Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece

A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5–1.5 μm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 μm in length. The strain grew optimally at 95°C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H₂S, CO₂, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota. Received: September 4, 1996 / Accepted: November 12, 1996     

Comparison of the effects of microwave irradiation with different intensities on the biodegradability of sludge from the dairy- and meat-industry

Microwave (MW) irradiation is a relatively new possibility of conditioning and pretreating for wastewater sludge. Following its application in the telecommunications and food-industries, the environmental use of this technique has come into the limelight in recent years, and has become increasingly popular. Various publications have dealt with the examination of the effects of MW irradiation in municipal sludge-handling processes. We focused on the effects of MW irradiation at different power levels on solubilization (sCOD/tCOD), biodegradation and anaerobic digestion of sludge from the food-industry. For evaluating the efficiency of MW pre-treatment, the changes in the soluble fraction of the organic matter, the VS/TS ratio, the biogas yield, the methane content in the biogas, and the rate of batch mesophilic digestion were used as control parameters. Additionally, the energetic efficiency of MW pre-treatment was also examined. The results were compared with those of conventional heat (CH) treatments of the same sludge. The MW treatment proved to increase both the sCOD/tCOD and the VS/TS ratio. Furthermore, the biogas and methane yields increased during the digestion of the MW-pretreated food-industry sludge. A higher MW power level generally enhanced the biogas and methane production. Energetically, the most economic pre-treatment of sludge from dairy and meat processing was at a power level of 1.5 Wg⁻¹ and 2.5 Wg⁻¹ MW respectively; the surplus energy content of the enhanced biogas product could not compensate the extra energy demand of the stronger MW pre-treatments.     

N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

Methylene-H₄MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F₄₂₀ as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K _m for methylene-H₄MPT of 16 M, a K _m for F₄₂₀H₂ of 4 M, and a V _max of 450 U/mg (K_cat=265 s^-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F₄₂₀ (0.2 mM), methylene-H₄MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H₄MPT reductase (F₄₂₀-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵,N ¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min     

Effects of different heat pre-treatments of wheat straw on its microbial activity and colonization by different tropical and sub-tropical edible mushrooms

Quantitative differentiation of microbial activity in wheat straw substrate is described after different heat pre-treatments and addition of water during solid-state fermentation. All the 28 tested strains of tropical and sub-tropical edible mushrooms colonized sterile wheat straw. Substrate pre-treated at 25°C was primarily colonized by Coprinus sp. and other competitive microorganisms, and had the highest pH values. With some exceptions, increasing rates of growth occurred with substrate pre-treatment at 60 and 90°C. Best growth and highest speed of colonization were on sterilized straw. Heat pre-treatment of cereal straw at 60 and 90°C should be sufficient for commercial cultivation processes. Thus, a short fermentative pre-treatment could reduce the risk of infection. The strains tested do not differ significantly from those of temperate climates.R. Maziero was with the Instituto de Botânica, CP 4005, 01061-970, São Paulo, SP, Brazil, and is now at DISTAM Section of Industrial Microbiology, University of Milano, Via G. Celoria 2, 20133, Milano, Italy. F. Zadrazil is with the Institut für Bodenbiologie, Bundesforchungsanstalt für Landwirtschaft, Bundesallee 50, D 38116 Braunschweig, Germany.     

Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against “quorum sensing signals”, and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers. Pompea Del Vecchio, Mikael Elias and Luigia Merone were contributed equally to this paper.     

A novel domain arrangement in a monomeric cyclodextrin-hydrolyzing enzyme from the hyperthermophile Pyrococcus furiosus

PFTA (Pyrococcus furiosus thermostable amylase) is a hyperthermophilic amylase isolated from the archaeon Pyrococcus furiosus. This enzyme possesses characteristics of both α-amylase- and cyclodextrin (CD)-hydrolyzing enzymes, allowing it to degrade pullulan, CD and acarbose—activities that are absent in most α-amylases—without the transferring activity that is common in CD-hydrolyzing enzymes. The crystal structure of PFTA revealed a unique monomeric subunit with an extended N-terminal region and an N′-domain folded into its own active site—a significantly altered domain configuration relative to that of the conventional dimeric CD-hydrolyzing amylases in glycoside hydrolase family 13. The active site is formed by the interface of the N′-domain and the catalytic domain and exhibits a broad and wide-open geometry without the concave pocket that is commonly found in the active sites of maltogenic amylases. The mutation of a residue (Gly415 to Glu) located at the domain interface between the N′- and catalytic domains yielded an enzyme that produced a significantly higher purity maltoheptaose (G7) from β-CD, supporting the involvement of this interface in substrate recognition and indicating that this mutant enzyme is a suitable candidate for the production of pure G7. The unique configuration of the active site distinguishes this archaic monomeric enzyme from classical bacterial CD-hydrolyzing amylases and provides a molecular basis for its enzymatic characteristics and for its potential use in industrial applications.     

