Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species.

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).

Isolation and characterization of sulphur-oxidizing Thiomonas sp. and its potential application in biological deodorization.

AIMS: To isolate and characterize a sulphur-oxidizing bacterial strain from activated sludge and to evaluate its potential application in biological deodorization. METHODS AND RESULTS: A dominant sulphur-oxidizing bacterial strain, designated as strain SS, was isolated from an enrichment culture using thiosulphate as a sole energy source and CO2 as a sole carbon source. The cells of this organism were aerobic, rod-shaped, Gram-negative and motile. Strain SS could grow autotrophically, heterotrophically as well as mixotrophically. Autotrophic growth was observed at pH values ranging from 2.3 to 9.0. Phylogenetic analyses revealed that strain SS belonged to Group 1 of the genus Thiomonas, closely related to Thiomonas perometabolis and Thiomonas intermedia. The thiosulphate oxidation rates of strain SS at different pH values were evaluated in terms of oxygen uptake using a Micro-Oxymax respirometer. The results showed that the maximum oxidation rate of 5.65 mg l(-1) h(-1) occurred at 56 h of growth and pH 6.0. Continuous H2S removal study demonstrated that strain SS could remove more than 99% of H2S when the inlet concentration was below 58.6 ppm. Further increase of the inlet concentration to 118 ppm gave rise to a decline in the removal efficiency to ca 90%. CONCLUSIONS: The strong acidification of the culture medium during the later period could result in the deterioration of the growth activity and the metabolism activity of strain SS. In practical application, the problems caused by the end-product inhibition and the acidification can be alleviated by periodical replacement of culture medium with fresh medium. Given the physiological flexibility and the ability to remove H2S rapidly and efficiently, strain SS could be a good 'deodorizing' candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that Thiomonas species has been reported for biological deodorization application.

Removal of odorous sulphur-containing gases by a new isolate from activated sludge.

Biological treatment of odorous sulphur-containing compounds is attracting attention due to its benign eco-friendliness, energy-savings and low operating costs. As the biological treatment efficiency of dimethylsulphide (DMS) reported was often low and variable, selection of useful DMS-degrading microorganisms is of importance for the enhancement of the biological deodorizing process. This paper reports the successful isolation of a DMS-degrading bacterium from activated sludge, using the enrichment isolation technique. The isolate was identified by 16S rRNA gene sequencing, and found to belong to the alpha group of Proteobacteria, with an identity of 99.4% and 99.1% to the 16S rRNA gene sequences of Afipia felis and Pseudomonas carboxydohydrogena, respectively. The isolate was able to metabolize DMS as well as hydrogen sulphide (H2S). A batch experiment was performed to assess the removal characteristics of DMS by the isolate. The results showed that over half of DMS could be removed by the isolate in 3 hours when the initial DMS amount was approximately 10 micromol and 25 micromol. Removal of H2S by the isolate was evaluated by a continuous test in a 2-L gas-bubbling bottle. Although part of the H2S removal by the mineral medium itself was observed in the control test, the majority of H2S removal was believed to be attributed to the metabolic activity of the isolate. In conclusion, the isolate might be potentially useful for the enhancement of the biological deodorizing processes.

Batch experiment on H2S degradation by bacteria immobilised on activated carbons.

Biological treatments of odorous compounds, as compared to chemical or physical technologies, are in general ecologically and environmentally favourable. However, there are some inefficiencies relative to the media used in biofiltration processes, such as the need for an adequate residence time; the limited lifetime, and pore blockage of media, which at present render the technology economically non-viable. The aim of the study is to develop novel active media to be used in performance-enhanced biofiltration processes, by achieving an optimum balance and combination of the media adsorption capacity with the biodegradation of H2S through the bacteria immobilised on the media. An enrichment culture was obtained from activated sludges in order to metabolise thiosulphate. Batch-wise experiments were conducted to optimise the bacteria immobilisation on activated carbon, so as to develop a novel "biocarbon". Biofilm was mostly developed through culturing the bacteria with the presence of carbons in mineral media. SEM and BET tests of the carbon along with the culturing process were used to identify, respectively, the biofilm development and biocarbon porosity. Breakthrough tests evaluated the biocarbon performance with varying gas resistance time, inlet H2S concentration, and type of support materials. Fundamental issues were discussed, including type of support material, mode of bacteria immobilisation, pore blockages, and biodegradation kinetics, etc. This batch-wise study provides a basis for our future research on optimisation of the biofiltration process using a bio-trickling reactor.

