Osami Yagi

Purification and Properties of a Soluble Methane Monooxygenase from Methylocystis sp. M

A soluble methane monooxygenase (sMMO: EC 1.14.13.25) was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three components, I, II, and III. Components II and III were purified to homogeneity and were essential for the sMMO activity. Components II and III had molecular masses of approximately 233,000 and 39,000, respectively. Component II consisted of three subunits with molecular masses of 55,000, 44,000, and 21,000, which appeared to be present in stoichiometric amounts, suggesting a (αβγ)2 configuration in the native protein. Component II contained 1-4 mol of iron and was considered to be a hydroxylase. Component III was a flavoprotein, which contained 1 mol of FAD as well as 1-2mol of iron. It catalyzed the reduction of K3Fe(CN)6 and 2,6-dichloroindophenol by NADH. Component I, which was partially purified and not essential for sMMO activity, stimulated the activity by about 11-fold. Its stimulation could be replaced by addition of Fe(2+). The molecular mass of the partially purified component I was estimated to be from 35,000 to 40,000 based on gel filtration, which suggested the presence of a new type of regulatory protein of sMMO.

Survival and Impact of Genetically Engineered Pseudomonas putida Harboring Mercury Resistance Gene in Aquatic Microcosms

The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms. We used lake, river, and spring water samples. The density of genetically engineered and wild-type P. putida decreased rapidly within 5 days (population change rate k -0.87 approximately -1.00 day-1), then moderately after 5 to 28 days (-0.10 approximately -0.14 day-1). The population change rates of genetically engineered and wild-type P. putida were not significantly different. We studied the important factors affecting the survival of genetically engineered and wild-type P. putida introduced in aquatic microcosms. Visible light exerted an adverse effect on the survival of the two strains. The densities of genetically engineered and wild-type P. putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-micron membrane filter, or treated with cycloheximide to inhibit the growth of protozoa. These results suggested that protozoan predation was one of the most important factors for the survival of two strains. We examined the impact of the addition of genetically engineered and wild-type P. putida on indigenous bacteria and protozoa. Inoculation of genetically engineered or wild-type P. putida had no apparent effect on the density of indigenous bacteria. The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P. putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms.

Trichloroethylene degradation by immobilized resting-cells of Methylocystis sp. M in a gas-solid bioreactor

SummarySemicontinuous and continuous biodegradation of gaseous trichloroethylene (TCE) was achieved in a batch reactor and in a glass-column reactor, containing the alginate-immobilized cells of a methane-utilizing bacterium,Methylocystis sp. M. Since gaseous TCE was degraded in both reactors, it is suggested that atmospheric contamination by TCE can be prevented through the use of bioreactors.

Novel Metabolite of Trichloroethylene in a Methanotrophic Bacterium, Methylocystis sp. M, and Hypothetical Degradation Pathway

Previously, a new type II methanotrophic bacterium, Methylocystis sp. M (strain M), was isolated in our laboratory [H. Uchiyama et al., Agric. Bioi. Chem., 53, 2903-2907 (1989)]. In this paper, metabolites resulting from the degradation of trichloroethylene (TCE) by strain M were studied with gas chromatography-mass spectrometry. Trichloroacetic acid, dichloroacetic acid, and a small amount of 2,2,2-trichloroethanol were detected in the water-soluble fraction of the reaction mixture. These results suggest that the conversion of TCE to trichloroacetaldehyde via a CI-shift reaction, followed by the formation of trichloroacetic acid and 2,2,2-trichloroethanol, as well as a spontaneous breakdown of TCE oxide, with subsequent formation of dichloroacetic acid, etc., is involved in the TCE degradation pathway of the methanotrophic bacterium.

Immobilization of trichloroethylene-degrading bacterium, Methylocystis sp. strain M in different matrices

Abstract The trichloroethylene (TCE) degradation activities of a TCE-degrading methanotroph, Methylocystis sp. strain M, which was immobilized in different matrices have been investigated to identify a suitable matrix. Cells immobilized in Ca-alginate, κ-carrageenan, and agarose showed higher or almost the same degradation activity in comparison with that of free cells, while low activity was observed in the cells immobilized in photo-crosslinkable resin, polyurethane, and polyelectrolyte complex. In repeated use, only the agarose-immobilized cells were not damaged and retained about 40% of the initial TCE degradation activity among the cells immobilized in the three agents. The fundamental degradation characteristics of the immobilized cells were also investigated using agarose-immobilized cells. The maximum degradation rate ( V max ) of TCE was 3.1 μg of TCE/mg of dry cells·h, while the saturation constant ( K s ) was 56.5 μM. The optimum pH was found to be 7.0 and the optimum temperature 35°C.

Studies on the succession of blue‐green algae, Microcystis, Anabaena, Oscillatoria and Phormidium in Lake Kasumigaura

Abstract This study was conducted to examine and determine the factors affecting dominance of the blue‐green algal species of genus Microcystis, Anabaena, Oscillatoria and Phormidium from 1988 to 1990 in eutrophic Lake Kasumigaura. The algal dominancy of the three Anabaena species, A. affinis,A. flos‐aquae and A. spiroides, took place in early summer, while the dominance of the Microcystis species, M. aeruginosa, M. viridis and M. wesenbergii, took place from summer to fall. Water samples were collected at Tsuchiura Harbour in Lake Kasumigaura. The correlations between cell numbers of blue‐green algal species and water qualities were investigated. The growth rates of algae in situ were determined. Af. aeruginosa showed the highest increasing growth rate, while Af. viridis was the lowest decay rate in the Microcystis species. M. aeruginosa exhibited high single correlations between water temperature, pH, total chemical oxygen demand (T‐COD) and dissolved chemical oxygen demand (D‐COD). The cell numbers of ...

