Haruo Mimura

Adsorption of tributyltin by tributyltin resistant marine Pseudoalteromonas sp. cells.

The isolate, Pesudoalteromonas sp. TBT1, could grow to overcome the toxicity of tributyltin chloride (TBTCl) up to 30 microM in the absence of Cl(-) in the medium until the cells reached an exponential phase of growth. The viability, however, was reduced after the cells reached a stationary phase. The degradation products, such as dibutyltin (DBT) and monobutyltin (MBT), were not detected in the growth medium, indicating that the isolate has no ability to degrade TBT into less toxic DBT and MBT. Up to about 10(7.5) TBT molecules were adsorbed by a single cell. The observation of morphological changes with an electron microscope showed that the cell surface became wrinkled after exposure to the lethal concentration of 10 mM TBTCl. These results indicate that the resistance of the isolate toward the toxicity of TBTCl is not related to the unique cell surface, which seems to play an important role in preventing the diffusion of TBTCl into the cytoplasm.

Accelerator analysis of tributyltin adsorbed onto the surface of a tributyltin resistant marine Pseudoalteromonas sp. cell.

Tributyltin (TBT) released into seawater from ship hulls is a stable marine pollutant and obviously remains in marine environments. We isolated a TBT resistant marine Pseudoalteromonas sp. TBT1 from sediment of a ship’s ballast water. The isolate (109.3 ± 0.2 colony-forming units mL−1) adsorbed TBT in proportion to the concentrations of TBTCl externally added up to 3 mM, where the number of TBT adsorbed by a single cell was estimated to be 108.2. The value was reduced to about one-fifth when the lysozyme-treated cells were used. The surface of ethanol treated cells became rough, but the capacity of TBT adsorption was the same as that for native cells. These results indicate that the function of the cell surface, rather than that structure, plays an important role to the adsorption of TBT. The adsorption state of TBT seems to be multi-layer when the number of more than 106.8 TBT molecules is adsorbed by a single cell.

Effect of hyper-salt stress on the heat resistance of a halotolerant Brevibacterium sp. JCM6894

Abstract The heat resistance of Brevibacterium sp. JCM6894 was examined as a function of externally added NaCl concentrations. About a 5-log cycle reduction of the viable cell numbers was observed to result from heat treatment for 30 min at 47°C in the absence of NaCl. When the cells were heated in the buffer containing 2 M NaCl, the viability was maintained within less than 1-log cycle reduction after incubation for 30 min at 56°C. During the heat treatment for 30 min at 47°C in the presence of 2 M NaCl, Na + and K + ions in the cells increased and decreased by 13 and 26 μg ions per mg of cell protein, respectively. Under this condition, the amount of free amino acids in the cells changed little except for glutamate and hydroxyproline, which were reduced by 72 and 43 nmol per mg cell protein, respectively. These results indicate that the salt stress itself and Na + ions existing in the cytoplasm are more important factors than in vivo protein synthesis for preventing the thermal death of the resting cells of this strain.

Study on application of shock waves generated by micro bubbles to the treatment of ships’ ballast water

This paper reports the fundamental study for development of a ships’ ballast water clarifying treatment technology using shock pressures generated by the collapse of micro bubbles. From the microscopic observation, it was confirmed that micro bubbles less than 65 μm in diameter became small in size and disappeared, and the shock wave generation by their collapse was observed by optical visualization method and pressure sheets. The bio-experiments using a marine Vibrio sp. were carried out under the three conditions: (1) only use of micro bubbles, (2) only use of 20 kHz ultrasonic waves, and (3) simultaneous use of them. The number of survival cells was investigated under the respective conditions for 12 hours. The inactivation effect on the marine Vibrio sp. by simultaneous use of micro bubbles and ultrasonic waves indicated better inactivation than that by the only use of micro bubbles or ultrasonic waves.

Sterilization of Ships’ Ballast Water

In this chapter, applied research on microbubble dynamics induced by shock waves is presented for the sterilization treatment of marine bacteria contained in ships’ ballast water. Destruction of the marine ecosystem caused by sea creatures carried in ships’ ballast water to different seas is a serious global issue in the field of maritime sciences. Therefore, we propose a new sterilization technique using shock pressure and microbubbles to develop more secure and environmentally friendly treatment methods for ships’ ballast water. We present our research and the problems that need to be solved using prospective approaches.

Experimental Study on Inactivation of Marine Bacteria Using Electrodischarge Shock Waves

The trans-boundary movement of the microorganism included in the ship ballast water causes the destruction of an marine ecosystem globally. The restrictions concerning the ballast water managements provided by International Maritime Organization (IMO) probably take effect for all new ships in the near future, so that enterprises and research institutions in the world have developed many kinds of ballast water treatment systems to obtain the IMO approval. In most of all systems, chemical treatment is used to kill marine bacteria such as cholera and colon bacillus. For marine environment convention, safe and economic and eco-friendly ballast water treatment systems are desired.

