Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s-1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

Spatial homogeneity of bacterial and archaeal communities in the deep eastern Mediterranean Sea surface sediments

The diversity of microorganisms inhabiting the deep sea surface sediments was investigated in 9 stations (700-1900 m depth) in the Levantine basin by 454 massive tag sequencing of the 16S rDNA V4 region using universal primers. In total, 108,811 reads (an average of 10,088 per sample) were assigned to 5014 bacterial and 966 archaeal operational taxonomic units (OTUs; at 97% cut off). The 55% of the reads were of archaea, indicating dominance of archaea over bacteria at eight of the stations. The diversity and estimated richness values were high (e.g., H´ ranged from 5.66 to 7.41 for bacteria). The compositions of the microorganisms at all stations were remarkably similar, with Bray-Curtis similarities of 0.53-0.91 and 0.74-0.99 for bacterial and archaeal orders respectively. At two stations, very high abundances of only a few genera (Marinobacterium, Bacillus, Vibrio, Photobacterium) were accountable for the dissimilarities documented compared to the other deep sea stations. Half of the bacterial reads (51%) belonged to the phylum Proteobacteria, comprising mainly Gammaproteobacteria (41-72% of the proteobacterial reads per sample), Deltaproteobacteria (12-29%), Alphaproteobacteria (7-18%) and Betaproteobacteria (3-14%). The most abundant bacterial family was Sinobacteraceae (order Xanthomonadales) with 5-10% of total bacterial reads per sample. Most abundant reads (15.4% of all microbial reads) were affiliated with Marine Group 1 archaea, putatively capable of ammonia oxidation (213 OTUs), and bacteria involved in nitrification were found in all samples. The data point to the significant role that chemolithotrophic carbon assimilation and nitrification fill in the oligotrophic deep sea Levant sediments (AU)

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Thermosulfidibacter takaii gen. nov., sp. nov., a thermophilic, hydrogen-oxidizing, sulfur-reducing chemolithoautotroph isolated from a deep-sea hydrothermal field in the Southern Okinawa Trough.

A novel thermophilic, sulfur-reducing chemolithoautotroph, strain ABI70S6(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain ABI70S6(T) were motile rods, 0.9-2.0 microm in length and 0.4-0.8 microm in width. Strain ABI70S6(T) was an obligately anaerobic chemolithotroph, exhibiting hydrogen oxidation coupled with sulfur reduction. Growth was observed at 55-78 degrees C (optimum, 70 degrees C), pH 5.0-7.5 (optimum, pH 5.5-6.0) and 0.5-4.5 % NaCl (optimum, 3.0 % NaCl). H(2) and elemental sulfur were utilized as electron donor and acceptor, respectively. The major fatty acids were C(16 : 0) (40.0 %) and C(20 : 1) (60.0 %). The G+C content of genomic DNA was 44.2 mol%. The physiological attributes of strain ABI70S6(T) are similar to those of species of genera within the family Desulfurobacteriaceae, most of which are thermophilic and chemolithoautotrophic sulfur reducers. However, 16S rRNA gene sequence similarities between the novel isolate and type strains of all species within the family Desulfurobacteriaceae were <87 %, which is close to the similarities found between the novel isolate and members of the family Thermodesulfobacteriaceae (<85 %). Based on physiological and phylogenetic features of the novel isolate, it is proposed that it represents a novel species in a novel genus, Thermosulfidibacter takaii gen. nov., sp. nov., within the phylum Aquificae. The type strain of T. takaii is ABI70S6(T) (=JCM 13301(T)=DSM 17441(T)).

Hydrogenivirga okinawensis sp. nov., a thermophilic sulfur-oxidizing chemolithoautotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough.

