Modification of water-soluble coal-derived products by dibenzothiophene-degrading microorganisms.

To study mechanisms by which microorganisms oxidize thiophenic sulfur in coal, we tested bacterial cultures for the ability to degrade dibenzothiophene (DBT), DBT-5-oxide, and DBT-sulfone and to modify water-soluble coal products derived from Illinois no. 6 and Ugljevik coals. In yeast extract medium, the majority of selected isolates degraded DBT and accumulated DBT-5-oxide in culture fluids; all but one of the cultures degraded DBT-5-oxide, and none of them degraded DBT-sulfone. Elemental analysis data indicated that the microbial cultures were able to decrease the amount of sulfur in soluble coal products derived from Illinois no. 6 and Ugljevik coals. However, these data suggested that microbially mediated sulfur removal from soluble Ugljevik coal occurred by nonspecific mechanisms. That is, extensive degradation of the carbon structure was concurrent with the loss of sulfur. This conclusion was supported by X-ray photoelectron spectroscopic data which indicated that the reduced sulfur forms in the soluble Ugljevik coal product was not oxidized by microbial treatment.

Protein and elemental analysis of contact lenses of patients with superior limbic keratoconjunctivitis or giant papillary conjunctivitis.

Soft contact lenses worn by six patients (12 eyes) diagnosed as having contact lens-induced superior limbic keratoconjunctivitis (SLK) and from four patients (5 eyes) with giant papillary conjunctivitis (GPC) were analyzed for protein concentration and for elemental content. Fifty-five percent water content, ionic lenses containing methacrylic acid had high protein concentration. Calcium was not a common element found on protein-coated lenses. Sulfur and iron were found on used and new lenses. An elevated level of mercury was detected in one gray lens that had been disinfected in thimerosal-preserved saline with a high heat disinfection unit by a patient with SLK.

Isolation and characterization of the facultative methylotroph Mycobacterium ID-Y.

A facultatively methylotrophic Mycobacterium was isolated from Cleveland Harbor, Ohio, USA. The isolate, designated ID-Y, used a wide range of carbon and energy sources including methane and several other hydrocarbons. It displayed a growth cycle from rod-shaped exponential-phase cells, with many cell pairs exhibiting V-formation, to cocco-bacillary stationary-phase cells. A fixation technique involving glutaraldehyde/alcian blue resulted in the observation of a three-layered cell wall. Isolate ID-Y has an ultrastructure similar to that of other mycobacteria, particularly Mycobacterium phlei and Mycobacterium flavum, which is presently classified as a Xanthobacter species.

Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. I. Preliminary experiments in controlled shaken flasks.

Changes of pH and sulfate concentration in high-sulfur coal refuse slurries are used as measurements of microbial pyrite oxidation in the laboratory. Sodium lauryl sulfate (SLS), alkylbenzene sulfonate (ABS), benzoic acid (BZ) and combinations of SLS plus BZ and ABS plus BZ effectively inhibited formation of sulfate and acid when added in concentrations greater than 50 mg/L to inoculated 20 or 30% coal refuse slurries. Here 25 mg/L concentrations of SLS, ABS, and ABS + BZ stimulated acid production. Formic, hexanoic, oxalic, propionic, and pyruvic acids at 0.1% concentrations were also effective inhibitors. Four different lignin sulfonates were only slightly effective inhibitors at 0.1% concentrations. It was concluded that acid formation resulting from microbial oxidation in high-sulfur coal refuse can be inhibited.

Prevention of formation of acid drainage from high-sulfur coal refuse by inhibition of iron- and sulfur-oxidizing microorganisms. II. Inhibition in "run of mine" refuse under simulated field conditions.

The combination of sodium lauryl sulfate and benzoic acid effectively inhibits iron- and sulfur-oxidizing bacteria in coal refuse and prevents the conversion of iron pyrite to sulfate, ferric iron, and sulfuric acid, thereby significantly reducing the formation of acidic drainage from coal refuse. The inhibitors were effective in a concentration of 1.1 mg/kg refuse, and data indicate that the SLS was in excess of the concentration required. The treatment was compatible with the use of lime for neutralization of acid present prior to inhibition of its formation.

Bacteria and acidic drainage from coal refuse: inhibition by sodium lauryl sulfate and sodium benzoate.

The application of an aqueous solution of sodium lauryl sulfate and sodium benzoate to the surface of high-sulfur coal refuse resulted in the inhibition of iron-and sulfur-oxidizing chemoautotrophic bacteria and in the decrease of acidic drainage from the refuse, suggesting that acid drainage can be abated in the field by inhibiting iron- and sulfur-oxidizing bacteria.

Exospore formation in Methylosinus trichosporium.

Formation of exospores in Methylosinus trichosporium was examined by electron microscopy; serial sectioning was used to visualize the shape and location of the developing exospore in relation to the vegetative cell. The initial stage was the formation of a budlike enlargement on one end of the vegetative cell. The enlargement was surrounded by the exospore capsule, and the cell wall was continuous around both the cell and the developing exospore. A constriction occurred in the area where the budlike structure was attached to the vegetative cell, and the constriction continued to form until the immature exospore was detached from the vegetative cell. The cup-shaped immature exospore was surrounded by the exospore capsule, which appeared to hold the exospore close to the vegetative cell. After separation from the vegetative cell, the immature exospore developed further by forming the exospore wall and by becoming spherical.

Plasmid DNA in acidophilic, chemolithotrophic thiobacilli.

Plasmid patterns were determined in 15 strains of iron-oxidizing Thiobacillus ferrooxidans. In four of these strains plasmid DNA was not detected. In the other strains the molecular weights of plasmids ranged from 5 x 10(6) to 50 x 10(6) and each strain had a different plasmid composition. The change of growth substrate from ferrous iron to tetrathionate did not affect the plasmid pattern in T. ferrooxidans nor did it in T. acidophilus, which was adapted from glucose to grow on tetrathionate.

