Gerhard Zellner

Isolation and characterization of a thermophilic sulfate reducing archaebacterium archaeoglobus fulgidus strain z

Summary An extremely thermophilic, strictly anaerobic sulfate reducing bacterium, strain Z, was isolated from sediments of a hydrothermal vent located near Mount Vulcanello, Vulcano, Italy. On the basis of 16S rRNA cataloguing, strain Z was shown to belong to the archaebacterial kingdom, representing another strain of Archaeoglobus fulgidus . The weakly motile, irregular coccoid cells showed the fluorescence regarded as typical for methanogens, when viewed under the fluorescence microscope, although no relationship with methanogens was seen from comparative analysis of antigenic fingerprints with antibody S-probes. Furthermore, methane was not produced in significant amounts. Dissimilatory sulfate reduction was found with lactate, pyruvate and 2,3-butandiol as substrates. With thiosulfate as electron acceptor H 2 /CO 2 , formate, lactate, pyruvate or fumarate were utilized as electron donors. Sulfur bloom was not used as electron acceptor. Growth was inhibited by molybdate, a typical inhibitor of sulfate reducers. Lactate was decarboxylated to acetate and part of the acetate was oxidized to CO 2 with the reducing equivalents serving for sulfate reduction. The cell envelope consisted of a hexagonally arranged S-layer. The apparent molecular weight of its subunits, staining PAS-positive, was 132000. No significant amounts of polyamines were detected. The DNA polymerase was sensitive towards aphidicolin, a specific inhibitor of DNA polymerases type a of eucaryotes and of the DNA polymerase of Methanococcus vannielii . The DNA base composition was 45 mol% G+C. Archaeoglobus fulgidus strain Z has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 4139.

Isolation and characterization of Methanocorpusculum parvum, gen. nov., spec. nov., a new tungsten requiring, coccoid methanogen

A new mesophilic, monotrichously flagellated methane-producing coccus of ≦1μm in diameter was isolated from an anaerobic sour whey digester, originally inoculated with sewage sludge. Growth and methane production were observed with H2/CO2, formate and — less effectively — with 2-propanol/CO2. The isolate grew at temperatures between 15° C and 45° C with the optimum at around 37° C. Acetate, yeast extract and tungstate were required in the medium. Clarified rumen fluid stimulated growth.The DNA of the new methanogen has a G+C content of 48.5 mol%. Comparative 16 S rRNA oligonucleotide cataloguing allows to define the new isolate as a member of a new genus of the order Methanomicrobiales. Further evidence for this is provided by the antigenic crossreactivity with anti-S probes and by metabolic features.Because of its small size the new methanogen is named Methanocorpusculum parvum.

Characterization of a new mesophilic, secondary alcohol-utilizing methanogen, Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H2/CO2 and formate in complex medium. It also grew autotrophically on H2/CO2. Furthermore, growth on 2-propanol/CO2 was observed. Methane was formed from CO2 by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO2 apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP+ was the only electron carrier that was utilized. No reaction was found with NAD+, F420, FMN and FAD.

Methanocorpusculaceae fam. nov., represented by Methanocorpusculum parvum, Methanocorpusculum sinense spec. nov. and Methanocorpusculum bavaricum spec. nov.

Two new methanogenic bacteria, Methanocorpusculum sinense spec. nov. strain DSM 4274 from a pilot plant for treatment of distillery wastewater in Chengdu (Province Sichuan, China), and Methanocorpusculum bavaricum spec. nov. strain DSM 4179, from a wastewater pond of the sugar factory in Regensburg (Bavaria, FRG) are described. Methanocorpusculum strains are weakly motile and form irregularly coccoid cells, about 1 μm in diameter. The cell envelope consists of a cytoplasmic membrane and a S-layer, composed of hexagonally arranged glycoprotein subunits with molecular weights of 90000 (Methanocorpusculum parvum), 92000 (M. sinense), and 94000 (M. bavaricum). The center-to-center spacings are 14.3 nm, 15.8 nm and 16.0 nm, respectively. Optimal growth of strains is obtained in the mesophilic temperature range and at a pH around 7. Methane is produced from H2/CO2, formate, 2-propanol/CO2 and 2-butanol/CO2 by M. parvum and M. bavaricum, whereas M. sinense can only utilize H2/CO2 and formate. Growth of M. sinense and M. bavaricum is dependent on the presence of clarified rumen fluid. The G+C content of the DNA of the three strains is ranging from 47.7–53.6 mol% as determined by different methods. A similar, but distinct polar lipid pattern indicates a close relationship between the three Methanocorpusculum species. The polyamine patterns of M. parvum, M. sinense and M. bavaricum are similar, but distinct from those of other methanogens and are characterized by a high concentration of the otherwise rare 1,3-diaminopropane. Quantitative comparison of the antigenic fingerprint of members of Methanocorpusculum revealed no antigenic relationship with any one of the reference methanogens tested. On the basis of the distant phylogenetic position of M. parvum and the data presented in this paper a new family, the Methanocorpusculaceae fam. nov., is defined.