A novel extremely thermophilic sulfur-oxidizing bacterium, strain LS12-2(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain LS12-2(T) were motile rods, 1.5-4.0 microm in length and 0.4-0.5 microm in width. Strain LS12-2(T) was an obligate chemolithoautotroph that could utilize elemental sulfur or thiosulfate as an electron donor and nitrate or oxygen as an electron acceptor. Growth was observed at 65-85 degrees C (optimum 70-75 degrees C), pH 5.8-8.3 (optimum pH 6.9-7.5), 1.0-4.0 % (w/v) NaCl (optimum 2.5 %) and 1.0-7.0 % O(2) in the gas phase (optimum 3.0 %). Fatty acids detected were C(16 : 0) (8.0 %), C(18 : 0) (9.0 %), C(18 : 1) (62.5 %) and C(20 : 1) (20.5 %). The genomic DNA G+C content was 51.3 mol%. 16S rRNA gene sequence analysis indicated that strain LS12-2(T) belonged to the genus Hydrogenivirga. Based on physiological and phylogenetic characteristics of the isolate, it is proposed that this strain represents a novel species in the genus Hydrogenivirga, Hydrogenivirga okinawensis sp. nov. The type strain of Hydrogenivirga okinawensis is LS12-2(T) (=JCM 13302(T)=DSM 17378(T)).

Venenivibrio stagnispumantis gen. nov., sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from Champagne Pool, Waiotapu, New Zealand.

A novel thermophilic, hydrogen-oxidizing bacterium, designated strain CP.B2(T), was isolated from a terrestrial hot spring in Waiotapu, New Zealand. Cells were motile, slightly rod-shaped, non-spore-forming and Gram-negative. Isolate CP.B2(T) was an obligate chemolithotroph, growing by utilizing H(2) as electron donor and O(2) as corresponding electron acceptor. Elemental sulfur (S(0)) or thiosulfate ( ) was essential for growth. Microbial growth occurred under microaerophilic conditions in 1.0-10.0 % (v/v) O(2) [optimum 4-8 % (v/v) O(2)], between 45 and 75 degrees C (optimum 70 degrees C) and at pH values of 4.8-5.8 (optimum pH 5.4). The DNA G+C content was 29.3 mol%. 16S rRNA gene sequence analysis demonstrated that strain CP.B2(T) belonged to the order Aquificales, with a close phylogenetic relationship to Sulfurihydrogenibium azorense (94 % sequence similarity to the type strain). However, genotypic and metabolic characteristics differentiated the novel isolate from previously described genera of the Aquificales. Therefore, CP.B2(T) represents a novel species in a new genus, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. The type strain of Venenivibrio stagnispumantis is CP.B2(T) (=JCM 14244(T) =DSM 18763(T)).

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26.

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments.

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved 02 and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment-water interface, and which support comparable numbers (10(5)-10(6) mL(-1)) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O2 input from the atmosphere and measurable dissolved O2 to a depth of 2 mm below the sand-water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(IllI)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe.

[The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1]. / Fermenty metabolizma ugleroda u termotolerantnoi sul'fobatsilly, shtamm K1.

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.

Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system.

AIMS: This study compares the ability of Thiosphaera pantotropha ATCC 35512 and the newly isolated Pseudomonas stutzeri SU2 to perform aerobic denitrification. METHODS AND RESULTS: Nitrate-supplemented basal medium in airtight crimp-sealed serum bottles containing an atmosphere of 92% oxygen was inoculated with Ps. stutzeri SU2 or T. pantotropha and incubated at 30 degrees C. During the 92-h incubation period, aerobic denitrification by Ps. stutzeri SU2 (NO3(-) - N removal 99.24%) was more efficient than that by T. pantotropha (NO3(-) - N removal 27.29%). CONCLUSION: Pseudomonas stutzeri SU2, which was isolated from the activated sludge of a sequencing batch reactor treating piggery wastewater, rapidly reduced the nitrate to nitrogen gas without nitrite accumulation. The nitrate removal rate of SU2 was 0.032 mmol NO3(-) - N g cell-1 h-1 after 44 h incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas stutzeri SU2 can be used in a full-scale sequencing batch system for efficient in situ aerobic nitrate removal from piggery wastewater.

[Carbon metabolism in Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41]. / Metabolizm ugleroda u Sulfobacillus thermosulfidooxidans subsp. Asporogenes, shtamm 41.