Use of activated carbon as a support medium for H2S biofiltration and effect of bacterial immobilization on available pore surface.

The use of support media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and a shelter that protects the microorganisms from inhibitory compounds. In this study, activated carbon is used as a support medium for the immobilization of microorganisms enriched from municipal sewage activated sludge to remove gas-phase hydrogen sulfide (H2S), a major odorous component of waste gas from sewage treatment plants. A series of designed experiments is used to examine the effect on bacteria-immobilized activated carbon (termed "biocarbon") due to physical adsorption, chemical reaction, and microbial degradation in the overall removal of H2S. H2S breakthrough tests are conducted with various samples, including microbe-immobilized carbon and Teflon discs, salts-medium-washed carbon, and ultra-pure water-washed carbon. The results show a higher removal capacity for the microbe-immobilized activated carbon compared with the activated carbon control in a batch biofilter column. The increase in removal capacity is attributed to the role played by the immobilized microorganisms in metabolizing adsorbed sulfur and sulfur compounds on the biocarbon, hence releasing the adsorption sites for further H2S uptake. The advantage for activated carbon serving as the support medium is to adsorb a high initial concentration of substrate and progressively release this for microbial degradation, hence acting as a buffer for the microorganisms. Results obtained from surface area and pore size distribution analyses of the biocarbon show a correlation between the available surface area and pore volume with the extent of microbial immobilization and H2S uptake. The depletion of surface area and pore volume is seen as one of the factors which cause the onset of column breakthrough. Microbial growth retardation is due to the accumulation of metabolic products (i.e., sulfuric acid); and a lack of water and nutrient salts in the batch biofilter are other possible causes of column breakthrough.

Inflight cabin ozone aboard long duration C-5 airlift missions: a historical issue revisited.

BACKGROUND: Past research has shown that U.S. Air Force C-5 aircrews have been occasionally exposed to ambient, stratospheric ozone concentrations in excess of Federal Aviation Administration (FAA) ceiling and time-weighted average (TWA) standards. Although strategies such as steering around meteorological areas of high ozone concentrations or outfitting the C-5 with ventilation system modifications were recommended by past investigators, review has shown that such changes were not adopted. Informal interviews with current C-5 aircrews indicate mucosal dryness and irritation are routinely encountered during long duration flights. Such symptoms could be due to low humidity or perhaps, ozone-induced irritation. With the advent of miniaturized, highly accurate, electronic air quality monitoring equipment, a repeat study of cabin ozone was believed to be warranted to better clarify exposure risk and to look for other possible explanations for the encountered symptoms. METHODS: A portable, electronic air quality monitor was used to monitor breathing zone cabin air during 47 h of flight. Ozone, carbon dioxide, carbon monoxide, temperature, and relative humidity were recorded for the duration. Ozone levels were corrected for altitude using a calibration curve derived by earlier, unpublished hypobaric chamber work. RESULTS: Ozone levels were not found to exceed the FAA ceiling limit. Two flights had 3-h time-weighted average values that minimally exceeded FAA standards. Carbon dioxide and carbon monoxide levels were well below health effect threshold. Relative humidity reached a nadir of 3% and could well have explained symptoms of mucosal irritation. CONCLUSIONS: Current evidence suggests that C-5 aircrews are exposed to relatively low concentrations of ozone. Review of the literature regarding observed short-term and long-term toxicity to low dose ozone exposure would indicate that no further operational or aircraft design action is required.

Reduction of Selenate and Selenite to Elemental Selenium by a Pseudomonas stutzeri Isolate.

A Pseudomonas stutzeri isolate rapidly reduced both selenite and selenate ions to elemental selenium at initial concentrations of both anions of up to 48.1 mM. Optimal selenium reduction occurred under aerobic conditions between pH 7.0 and 9.0 and at temperatures of 25 to 35 degrees C. Reduction of both selenite and selenate was unaffected by a number of anions except for sulfite, chromate, and tungstate ions, which inhibited both growth and reduction.

Rhizobacteria of cotton and their repression of seedling disease pathogens.

