Nitrite oxidation is a key step in nitrogen removal in biological wastewater treatment plants. Recently, two phylogenetically different Nitrospira (sublineages I and II) have been recognized as the numerically dominant nitrite-oxidizing bacteria in wastewater treatment plants. However, Nitrospira sublineage II inhabiting activated sludge was not isolated and its detailed properties were unclear. In this study, we developed a new method for the isolation of Nitrospira forming micro-colonies using a cell sorter. We obtained a novel pure strain “Nitrospira japonica” from the activated sludge. Subsequently, phylogenetic and physiological analyses revealed that Nitrospira japonica belongs to sublineage II and grew in medium containing formate. This method has the potential to isolate other uncultured microorganisms forming micro-colonies.

This study evaluates the community structure in nitrifying granules (average diameter of 1600 mum) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 mum below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 mum, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH(4)(+), NO(2)(-), NO(3)(-) and O(2) concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria-substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.

A hollow-fiber membrane chamber (HFMC) was developed as an in situ cultivation device for environmental microorganisms. The HFMC system consists of 48 to 96 pieces of porous hollow-fiber membrane connected with injectors. The system allows rapid exchange of chemical compounds, thereby simulating a natural environment. Comparative analysis through the cultivation of three types of environmental samples was performed using this newly designed device and a conventional agar-based petri dish. The results show that the ratios of novel phylotypes in isolates, species-level diversities, and cultivabilities in HFMC-based cultivation are higher than those in an agar-based petri dish for all three samples, suggesting that the new in situ cultivation device is effective for cultivation of various environmental microorganisms.

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression, and friction force, in the fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, we developed the Tilting Embryo Culture System (TECS). The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P<0.05). Although not significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared to static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low quality embryos and those embryos cultured under suboptimal conditions (P<0.05). Here, we propose that the TECS could be a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to in the fallopian tube.