity; 10-fold productivity increase was achieved compared to chemostat continuous cultures. Circulation of the broth between the bioreactor and the membrane unit can be however challenging from physiological points-of-view. Hence, operating the system with external cell retention at optimal cross flow rate is physiologically essential: at lower cross flow rates, higher extracellular protein concentrations were measured due to oxygen limitation. In turn, at higher cross flow rates, shear stress reasoned higher concentrations of DNA fragments. This work contributes in a pioneering way to the bioprocess development of extreme halophilic Archaea by optimizing continuous laboratory scale processes regarding robustness and scalability.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Carbon monoxide production by Methanobacterium thermoautotrophicum

Abstract Methanobacterium thermoautotrophicum was grown in a fermenter gassed with an 80% H₂/20% CO₂ mixture. The effluent gas was found to contain between 30 ppm and 90 ppm carbon monoxide. Approx. 5 nmol CO were produced per min and mg cells (dry weight) by the culture. This is to our knowledge the first report on biological carbon monoxide formation under strictly anaerobic conditions.     

Metabolism in hyperthermophilic microorganisms

Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H₂S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H₂ and CO₂, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO₂ do so via a reductive citric acid cycle. Two S^o-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S^o. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S^o has been proposed. However, the mechanisms by which S^o reduction is coupled to energy conservation in this organism and in obligate S^o-reducing hyperthermophiles is not known.Abbreviations ADH alcohol dehydrogenase (ADH) - AOR aldehyde ferredoxin oxidoreductase - FMOR formate ferredoxin oxidoreductase - FOR formaldehyde ferredoxin oxidoreductase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - GluOR glucose ferredoxin oxidoreductase - KGOR 2-ketoglutarate ferredoxin oxidoreductase - IOR indolepyruvate ferredoxin oxidoreductase - LDH lactate dehydrogenase - MPT molybopterin - POR pyruvate ferredoxin oxidoreductase - PLP pyridoxal-phosphate - PS polysulfide - TPP thiamin pyrophosphate - S^o elemental sulfur - VOR isovalerate ferredoxin oxidoreductase     

The phylogenetic position of the Thermococcus isolate AN1 based on 16S rRNA gene sequence analysis: a proposal that AN1 represents a new species, Thermococcus zilligii sp. nov.

The 16S rRNA gene from the Thermococcus New Zealand isolate AN1 was cloned and sequenced. Analysis of the gene revealed the presence of signature sequences, indicating that strain AN1 represents a new species of the genus Thermococcus. Since the isolate AN1 differed from other thermococci in both its lower optimal NaCl concentration and generally lower optimal temperature for growth, in its unusual lipid membrane composition, and in its sensitivity to antibiotics, we propose that strain AN1 represents a new species of Thermococcus. The proposed name is Thermococcus zilligii, and the type strain is DSM 2770. Received: 13 February 1997 / Accepted: 30 May 1997     

Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K₂HPO₄ (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases. Received: 20 November 1997 / Accepted: 28 February 1997