Phylum All. Euryarchaeota phy. nov.

The phylum currently consists of seven classes: the Methanobacteria, the Methanococci, the Halobacteria, the Thermoplasmata, the Thermococci, the Archaeoglobi, and the Methanopyri. With the sole exception of the Methanococci, which is subdivided into three orders, each class contains a single order. The Euryarchaeota are morphologically diverse and occur as rods, cocci, irregular cocci, lancet-shaped, spiral-shaped, disk-shaped, triangular, or square cells. Cells stain Gram-positive or Gram-negative based on the presence or absence of pseudomurein in cell walls. In some classes, cell walls consist entirely of protein or may be completely absent (Thermoplasmata). Five major physiological groups have been described previously: the methanogenic Archaea, the extremely halophilic Archaea, Archaea lacking a cell wall, sulfate reducing Archaea, and the extremely thermophilic S0 metabolizers.

Methanogenium liminatans spec. nov., a new coccoid, mesophilic methanogen able to oxidize secondary alcohols

A new mesophilic, coccoid methanogen, assigned as Methanogenium liminatans spec. nov. strain DSM 4140, was isolated from effluent of a reactor for the anaerobic treatment of industrial waste water. Cells of M. Liminatans formed irregular cocci, about 1.5 μm in size, and occurred singly. The cell envelope was an S-layer with hexagonally arranged glycoprotein subunits (Mr=118000). The center-to-center spacings were 15.4 nm. The polar lipid pattern was similar to that of Methanogenium tationis, the polyamine content similar to that found in several Methanogenium species. Strain DSM 4140 grew with H2/CO2, formate, 2-propanol/CO2, 2-butanol/CO2 and cyclopentanol/CO2. For growth with the different substrates acetate was required as an additional carbon source. Growth on H2/CO2 was stimulated by the addition of tungstate. The optimal concentration was 1–2 μM Na2WO4. 185WOinf4sup2-was incorporated into cells. Growth was not influenced by 0–600 mM NaCl, but no growth occurred in the presence of ≥800 mM NaCl. Increasing concentrations of KCl up to 100 mM were slightly inhibitory for growth. The optimal growth temperature was around 40°C. The G+C content of the DNA was 59.3 mol% (Tm) or 60.5 mol% (HPLC).

Anaerobic digestion of whey and whey permeate with suspended and immobilized complex and defined consortia

SummaryWhey could be anaerobically digested at space loadings up to 36 kg COD/m3·d in an upflow digester containing porous clay beads for immobilization of microorganisms. In a parallel fermenter without immobilization a space loading of only 8 kg/m3·d was reached. The start-up time was very much reduced by the support material. The COD-reduction in both reactors was 95% and volatile fatty acids in the effluent were below 10 mmol/l. During the digestion of whey a thick layer of Methanothrix soehngenii and occasionally Methanobrevibacter arboriphilus was immobilized on the clay beads. The Methanothrix soehngenii layer disappeared, when whey permeate was fed. Methanosarcina spec. became the predominant acetotrophic methanogen, probably due to the lower pH resulting from digestion of whey permeate. Methanosarcina spec., however, was suspended and only occasionally trapped in the pores of the clay beads. No significant adhesion of other bacteria occurred.In a chemostat a consortium of 5 isolates from digested whey and a strain of Methanosarcina barkeri was able to degrade all components of whey, although at a slightly lower conversion rate than by the complex natural consortium. The total population in the whey digester was more than twice as numerous as that in the whey permeate digester. The lower number of acetotrophic methanogens seemed to be the rate-limiting step in the whey permeate digester and seemed to be responsible for the lower overall conversion rates.

