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Factors affecting the freeze-fracture morphology of in vivo polyhydroxyalkanoate granules.

Interesting morphologies were observed when Comamonas acidovorans containing polyhydroxyalkanoates (PHA) of various compositions was freeze-fractured at temperatures far below the glass transition temperatures of PHA. In vivo granules of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comparatively showed the most ductility, and could be stretched extensively. Contrary to the uniform needle-type deformation shown by the poly(3-hydroxybutyrate) homopolymer when fractured at -110 degrees C, copolymers containing 3-hydroxyvalerate units showed various deformation structures. Similar observations were made when in vivo granules of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) were freeze-fractured, although the ductility of the latter was much reduced. In addition, it was found that fracturing at -160 degrees C resulted in decreased ductility of the PHA granules with the concomitant increase in the number of mushroom-type deformation structures. Our results suggest that PHA granules with higher resistance to freeze-fracture deformation show less ductility, and therefore produce the mushroom-type morphology. This is the first report on the freeze-fracture morphology of PHA copolymers containing short-chain-length monomers.

Cupriavidus necator↗

[Physico-chemical and microbiological characteristics of groundwater from observation boreholes of a deep radioactive liquid waste repository].

A radioactive liquid waste repository was found to be a habitat of the rich microbial community with a high catabolic potential. Groundwater from a depth of 162-189 m contained aerobic saprotrophic and anaerobic fermentative, sulfate-reducing, and denitrifying bacteria. Nitrate-reducing bacteria residing in this groundwater were isolated in pure cultures. Based on the results of their physiological studies, 16S rRNA sequencing, and phylogenetic analysis, the microorganisms isolated were ascribed to one phylogenetic branch, the gamma-subclass of gram-negative bacteria. Among six isolates, four belonged to the genus Acinetobacter, whereas two others belonged to the genera Comamonas and Aeromonas. The data obtained indicate that the microflora of the repository can exert a certain effect on the chemical composition of the formation fluids and bearing rocks, as well as on the migration of radionuclides.

Bacteria↗

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part I: characterization of water isolates for potential use as standard challenge organisms to qualify 0.1 micron rated filters.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.

Acholeplasma↗

[Auxotrophy and utilization of oxidized and reduced mineral sulfur forms by Brevundimonas diminuta strains].

Connection between auxotrophy of Brevundimonas diminuta strains for sulphur-containing amino acids and presence of oxidized or reduced sulphur forms in the medium has been shown. When grown in synthetic medium supplied with sulphate or sulphite B. diminuta strains required cystine, biotin, pantothenate and vitamin B12 for growth and utilized very limited spectrum--from 8 to 12--carbon sources. In the presence of 5 g/l of thiosulphate in the medium instead of mentioned sulphur-containing salts strains of this species did not require cystine and assimilated the ammonium nitrogen. The intensity of their growth in these conditions was lower and carbon nutrition spectra more limited than in custine-containing medium. Tetrathionate, elemental sulphur, sulphide were less efficient than thiosulphate, but they also provided growth of B. diminuta strains in the media supplied with ammonium salts, necessary vitamins and such universal C-sources as aspartate, glutamate, proline. Neither oxidized nor reduced mineral sulphur forms were able to substitute sulphur-containing amino acids for Comamonas terrigena strain requiring methionine.

Amino Acids, Sulfur↗

Microbial polyhydroxyalkanoates (PHAs): an emerging biomaterial for tissue engineering and therapeutic applications.

Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates (PHAs) offer the most diverse range of thermal and mechanical properties. Of particular interest are the PHAs that contain 4-hydroxybutyrate such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB) and poly(4-hydroxybutyrate) [P(4HB)]. These polyesters can only be synthesized by a few types of bacteria, among which Comamonas acidovorans has the most efficient metabolic pathways to channel 4HB monomers. The resulting polyesters are bioabsorbable and are being developed as a new biomaterial for medical applications. By controlling the molar ratio of the monomers, it is possible to produce materials that are as tough and elastic as rubber.

3-Hydroxybutyric Acid↗

Diversity of denitrifying bacteria isolated from Daejeon Sewage Treatment Plant.

