【 Project 】
task02N₂O Recycling
01 Establishment of N₂O detoxification with root nodule symbiosis
Root nodule symbiosis between rhizobia and legumes can fix atmospheric nitrogen, but it is also known to release N₂O during the late growth stages of legumes. In this project, we will establish symbiosis systems in which N₂O-reducing rhizobia preferentially infect the host leguminous plants, by (i) searching for rhizobia with higher activities of N₂O reduction and establishing new technology to improve their N₂O-reducing activities, and (ii) optimizing host plants that preferentially accommodate N₂O-reducing rhizobia.

Ⅱ-1-a: N₂O reduction by strains with higher N₂O reducing activity.
N₂O is generated from agricultural land due to nitrogen fertilization and crop residues, causing global warming. However, N₂O generated from agricultural land can be reduced by inoculating microorganisms such as rhizobia that are able to reduce N₂O. Therefore, we will search for rhizobia with higher N₂O-reducing ability and investigate their behavior in soil.
1) Search for Bradyrhizobium root nodule bacteria with higher N₂O reducing activity.
We are exploring bacterial strains possessing N₂O reductase gene (nos) and high N₂O reducing activity in various soil ecosystems (nodules, soil, rhizosphere, roots).
2) Behavior of N₂O reducing rhizobia in soil.
Little is known about the behavior of rhizobial survivability in soil when inoculated. Therefore, we are analyzing the dynamics of rhizobia in soil microcosm experiments, and clarify the environmental factors and microbial factors that affect the survivability of inoculated microorganisms. In addition, we will assess the impact of microbial inoculation on soil biodiversity.


Ⅱ-1-b: Optimizing of symbiotic interactions for successful rhizobial inoculation.
Since a wide variety of rhizobia exist in the soil, even if useful N₂O-reducing rhizobia are inoculated, about 80% of the nodules formed on soybean root are occupied by indigenous rhizobia that do not have N₂O-reducing activity. To maximize the N₂O reducing ability of N₂O-reducing bacteria, it is necessary to develop technology to improve the infection prevalence of N₂O-reducing bacteria.
Infectivity of rhizobia is based on "compatibility" between rhizobia and host plants. It is controlled by "effector proteins" that rhizobia inject into host plant cell during infection, and "incompatibility genes" of host plants that recognize specific effectors. Therefore, our group has been studying
1) Breeding soybean cultivars with high compatibility for N₂O-reducing bacteria by searching for and integrating various incompatibility genes of soybean.
2) Selection of strains of N₂O-reducing bacteria with high compatibility to the soybean cultivars described in 1) based on the mechanism of incompatibility system.
The goal of this project is to establish a soybean/rhizobia symbiosis system in which N₂O-reducing bacteria preferentially coexist and express high N₂O-reducing activity.
02 Detoxification and recycling of N₂O in upland and paddy soil
To establish the N₂O detoxifying/recycling/absorbing upland and paddy field technology, (i) isolation and characterization of N₂O detoxifying/recycling microorganisms and (ii) functional analysis of N₂O-transforming soil microbial community will be performed.

Ⅱ-2: Detoxification of N₂O by rhizospheric/endohytic microorganisms
Since the Industrial Revolution, atmospheric CO₂ level has raised to 410 ppm, which has induced approximately 1.09 ℃ increasing in the global surface temperature. An environmental-friendly agronomy using the combination of cover crop and no-tillage managements results in a massive CO₂ capture and storage into the soil carbon pools, therefore, it has been expected to mitigate the global warming and climate change. However, the increased soil organic carbon enhances the N₂O emission from soil. Our study is aimed to 1) unveil the microbial process involved in the nitrogen cycle in the field soil managed with the long-term cover crop and no-tillage treatments; 2) obtain high-affinity N₂O-reducing microbes and develop their application in the sustainable crop cultivation.
Endophytic bacteria are derived from diverse soil bacteria, but which are limited to specific bacteria by strong effects of plant such as supply of specific substances from plant and protective responses of plant. Therefore, it is expected that endophytic bacteria with high N₂O detoxification ability can be used sustainably and effectively to N₂O detoxification in crops. In this study, we are searching for endophytic bacteria with high N₂O detoxification ability from the forage crop.

Ⅱ-3: N₂O recycling by N₂O fixing bacteria and iron-reducing bacteria
Recently, we isolated the iron-reducing bacteria from paddy soil, which harbors the potential not only to reduce N₂O but also to convert into organic nitrogen, i.e., to fix N₂O. In this project, we verify the N₂O fixation activity of iron-reducing bacteria and develop basic technology to enhance their N₂O activity in soils. In addition, we work on researches to elucidate the dynamics of N₂O-fixing iron-reducing bacteria in soils based on microcosmic experiments and global trends for diversity of N₂O-fixing microbiome in soils by analyzing soil metagenomic data around the world.
02 Construction of the rhizosphere cultivation system for designing and evaluating the soil ecosystem for N₂O recycling
When we encounter something unknown, we try to “observe” it carefully. The well-known phrase “seeing is believing” is undoubtedly applicable to biology. Recent advances in various microscopic imaging techniques using state-of-the-art instruments have enabled novel insights into a wide range of biological processes. In our project aiming at “Cool Earth via Microbes in Agriculture”, there still exist many fundamental questions, for instance, where the N₂O-/CH₄-detoxifying microbes actually reside in the soil structure and how they colonize rhizosphere of plant roots. Revealing these key points under natural conditions should accelerate our project as well as the practical application of these microbes, and microscopy can help it a lot.
In the subject II-4, we will construct a rhizosphere environmental chamber system for in situ imaging analyses of plant-microbe interactions in the soil. The system that is able to reconstitute adequately natural soil ecosystems will be used to investigate, evaluate and optimize greenhouse gas mitigation by synthetic soil-microbe-plant interactions. “Seeing is believing, but sometimes the most real things in the world are the things we can't see (Chris Van Allsburg, The Polar Express)” UNDER THE DARK SOIL. We will make it VISIBLE!