Novel structure of an N-terminal domain that is crucial for the dimeric assembly and DNA-binding of an archaeal DNA polymerase D large subunit from Pyrococcus horikoshii

Archaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. The N-terminal (1–300) domain structure of the large subunit was determined by X-ray crystallography, although ∼50 N-terminal residues were disordered. The determined structure consists of nine alpha helices and three beta strands. We also identified the DNA-binding ability of the domain by SPR measurement. The N-terminal (1–100) region plays crucial roles in the folding of the large subunit dimer by connecting the ∼50 N-terminal residues with their own catalytic region (792–1163).

Structured summary

DP2binds to DP2 by molecular sieving(View interaction)DP2binds to DP2 by fluorescence technology(View interaction)DP2binds to DP2 by circular dichroism(View interaction)     

Alkaline pre-treatment of oilseed rape straw for bioethanol production: evaluation of glucose yield and pre-treatment energy consumption

The objective of the research was to investigate the effect of biomass loading, alkali (NaOH) concentration and pre-treatment time on the yield of glucose obtained following alkaline pre-treatment and enzymatic hydrolysis of oilseed rape (OSR) straw. A maximum glucose yield of (440.6 ± 14.9) g glucose kg⁻¹ biomass was obtained when OSR straw was pre-treated at a biomass loading of 50 g kg⁻¹ and an alkali concentration of 0.63 mol dm⁻³ NaOH for 30 min. The energy efficiency of glucose extraction (0.39 kg glucose MJ⁻¹ consumed) was highest when OSR straw was pre-treated at a biomass loading of 50 g kg⁻¹ and an alkali concentration of 0.63 or 0.75 mol dm⁻³ for 30 min. The study demonstrated alkaline pre-treatment of OSR straw is superior to acid pre-treatment in terms of glucose yield and energy efficiency.     

Promotion of polylactide degradation by ammonia under hyperthermophilic anaerobic conditions

The objective of this study was to evaluate the promotion effect of ammonia on the biodegradation of polylactide (PLA) under hyperthermophilic (80 °C) and thermophilic (55 °C) anaerobic condition. The results showed that PLA was transformed to lactic acid under hyperthermophilic conditions, but that the transformation ratio was negligible under thermophilic conditions. The hydrolysis process can be markedly increased with ammonia addition and microorganism activity. The maximum transformation ratios of the two kinds of PLA used in this study were 65.2% and 51.8%, respectively, with ammonia addition of 4 g N/L over 3 days treatment of anaerobic sludge. After the hyperthermophilic pretreatment, the hydrolysis products were converted to methane by methanogens under the thermophilic and anaerobic conditions. The final methane conversion ratios of the two kinds of PLA after 22 days treatment were 81.8% and 77.0%, respectively.     

Influence of antifungal pre-treatment in preparing test mycelium on MIC values of several antifungal agents againstAspergillus niger

Antifungal activity of two imidazoles (miconazole and ketoconazole) and one polyene (amphotericin B) was evaluated using an automatic growth analysis system. Spores ofAspergillus niger were inoculated on the polylysine-coated glass bottom of a culture vessel. A colony formed in liquid medium was exposed to an antifungal agent and subsequently washed. Based on the dynamic growth rate of a test hypha selected from the colony in response to the antifungal agent, the minimum inhibitory concentration (MIC) was evaluated. The influence of time of reading (1, 2 and 3 h after washing) on the MIC determined was investigated. MICs for test hyphae subjected to antifungal pre-treatment were compared with those for hyphae without pre-treatment. Hyphae pre-treated with an antifungal agent for 1 h were found to become adapted and tolerant to that antifungal agent. Hyphae exposed and adapted to an imidazole obtained tolerance to amphotericin B as well as to the other imidazole.