During the 1983 field season, the rhizobacteria (including organisms from rhizosphere soil and the root rhizoplane) of cotton plants at one location in Mississippi were inventoried at different plant growth stages. Isolates (1,000) were identified to the genus level and characterized for repression of Pythium ultimum and Rhizoctonia solani. Cotton seedlings were initially colonized by bacteria of many different genera, and populations quickly reached 10 CFU/g of root tissue. As the season progressed, the bacterial populations declined as root mass increased and the roots became more woodlike in consistency. Fluorescent pseudomonads were the most numerous gram-negative rhizobacterial isolates of those that were randomly collected and identified, and they provided the largest number of isolates with fungal repressive activity. Several other gram-negative bacterial genera were recovered throughout the growing season, and some gram-positive bacteria were also isolated routinely, but at lower numbers. There was no correlation between the proportion of rhizobacterial isolates that possessed fungal repressive activity and the plant growth stage from which the isolates were obtained. Approximately twice as many bacterial isolates demonstrated fungal repression in the agar assay compared with the inplanta assay, and isolates were found more frequently with fungal repressive activity against P. ultimum than against R. solai.

A selective medium for enumeration and recovery of Pseudomonas cepacia biotypes from soil.

TB-T medium provides a high degree of selectivity for and detection of Pseudomonas cepacia biotypes upon initial plating from soil. TB-T medium consists of a basal medium with glucose as the sole carbon source and asparagine as the sole nitrogen source. The selectivity of TB-T medium is based on the combination of trypan blue (TB) and tetracycline (T) (pH 5.5). On TB-T medium, 216 of 300 isolates (72%) from five different soil types were identified as P. cepacia. The remaining 28% were facultative organisms that could be separated readily from P. cepacia by anaerobic glucose fermentation and by their inability to grow at 41 degrees C. Molds were controlled on low soil dilutions by adding crystal violet, nystatin, or both. Elimination of either ingredient or elevation of the pH to 7.5 resulted in a pronounced loss of selectivity. The efficiency of recovery varied considerably among P. cepacia strains but was high enough for some strains (76 to 86%) to permit quantitative studies. TB-T medium combines a defined formulation with high selectivity and allows recovery of P. cepacia biotypes from low soil dilutions (10(1) to 10(3)).

Histochemical and biochemical urease localization in the periplasm and outer membrane of two Proteus mirabilis strains.

Proteus mirabilis, a gram-negative bacillus, is often implicated in the formation of infectious kidney stones. As ureolytic activity of this organism is thought to play a major role in its pathogenesis, we adapted our recently described urease localization technique to visualize urease activity in vivo. Urease activity was ultrastructurally localized in two clinically isolated P. mirabilis strains by precipitating the enzymatic reaction product (ammonia) with sodium tetraphenylboron. Subsequent silver staining of the cells revealed urease activity to be predominantly associated with the periplasm and outer membranes of each strain. Biochemical measurements of urease activity in P. mirabilis cell fractions correlated well with histochemical observations in that the majority of urease activity was associated with the periplasm. Membrane-bound urease activity of these strains was associated mainly with the peptidoglycan in the detergent-insoluble (outer membrane) fraction.

Cytochemical Localization of Urease in a Rumen Staphylococcus sp. by Electron Microscopy.

We describe a technique whereby intracellular urease activity can be localized by transmission electron microscopy. The ammonia produced from the enzymatic hydrolysis of urea is first precipitated with sodium tetraphenylboron and then replaced with silver to produce electron-dense silver tetraphenylboron. This direct reaction product deposition procedure was used to demonstrate the presence of membrane-bound urease of Staphylococcus sp. H3-22, a gram-positive ruminal bacterium.

New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats.

New media (S1 and S2) were formulated that provide a high degree of selectivity and detection of fluorescent pseudomonads on initial plating. The selectivity of the S-type media was based on a detergent, sodium lauroyl sarcosine, and an antibiotic, trimethoprim. A total of five soils from different geographical locations and one sewage sludge sample were examined. On S1 medium, isolates from two soils with low fluorescent pseudomonad populations exhibited a high frequency of arginine dihydrolase (78%) and oxidase-positive (95%) phenotypes, but no fermentative isolates were recovered. Medium S2 was more defined and selective than S1, but lower numbers of fluorescent pseudomonads were recovered on S2. In soils in which fluorescent pseudomonads represent a small proportion of the total population, S1 medium consistently recovered high percentages of fluorescent phenotypes (82.5%).

Effect of bacteria and amoebae on rhizosphere phosphatase activity.

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.