The diversity of the denitrifying bacterial populations in Daejeon Sewage Treatment Plant was examined using a culture-dependent approach. Of the three hundred and seventy six bacterial colonies selected randomly from agar plates, thirty-nine strains that showed denitrifying activity were selected and subjected to further analysis. According to the morphological and biochemical properties, the thirty nine isolates were divided into seven groups. This grouping was supported by an unweighted pair group method, using an arithmetic mean (UPGMA) analysis with fatty acid profiles. Restriction pattern analysis of 16S rDNA with four endonucleases (AluI, BstUI, MspI and RsaI) again revealed seven distinct groups, consistent with those defined from the morphological and biochemical properties and fatty acid profiles. Through the phylogenetic analysis using the 16S rDNA partial sequences, the main denitrifying microbial populations were found to be members of the phylum, Proteobacteria; in particular, classes Gamma proteobacteria (Aeromonas, Klebsiella and Enterobacter) and Beta proteobacteria (Acidovorax, Burkholderia and Comamonas), with Firmicutes, represented by Bacillus, also comprised a major group.

Bacterial Typing Techniques↗

[Effects of the nitrogen nutrition conditions on the growth and protein synthesis of carboxydobacteria].

The rate of growth of bacterial strains oxidizing carbon monoxide (Pseudomonas gazotropha Z-1156, Comamonas compransoris Z-1155, and Seliberia carboxydohydrogena Z--1062) was studied as a function of the concentration of NH4Cl in the medium. The bacteria could grow on media containing various nitrogen sources (NH4Cl, KNO3, CO(NH2)2). Changes in the amino acid content and biochemical composition of the biomass were studied during growth of the bacteria on these media. The biological value of proteins of the bacteria was estimated.

Amino Acids↗

[Comparison of antibacterial activity of cefmenoxime with other cephalosporins against clinically isolated bacteria (author's transl)].

We examined the antibacterial activity of MX in comparison with those of other CEPs, using aerobic Gram-positive cocci, aerobic Gram-negative bacilli and anaerobic bacteria, 870 strains in total, all isolated from clinical specimens, in 1979 and 1980. Against Streptococcus, CMX showed superior antibacterial activity than those of CFX, CMZ, CXM and CTM. Against H. influenzae, CMX also showed superior antibacterial activity than those of CFX, CMX, CXM, CTM and CEZ. ABPC-and PIPC-resistant strains were sensitive to CMX. CTX, CPZ and CZX also showed antibacterial activities equivalent to that of CMX. Against enteric bacteria, E. coli, Klebsiella, E. cloacae, Serratia, C. freundii and Proteus, CMX showed superior antibacterial activity than those of CFX, CMZ, CXM, CTM and CEZ. Especially, against E. coli, Klebsiella, P. mirabilis, P. rettgeri and P. inconstans, CMX showed strong antibacterial activity. As to non-fermentation bacteria, CMX's antibacterial activity was relatively weak except P. putrefaciens, Alcaligenes and Comamonas. However, it was superior than that of CEZ. In comparison with other CEPs, the strength of CMX varied according to the kinds of bacteria. As to anaerobic bacteria, CMX showed strong antibacterial activity against Peptococcus, Peptostreptococcus, Lactobacillus, Propionibacterium, C. perfringens, Veillonella and Fusobacterium. However, its antibacterial activity against Bacteroides was similar to those of other CEPs.

Bacteria↗

[Metabolism of organic nitrogen compounds by carboxydobacteria].

The capability to hydrolyze and assimilate creatinine, uric and hippuric acids as sources of nitrogen, carbon and energy was studied in Comamonas compransoris Z-1155, Seliberia carboxydohydrogena Z-1062 and Pseudomonas gazotropha Z-1156. The organisms effectively hydrolyzed these compounds and assimilated their nitrogen under the conditions of autotrophic growth. The carboxydobacteria were also capable of growing in media containing creatinine, uric and hippuric acids and assimilated these compounds as carbon sources. In the absence of extraneous energy sources, none of the organisms grew in media with organic nitrogen compounds though the latter were hydrolyzed.