The activities of carbon metabolism enzymes were determined in cellular extracts of the moderately thermophilic, chemolithotrophic, acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes, strain 41, grown either at an atmospheric content of CO2 in the gas phase (autotrophically, heterotrophically, or mixotrophically) or autotrophically at a CO2 content increased to 5-10%. Regardless of the growth conditions, all TCA cycle enzymes (except for 2-oxoglutarate dehydrogenase), one glyoxylate cycle enzyme (malate synthase), and some carboxylases (ribulose bisphosphate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase) were detected in the cellular extracts of strain 41. During autotrophic cultivation of strains 41 and 1269, the increase in the CO2 content of the supplied air to 5-10% resulted in the activation of growth and iron oxidation, a 20-30% increase in the cellular content of protein, enhanced activity of the key TCA enzymes (citrate synthase and aconitase), and, in strain 41, a decrease in the activity of carboxylases.

Halothiobacillus kellyi sp. nov., a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium isolated from a shallow-water hydrothermal vent in the Aegean Sea, and emended description of the genus Halothiobacillus.

A new mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain Milos-BII1T, was isolated from a sediment sample taken from a shallow-water hydrothermal vent in the Aegean Sea with thiosulfate as electron donor and CO2 as carbon source. Based on the almost complete sequence of the 16S rRNA gene, strain Milos-BII1T forms a phylogenetic cluster with Thiobacillus hydrothermalis, Thiobacillus neapolitanus, Thiobacillus halophilus and Thiobacillus sp. W5, all of which are obligately chemolithoautotrophic bacteria. Because of their phylogenetic relatedness and their physiological similarities it is proposed to transfer these organisms to a newly established genus within the gamma-subclass of the Proteobacteria, Halothiobacillus gen. nov. (Kelly and Wood 2000). Strain Milos-BII1T represents a new species of this genus, named Halothiobacillus kellyi. Cells were Gram-negative rods and highly motile. The organism was obligately autotrophic and strictly aerobic. Nitrate was not used as electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate, sulfur and sulfide. Growth was observed between pH values of 3.5 and 8.5, with an optimum at pH 6.5. The temperature limits for growth were 3.5 and 49 degrees C, with an optimum between 37 and 42 degrees C. Growth occurred between 0 and 2 M NaCl, with an optimum NaCl concentration between 400 and 500 mM. The mean maximum specific growth rate on thiosulfate was 0.45 h(-1).

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N2O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N2O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N2O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N2O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N2O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH4 oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH4 in soils.

Preservation of some extremely thermophilic chemolithoautotrophic bacteria by deep-freezing and liquid-drying methods.

Long-term preservation methods for extreme thermophilic chemolithoautotrophic bacteria representing various species are described. The cultures were cryopreserved in liquid nitrogen under anaerobic conditions using 5% dimethylsulfoxide as a cryoprotectant. For easy storage and transport, the cultures were successfully liquid-dried, directly from the liquid phase without involving freezing under semiaerobic conditions using effective protective agents such as ethylenediamine and meso-inositol. The tested cultures showed good stability and survival rates after drying, after cryopreservation and on long-term storage. All tested strains were successfully preserved and reactivated within relatively short time. The viability, stability and ability of chemolithoautotrophic growth was not affected. Cryopreservation, liquid-drying and reactivation under microaerobic conditions proved very effective for these oxygen sensitive cultures.

Spontaneous mutation of Thiosphaera pantotropha enabling growth on methanol correlates with synthesis of a 26 kDa c-type cytochrome.

Thiosphaera pantotropha grows on methanol as carbon and energy source following spontaneous mutation to a Mox+ phenotype after incubation in media containing methanol. Acquisition of ability to grow on methanol correlates with the appearance of a c-type cytochrome, molecular mass 26 kDa, which is suggested to substitute for the product of the moxG gene, which is the electron acceptor from methanol in related bacteria, but which is absent from T. pantotropha. Mutation leading to growth on methylamine as carbon and energy source was not observed despite the presence of in vitro methylamine dehydrogenase activity in cells grown on choline. Lack of growth on methylamine may correlate with the absence of amicyanin, which is the obligatory electron acceptor from methylamine dehydrogenase in other organisms.