Adenosine 5'-triphosphate formation in Thiobacillus ferrooxidans vesicles by H+ ion gradients comparable to those of environmental conditions.

Vesicles prepared from iron-grown Thiobacillus ferrooxidans, and subsequently loaded with adenosine 5'-diphosphate and inorganic phosphate, produced adenosine 5'-triphosphate when subjected to H+ gradients comparable to those in the cells' normal environment (i.e., an internal pH in the range of 6.0 to 8.0 with an optimum of 7.0 to 7.8 and an external pH in the range of 2.1 to 4.1 with an optimum of 2.8). Nigericin, dicyclohexylcarbodiimide, and pentachlorophenol decreased adenosine 5'-triphosphate synthesis. Valinomycin at concentrations of 2.5 and 5.0 micrograms/ml increased adenosine 5'-triphosphate formation by 25 and 30%, respectively.

Structure of Methylosinus trichosporium exospores.

Methylosinus trichosporium exospores did not display a well-defined cortex or an exosporium. A thick, electron-dense exospore wall was characteristic of the exospores. Located on the exterior of the exospore wall was a cell wall to which a well-defined capsule was attached. An extensive lamellar intracytoplasmic membrane system characteristic of the kind in vegetative cells of this bacterium was present along the interior periphery of the exospore wall. Upon germination of M. trichosporium exospores, the thick exospore wall gradually disappeared and a germ tube formed. The intracytoplasmic membranes of the exospores extended into the germ tube which did not possess the extensive fibrillar capsule observed on the dormant exospore. Cup-shaped exospores which have an ultrastructure similar to that of mature exospores except that they are invaginated also germinated upon exposure to methane.

Study of Developmental Stages of Methylosinus trichosporium with the Aid of Fluorescent-Antibody Staining Techniques.

When stained by using an indirect fluorescent-antibody technique, Methylosinus trichosporium displayed an uneven fluorescence. Exospores and the polar tips of some vegetative cells displayed a more intense fluorescence than the other cells. Cross-absorption of the specific anti-M. trichosporium immunoglobulin G with exospores resulted in no fluorescence of exospores or exospore regions of sporulating vegetative cells. This demonstrated that antigens were present on exospores and exospore regions of vegetative cells that are different from vegetative cell antigens. Taking advantage of this phenomenon, three fluorescentantibody staining techniques were developed which were used to study the life cycle of M. trichosporium.

Distribution of Methylomonas methanica and Methylosinus trichosporium in Cleveland Harbor as Determined by an Indirect Fluorescent Antibody-Membrane Filter Technique.

An indirect fluorescent antibody-membrane filter staining technique, which permitted the autecological study of Methylomonas methanica and Methylosinus trichosporium, was developed. This technique was used to assay the numbers of these two organisms in Cleveland Harbor. The concentrations of M. methanica and M. trichosporium were found to be inversely proportional to the sampling depth, with the highest cell counts observed in the sediments. M. methanica was observed at every sampling station, whereas M. trichosporium was found at only two of the stations.

Tentative identification of methanogenic bacteria by fluorescence microscopy.

Methanogenic bacteria, which are presently identified on the basis of cell morphology and substrate conversion to CH(4), can be differentiated from nonmethanogens and identified in pure or mixed culture on the basis of their autofluorescence under ultraviolet illumination.

Leakage of cellular material from Thiobacillus ferrooxidans in the presence of organic acids.

Thiobacillus ferroodixans cells released varying amounts of iron, phosphate, sugar, ribonucleic acid, deoxyribonucleic acid, and substances that absorbed light at both 260 and 280 nm, when exposed to 10(-2) to 10(-1) M concentrations of these organic acids: propionic, butyric, valeric, hexanoic, and oxalacetic. These acids also retarded iron oxidation by the cells. Electron microscope observation of cells after exposure to the organic acids showed varying degrees of cell envelope disruption, suggesting that the mode of inhibition of autotrophic iron oxidation in the cell involves interference with the function of the cell envelope, possibly the cell membrane.

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.

Inhibition of growth, iron, and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.

Iron and sulfur oxidation by Thiobacillus ferrooxidans as well as growth on ferrous iron were inhibited by a variety of low molecular weight organic compounds. The influences of chemical structure of the organic inhibitors, pH, temperature, physical treatment of cells, and added inhibitory or stimulatory inorganic ions and iron oxidation suggest that a major factor contributing to the inhibitory effects on iron oxidation is the relative electronegativity of the organic molecule. The data also suggest that inhibitory organic compounds may (i) directly affect the iron-oxidizing enzyme system, (ii) react abiologically with ferrous iron outside the cell, (iii) interfere with the roles of phosphate and sulfate in iron oxidation, and (iv) nonselectively disrupt the cell envelope or membrane.

Whole-cell and membrane lipids of the methylotrophic bacterium Methylosinus trichosporium.

The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed.

Methyltrophic enzyme distribution in Methylosinus trichosporium.

Key enzymes involved in the oxidation and fixation of methane by Methylosinus trichosporium were examined for localization within the bacterial cells. A differential centrifugation scheme following cell disruption was used to provide membrane and soluble fractions for the enzyme assays. All the methylotrophic enzymes examined were found to be soluble with this fractionation scheme. Electron transport involving a cytochrome c2 with absorption peaks at 416, 522, and 550 nm and oxidative phosphorylation were found in the membrane fractions. Mixed soluble and membrane fractions coupled the oxidation of emthanol and formate with cytochrome reduction.