Bacteria↗

[Lipid composition of carboxydobacteria].

The composition of lipids was studied in three strains of carboxydobacteria, viz. Seliberia carboxydohydrogena Z-1062, Pseudomonas gazotropha Z-1156 and Comamonas compransoris Z-1155. The content of lipids extracted with an alcohol--chloroform mixture varied in these strains from 9 to 13.2%. The main classes were phospholipids (from 62.6 to 77.2% of the overall lipid content). The major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline. Moreover, the strains Z-1062 and Z-1156 contained three unidentified phospholipids which constituted 40 and 20% of the total content respectively. The neutral lipids of the carboxydobacteria comprised diglycerides, triglycerides, free fatty acids, methyl esters of fatty acids and a number of compounds which were not identified. The fatty acids of the strains were represented by saturated, monoene and cyclopropanoic acids. The main acids were palmitic, palmitolenic, C17-cyclopropanoic, stearic and octadecenic acids. Polar lipids differed from neutral lipids in their fatty acid composition. The fatty acid composition of firmly bound lipids was characterized by a high degree of saturation, the absence of cyclopropanoic acids and the presence of two unidentified acids that were not found in free lipids; apparently, they belonged to hydroxy acids.

Bacteria↗

Characterization of monoclonal antibodies against Erwinia carotovora subsp. atroseptica serogroup I: specificity and epitope analysis.

The characteristics of two monoclonal antibodies (Mabs), A23/1221.59.44.d.3 (1221) and A23/1239.36.64.e.2 (1239), against Erwinia carotovora subsp. atroseptica serogroup I produced in this study were compared with those of two other independently obtained Mabs, 4G4 in Spain and 4F6 in Canada, using different strains as immunogen and different screening procedures. The reaction pattern of Mabs 1221 and 1239 determined by indirect ELISA on over 200 bacterial strains including five E.c. atroseptica and 36 E.c. carotovora serogroups, seven Erw. chrysanthemi biovars, 23 other plant bacterial pathogens and 33 saprophytic bacteria from potato was similar to that of 4G4. Specificity for E.c. atroseptica serogroup I was improved, especially when skimmed milk (Marvel) was used instead of bovine serum albumin as blocking agent. Mabs 1221, 1239 and 4G4 reacted positively with all 22 E.c. atroseptica serogroup I, the dominant E.c. atroseptica serogroup on potato, strains tested and only with two out of five E.c. atroseptica serogroup XXII strains, one E.c. carotovora serogroup XXI strain and one strain of a saprophytic bacterium, Comamonas sp. Essentially similar results were obtained when examined by immunofluorescence. Characterization of the four Mabs showed that they were IgG3 and SDS-PAGE/immunoblot results suggested that they were probably against the O-side chain of bacterial cell wall lipopolysaccharides. In competition ELISA between biotin-labelled and unlabelled Mabs, the competition pattern of the four Mabs was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Attempt to establish genome similarity between Alcaligenes faecalis and several Gram-negative bacteria using the method of molecular hybridization of DNA to DNA].

The technique of DNA-DNA molecular hybridization was used to demonstrate the absence of genome similarity between Alcaligenes faecalis and gram-negative bacteria belonging to the genera Pseudomonas, Enterobacterium, Comamonas producing alkalis, and vibrions. Some similarity in the polynucleotide sequences was found between the DNA of the reper strain of Alcaligenes faecalis 45 (5--10% of homology) and the DNA of the culture belonging to the Achromobacter genus; therefore, the two genera may belong to one and the same family.

Alcaligenes↗

[Content of nitrogen bases in the DNA of carboxide bacteria].

The composition of nitrogen bases was determined in total DNA of live strains of carboxide bacteria belonging to the genera Pseudomonas, Comamonas, Seliberia (Agrobacterium), Achromobacter. Considerate differences in the nucleotide composition of DNA of the studied bacteria confirm that they indeed belong to different genera. At the same time, the content of GC pairs in DNA of these bacteria is within the range found for the corresponding genera by other authors.

Bacteria↗

[Development of microbiological technology of air deodoration in laboratory-industrial conditions using a pilot plant].