Microbial degradation of mussels removed from the surface of marine structures

After being crushed, a net weight 218 kg of mussels was reduced to 120.5 kg by discharge of seawater from the shells. The sample containing some of broken shells was applied to the degradation by Bacillus sp. The initial weight of 120.5 kg was reduced to 82.5 kg after 76 h of microbial degradation. Concentrations of the produced carbon dioxide and ammonia showed peak values of 15,000 and 720 ppm after 12.5 and 58 h, respectively. At 12.5 h, temperature in the sample reached to 65 degrees Celsius by the fermentation temperature. The ratio of total amounts of carbon and nitrogen, C/N ratio, was 22.6 after 76 h. Some of the heavy metals and toxic chemical compounds, which cause environmental pollution, were analyzed in the sample for the recycle of compost. The concentrations of such compounds detected were much lower than those recommended by the Japanese Ministry of the Environment. Therefore, the recycling of compost seems to be legal and possible. The strain used in this experiment was identified as Bacillus subtilis subsp. subtilis based on the biochemical and physiological properties as well as the homology analysis for the partial sequences of 16S rDNA.

Prevention effect of LED blue light irradiation on the settlement of cyprids

We applied blue LEDs to the prevention of the settlement of cyprids. The relatively strong intensity of the irradiation of ca. 500 (W m−2) could prevent the settlement of cyprids on the area of the bottom of a tub, above which the light source was adjusted. On the other hand, cyprids settled on the area on which the relatively weak intensity of the irradiation, < 100 (W m−2), was reached. In the field experiments, however, the number of cyprids settled on the adhesion plate, on which the relatively weak irradiation of blue light was reached from the relatively strong light source located 40 cm far from the adhesion plate, was reduced. Moreover, the surface of the transparent acrylic plate used for the LEDs cover was prevented from biofouling in the field. These results indicate that the relatively strong intensity of irradiation of blue light is actually useful for the prevention of biofouling on a ships bottom.

Environmental Impact of Tributyltin-Resistant Marine Bacteria in the Indigenous Microbial Population of Tributyltin-Polluted Surface Sediments

　We compared the TBT-resistant ability of resting cells prepared from isolates that formed colonies on nutrient agar plates containing 100 µM tributyltin (TBT) chloride, such as Photobacterium sp. TKY1, Halomonas sp. TKY2, and Photobacterium sp. NGY1, with those from taxonomically similar type strains. Photobacterium sp. TKY1 showed the highest ability among those three isolates. The number of surviving Photobacterium sp. TKY1 cells was hardly decreased after 1 h of exposure to 100 µM TBTCl, regardless of the number of resting cells in the range from 109.4 to 104.2 CFU mL-1. In such an experimental condition, the maximum number of TBT molecules available to associate with a single cell was estimated to be approximately 6.0 x 1011.8. Resting cells prepared from type strains Photobacterium ganghwense JCM 12487T and P. halotolerans LMG 22194T, which have 16S rDNA sequences highly homologous with those of Photobacterium sp. TKY1, showed sensitivity to TBT, indicating that TBT-resistant marine bacterial species are not closely related in spite of their taxonomic similarity. We also estimated the impact of TBT-resistant bacterial species to indigenous microbial populations of TBT-polluted surface sediments. The number of surviving TBT-sensitive Vibrio natriegens ATCC 14048T cells, 106.2±0.3 CFU mL-1, was reduced to 104.4±0.4 CFU mL-1 when TBT-resistant Photobacterium sp. TKY1 cells, 109.1±0.2 CFU mL-1, coexisted with 109.4±0.2 CFU mL-1 of V. natriegens ATCC 14048T cells in the presence of 100 µM TBTCl. These results indicate that the toxicity of TBT to TBT-sensitive marine bacterial populations might be enhanced when a TBT-resistant marine bacterial species inhabits TBT-polluted surface sediments.

Study of mechanical and chemical effects induced by shock waves on the inactivation of a marine bacterium

The present paper reports a study on the shock sterilization technique of ships’ ballast water. In order to expose cell suspension to strong shock waves, a gas gun experiment was carried out. In this experiment, shock waves in the suspension of a marine Vibrio sp. were generated by collision between an aluminum suspension container and an impactor plate accelerated by the gas gun. The changes of shock pressure in suspension were measured by piezofilm gauges and compared them to computational results. The visualization of shock waves in water was carried out by shadowgraph method, and the marine Vibrio sp. was observed with an electron microscope. In addition, the affect of free radicals was also investigated using VitC-Na 2%. It was found that both the dynamic action of shock pressure and the chemical reaction in suspension induced by shock waves were closely related to inactivation of the marine Vibrio sp. at an impact velocity lower than about 200 m/s.