Development and application of a bioluminescence ATP assay method for rapid detection of coliform bacteria

Abstract The conventional bacterial quantitative and qualitative methods are complex and require a lot of skills and time. Therefore a rapid microorganism detection system (RMDS) method was developed. With this method, yeast cells can be detected without cultivation, while some other microorganisms can be often detected after only 4–5 h of cultivation. The food sample is placed on a special membrane filter and cultivated on a plate until a sufficient amount of ATP is obtained. The membrane filter containing the microorganisms is then sprayed with the extractive and luminescent reagents and luciferin-luciferase reacts with the living cells. By this method, only the bioluminescent living cells are detected as spots. Thus, the RMDS method can detect the number but not the type of bacteria. An attempt to develop a method for the identification of coliforms, which is an index bacteria in food hygiene inspection, was made. In order to inhibit the growth of microorganisms which are closely related to the Pseudomonas genus and which form red colonies similar to coliforms as well as the germination of fungi spores, they were cultivated under anaerobic conditions in a new selective medium. Typical microorganisms which are often detected in foods, including yeasts, fungi, Gram-positive bacteria, Gram-negative bacteria (containing coliforms) and bacterial spores, were used. The sample was cultivated in an anaerobic L-shaped test-tube using the new coliform selective medium and then detected by the RMDS method. The results show that under this condition, the growth of both Pseudomonas sp. and fungi are suppressed, and coliforms could be detected within 6–7 h. This method is more rapid than the conventional method and has great potential for practical hygiene testing of both food beverages and water.

Biodegradation of trichloroethylene by Methylocystis sp. strain M immobilized in gel beads in a fluidized-bed bioreactor

Abstract A fluidized-bed bioreactor (FBB) containing Methylocystis sp. strain M (strain M) isolated from Japanese soil was developed for treating synthetic groundwater containing trichloroethylene (TCE). Strain M bacteria were immobilized in 2% calcium alginate gel beads and introduced into the reactor, which was supplied with a methane/air gas mixture. TCE concentrations in the reactor were reduced from 0.9–1.6 to 0.1–0.2 mg/litre after a residence time of 2.56 h. Thus, 80–90% of the influent TCE was degraded in the reactor, and the resulting effluent gas contained only 0.02–0.04 mg/litre TCE. The ability of the gel-immobilized cells to degrade TCE declined rapidly during the TCE degradation process. To compensate for this decrease in activity, the reactor was operated for TCE degradation and reactivation of the immobilized strain M cells on alternate days. TCE degradation activity was completely recovered within 24 h after supplying a methane/air mixture (2:8 v/v) and a mineral salt solution to the bacteria. The degradation capacity of the reactor was kept in a steady state for 10 days in this way, but the rate of recovery of TCE degradation activity gradually decreased. TCE degradation activity fell from 6.0 litres/g dry weight (gdw) per hour on the sixth day to 1.4 litres/gdw per hour on the 24th day. Interestingly, there was no corresponding decrease in methane consumption by the bacteria

Seasonal and longitudinal changes in copper and iron in surface water of shallow eutrophic Lake Kasumigaura, Japan

Abstract The concentrations of copper and iron in surface water of the eutrophic Lake Kasumigaura, the second largest lake in Japan, have been monitored at three sites monthly from April 1989 until March 1994. The metals were analyzed by a graphite furnace AAS after filtration with a 0.45-μm membrane filter. The concentrations of copper in the water were in the range 10 −9 to 5 × 10 −8 M and showed clear seasonal changes, being higher in summer and lower in winter. The concentrations of copper did not decrease on passage through the lake. The values of dissolved chemical oxygen demand (COD Mn ) showed a similar behavior, an interaction of copper in the lake water with organic matter was estimated. The concentration of copper in the lake became sometimes higher than the value of EC 50 for Microcystis , however, the metal forms stable complex species with organic matter and the toxic effect may be reduced. The concentrations of iron, on the other hand, showed wide variation, but no obvious seasonal change. The concentrations of iron decreased very markedly during flow of water through the lake.

Trichloroethylene degradation by cells of a methane-utilizing bacterium, Methylocystis sp. M, immobilized in calcium alginate

When Methylocystis sp. M cells were immobilized in calcium alginate, the resulting cell beads showed optimum trichloroethylene (TCE) degradation activity at pH 7.0 and 35°C. In comparison with free cells, the immobilized cells were more stable at low pH, and to some extent, at higher temperatures. Studies on the kinetics and the influence of cell density suggest that oxygen permeation was a rate-limiting step. Investigation of the storage stability and the optimum concentration of dissolved oxygen revealed that the TCE degradability was greater under anaerobic than aerobic conditions. Although a toxic effect caused by TCE was observed, methane seemed to restore activity, suggesting that the development of a two-step reactor system might be advantageous. The finding that the immobilized cells showed TCE degradation activity in actual groundwater suggests that TCE bioremediation could be achieved through the use of bioreactors with such cells.