Laboratory tests were performed to select a complex of bacterial strains capable of effective deodoration of waste air produced by an animal formulated feed works under elevated temperature with the presence of numerous organic pollutants. The complex included species from the general Nocardia, Rhodococcus, and Comamonas. The biocatalyst was tested in a real industrial process with the use of a pilot plant for microbiological deodoration of waste air. The test lasted for over six months and confirmed the efficiency of the development method of deodoration.

Air Pollution↗

An attempt to use selected strains of bacteria adapted to high concentrations of petroleum oil to increase the effective removal of petroleum products in excess activated sludge in laboratory conditions.

Forty two strains of bacteria were isolated from excess activated sludge from petroleum wastewater treatment plant. The strains were identified and classified to the following groups: Enterobacteriaceae (7 strains), Anitratum (3 strains), Pseudomonas (13 strains), Micrococcus (12 strains), Comamonas (2 strains), Xanthomonas (2 strains), Achromobacter (1 strain) and Vibrio-Aeromonas (1 strain). One of the isolates was found to be a yeast strain. Following preliminary selection ten strains, showing the best growth in medium with oil fraction as sole carbon source, were chosen for further studies. The selected strains belong to Pseudomonas (6 strains), Xanthomonas (2 strains), Micrococcus (1 strain) and Saccharomyces (1 strain). The strains were adapted to high oil concentration (500-2000 mg/L) and an attempt to use them to intensify removal of petroleum products from excess activated sludge was made. The sludge was inoculated with a mixture of the isolated strains. The experiment was carried out three times, each time with a fresh sample of the excess sludge. The obtained results show that the inoculation of activated sludge with the strains active against oil reduced the petroleum products content by 20% in 14 days. The greatest reduction of oil was observed in sludge with the lowest dry weight, that is with the greatest degree of hydration. The dry weight of the excess sludge did not significantly decrease during the course of the experiment, after having been inoculated with the mixture of strains.

Adaptation, Physiological↗

Mathematical analysis of solid-liquid mass transfer in a batch reactor for the enzymatic transformation of testosterone to 4AD.

A previous mathematical analysis of mass transfer in a two-phase (solid-liquid) batch reactor for enzymatic transformation of testosterone to 4AD (Pereira et al., 1987) is extended to incorporate the effect of convective mixing. The results of the analysis showed that for a given enzyme loading, the mass transfer resistance in the solid (a function of the bead size) and the intensity of convective mixing (as embodied in the mass transfer coefficient) are two parameters that can be varied such that the overall mass transfer rate from the solid to the liquid phase ensures optimal reactor performance.

17-Hydroxysteroid Dehydrogenases↗

Hydrodynamic characteristics of immobilized cell beads in a liquid-solid fluidized-bed bioreactor.

This study examined the hydrodynamic characteristics of a liquid-solid fluidized-bed bioreactor using elastic particles (PVA gel beads) of various diameters as carriers. The drag coefficient-Reynolds number, velocity-voidage, and expansion index-Reynolds number relationships observed during fluidization of PVA gel beads in a fluidized bed in our experiments were compared with the published results. Predictions made from previous correlations were examined with our new experimental findings, revealing the inadequacy of most of these correlations. Thus, new correlations describing the above-mentioned relationships are suggested. The drag coefficient of immobilized cell beads is larger than that of free cell ones at the same Reynolds number because the surface of the immobilized cell beads is rougher. For multiparticle systems, the correction factor, f(epsilon), is a function of the falling gel bead properties (Reynolds number) as well as the fluidized gel bead properties (Archimedes number), and depend strongly on the bed voidage (epsilon). A new simple relation was developed to predict easily the epsilon value from 0.5-0.9 at 4,986 < A(r) < 40,745 or 34 < Re(t) < 186. For all the immobilized cell beads used in this study, the prediction error of the bed voidage was less than 5% at epsilon > 0.5. The prediction equations in this study can be further applied to analyzing the hydrodynamic characteristics of a fluidized-bed reactor using similar entrapped elastic particles as carriers.

Acinetobacter baumannii↗

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli.

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.

Chromatography, Liquid↗