Desulfovibrio simplex spec. nov., a new sulfate-reducing bacterium from a sour whey digester

Desulfovibrio simplex spec. nov. strain XVI was isolated from an anaerobic sour whey digester. Single cells had a vibrioid shape and were motile by a single, polar flagellum. The size of cells was 0.5–1.0 μ×1.5–3.0 μm. The G+C content of the DNA of D. simplex strain XVI was 47.5 mol%. The only other Desulfovibrio species with a similar G+C content of the DNA was Desulfovibrio salexigens (46.1 mol%. D. simplex grew on H2/CO2, formate, pyruvate, L(+)-lactate, fumarate, malate, ethanol, 1-propanol and 1-butanol as electron donors, while Desulfovibrio salexigens grew in addition on methanol, 2-propanol, 2-butanol, glycerol, succinate, citrate, choline and glucose. Electron acceptors for D. simplex were sulfate, thiosulfate and nitrate. L(+)-Lactate was incompletely oxidized to acetate and CO2 during sulfate reduction. Furthermore, both species could be distinguished by the ability of D. simplex but not of D. salexigens to grow on and to oxidize benzaldehyde derivatives to the respective acids, including vanillin, p-anisaldehyde and syringaldehyde. Moreover, D. simplex could grow in the presence of trace amounts of NaCl, while D. salexigens had an obligate requirement of 25 g/l NaCl. In addition, D. simplex can be distinguished from D. salexigens by its differing polyamine pattern. On the basis of the presented data the description of strain XVI as Desulfovibrio simplex spec. nov. is proposed.

Analysis of a highly efficient methanogenic consortium producing biogas from whey

Summary Ninety % of the bacterial population of an anaerobic whey digester were isolated and characterized. Fifty-five % of the isolates were fermentative, 5% acetogenic and 40% methanogenic organisms. Of the fermentative isolates, one strain of Lactobacillus , three strains of Eubacterium and 2 strains that could not be assigned to any known genus were predominant, accounting for 45% of the culturable population. The remaining 10% of the fermentative strains comprised Clostridia, Fusobacterium and Bacteroides . Each of these isolates represented less than 2% of the culturable population. The isolates probably belonging to the acetogenic stage could be assigned to Desulfovibrio and constituted approximately 5% of the culturable population. The methanogens consisted of 5 morphologically different groups. One group had the characteristic morphology of Methanospirilium , but no strains were isolated. Isolates of the other 4 groups were identified as strains of Methanobacterium formicicum, Methanobrevibacter arboriphilus, Methanothrix soehngenii and Methanocorpusculum parvum ( Zellner et al., 1987). On the basis of the metabolic capabilities of the isolates a microbial food chain for the degradation of the whey components to methane and CO 2 is proposed, fitting the theoretical model of Bryant (1979).

Reclassification of Methanogenium tationis and Methanogenium liminatans as Methanofollis tationis gen. nov., comb. nov. and Methanofollis liminatans comb. nov. and description of a new strain of Methanofollis liminatans

Sequencing of 16S rRNA genes and phylogenetic analysis of Methanogenium tationis DSM 2702T (OCM 43T) (T = type strain) and Methanogenium liminatans GKZPZT (= DSM 4140T) as well as other members of the family Methanomicrobiaceae revealed that both species belong to a separate line of descent within this family. In addition, a new strain of Methanogenium liminatans, strain BM1 (= DSM 10196), was isolated from a butyrate-degrading, fluidized bed reactor and characterized. Cells of both species are mesophilic, highly irregular cocci that use H2/CO2 and formate for growth and methanogenesis. In addition, Methanogenium liminatans strains GKZPZT and BM1 used 2-propanol/CO2, 2-butanol/CO2 and cyclopentanol/CO2. Both species contained diether and tetraether lipids. The polar lipids comprised amino-phosphopentanetetrol derivatives, which appear to be characteristic lipids within the family Methanomicrobiaceae. The pattern of glycolipids, phosphoglycolipids and amino-phosphoglycolipids was consistent with the assignment of these two species to a taxon within the family Methanomicrobiaceae, but also permitted them to be distinguished from other higher taxa within this family. The G+C contents of the DNA of Methanogenium tationis and Methanogenium liminatans were 54 and 60 mol% (Tm and HPLC), respectively. On the basis of the data presented, the transfer of Methanogenium tationis and Methanogenium liminatans to the genus Methanofollis gen. nov. as Methanofollis tationis comb. nov. and Methanofollis liminatans comb. nov., respectively, is proposed, with Methanofollis tationis as the type species.