Restriction endonuclease Aor13HI from Acidiphilium organovorum 13H, a new isoschizomer of BspMII: purification and characterization.

A restriction endonuclease, Aor13HI, an isoschizomer of BspMII, was purified to homogeneity from cell extracts of Acidiphilium organovorum strain 13H. The enzyme has a molecular mass of 60,000 daltons and consists of two subunits identical in molecular mass of 30,000 daltons. Aor13HI endonuclease, like BspMII, recognizes the palindromic six-base sequence 5'-TCCGGA-3', and cleaves between the T and C to produce a four-base 5' extension. Aor13HI is not inhibited by dam-dependent methylation. The isoelectric point of the enzyme is 5.7. Aor13HI activity was maximum at pH 7.5, 100 mM KCl, 7.5-10 mM MgCl2, and 55 degrees C. The enzyme was stable up to 60 degrees C. The N-terminal amino acid sequence (30 residues) of Aor13HI did not show any similarity with the sequence of other restriction endonucleases reported.

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha.

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N2H5+) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.

Effect of organic substrates on biological sulphide oxidation.

Neither acetate nor higher fatty acids and glucose have a significant effect on the biotechnological process for sulphide removal at 20 degrees C, in which sulphide is oxidized to sulphur using oxygen. The oxidation of acetate and propionate with oxygen is mainly dependent on the sulphide and oxygen concentrations in the reactor. The occurrence of Thiothrix filaments in sulphide-removing waste-water treatment systems has been investigated using a fixer-film upflow reactor. The influent of this reactor consisted of anaerobically treated paper-mill waste-water, with a sulphide concentration of 140 mg/l. It was found that sulphide loading rate is the decisive parameter as to whether or not Thiothrix will develop in a sulphide-removing reactor.

Biosynthesis of caldariellaquinone in Sulfolobus spp.

The biosynthesis of caldariellaquionone (CQ) was studied in species of Sulfolobus by measuring the incorporation of stable isotopically labeled tyrosines into CQ. By feeding a series of tyrosines labeled with deuterium or 13C and then measuring the extent and position at which label was incorporated into CQ by mass spectrometry, it was shown that more than 95% of the label was incorporated into the benzo[b]thiophen-4,7-quinone moiety of CQ. From the labeling experiments, it is concluded that the benzo[b]thiophen-4,7-quinone is derived as an intact unit from all of the carbons of tyrosine except C-1.

Microbiological quality and shelf life prediction of chilled fish.

A number of storage experiments have been carried out with whole cod and vacuum-packed cod fillets stored in ice. The microbiological quality of the fish was determined on the basis of detection time estimated rapidly by conductance assays in a TMAO-containing medium at 25 degrees C. Detection time and sensory data have been incorporated into a predictive linear model to estimate the remaining shelf life of the products. It is concluded that the shelf life of iced whole cod can be predicted using this model but not that of vacuum-packed fillets because of the greater variability of bacterial activity in packaged fish.

Structure, partial elemental composition, and size of Thiopedia rosea cells and platelets.

The phototrophic purple sulfur bacterium Thiopedia rosea forms multicellular, gas-vacuolate, regular, flat aggregates (platelets, sheets) held together by slime. Platelets found in eutrophic water consisted of slime (85% of the total wet volume) and 16 cells, while the gas-filled vacuole occupied 44% of the volume of a single wet cell. Individual platelet cells contained central spindle-shaped gas vesicles (which together constitute the cell's gas vacuole), intracytoplasmic membrane vesicles (chromatophores), and peripheral sulfur globules. Cells were surrounded by a Gram-negative type cell envelope and were connected to neighboring cells of the same platelet by mostly unstructured slime. Cells contained detectable amounts of magnesium, phosphorus, sulfur, and potassium as determined by wavelength-dispersive X-ray microanalysis. The large size and relatively low slime density of the platelet, as well as the flat shape, could greatly decrease platelet sedimentation and so stabilize the position of T. rosea within its water column.