Bio-augmentation implies bioremediation which introduces exogenous microbes cultivated in the laboratory. Bio-stimulation, on the other hand, activates indigenous microbes living in a remediation site by adding nutritional substance and oxygen
Subjected contaminated substances in bio-augmentation are persistent substances which cannot be easily degraded by methods such as bio-stimulation. Regarding trichloroethylene and tetrachloroethylene, anaerobic dechlorination digestion techniques using in-situ bio-stimulation by adding usable material in contaminated groundwater have already been applied. However, the application of bio-augmentation has begun for the purpose of substantially shortening the processing period.
Regarding bio-augmentation technology, a certain standard needs to be imposed on the use of bio-augmentation in order to ensure safety for the environment and humans. The Ministry of International Trade and Industry (The Ministry of Economy, Trade and Industry, presently) revised “Recombinant DNA Technology Industrialization Guidelines（※「組換えDNA技術工業化指針」）” in 1998. Thus, bio-augmentation should correspondingly apply the guidelines for the use of the open system. Even for the use of nongenetically-modified microbes we should follow the guidelines. Subsequently, the Ministry of Economy, Trade and Industry and the Ministry of the Environment established joint guidelines for the use of microbes in bioaugmentation as the “Guidelines on Biorementation by Microbes（※「微生物によるバイオレメディエーション利用指針について」）” in February 2005. Consequently, the items which need consideration when bioaugmentation is commercialized were well organized. Incidentally, the items for safety assessment are largely similar to the “Recombinant DNA Technology Industrialization Guidelines” mentioned above.
As a case study indicating the trend toward the era of commercializing bio-augmentation, the soil remediation technology of organochlorine compounds using microbes like the MO7 strain is described in this paper. The technology was jointly developed by Obayashi Corporation and Toyota Motor Corporation.

It is known that trichloroethylene (TCE) is degraded by aerobic co-metabolism. TCE is effectively degraded by microbes using chemical substances such as methane, phenol and toluene. The MO7 strain with a high TCE degrading ability was selected because using microbes with a high degrading ability for soil remediation is effective. The MO7 strain does not use gene recombination microbes, and is not genetically recombined, only isolated from nature (Fig. 1). Genetic lineage analysis of 16SrRNA indicates that the MO7 strain belongs taxonomically to Janibacter. It grows vigorously in the range of neutral pH under conditions between 20- 30°C. It is a phenol-using bacteria which can be cultivated by using nutrition or inorganic medium.
The MO7 process applied under the “Recombinant DNA Technology Industrialization Guidelines” in April 2004, and it was confirmed that the MO7 strain is the first example in Japan applicable to the guidelines as an application which does not pinpoint a site. This was concluded after deliberations of a special committee and the invitation of comments from the public. Thus, the MO7 strain can respond to any contaminated site in the country.

Fig. 1 Electron Micrograph of MO7 Strain

The safety in soil remediation technology using the MO7 strain and its microbes are identified in detail in “Recombinant DNA Technology Industrialization Guidelines”. The safety in pathogenicity and physiological property of the MO7 strain itself on humans and animals and plants is confirmed based on the data verified by non-toxicity tests shown in Table 1. The soil remediation technology specifically noted that there are no problems concerning safety in the introduction to work area, behavior in work area and every process after remediation. It has been confirmed that this soil remediation technology meets the guidelines. One of the characteristics of this soil remediation technology is the utilization of microbes. In the soil remediation technology, the microbes cultivated are harvested and washed. Next, resting cells remove phenol, an inducer of TCE- degrading enzymes, and are deposited into the soil. Therefore, harmful phenol itself is not deposited into the soil. Moreover, there is no secondary contamination caused by nutrient salt because there is no additional nutrient salt deposited into soil. There is a safety benefit: less possibility that microbes in the soil increase after remediation because nutrient salt is removed by washing. Also, postulated degradation pathways of this technology are shown in Fig. 2. The compounds such as dichloroacetic acid, trichloroacetic acid and chloral expected as an intermediate of trichloroethylene degradation, each of which was not detected.
　 The decrease of viable count of the MO7 strain was also confirmed. It is concluded that there is no survival of the MO7 strain or residue of harmful substances in the soil.

Table 1 Non-toxicity Test Items of MO7 Strain

Fig.２ Postulated Degradation Pathways of Trichloroethylene by MO7 Strain

The soil remediation method was developed with a soil environment feasible for bio-augmentation obtained by mixing a soil enriching agent of an inorganic soil, based on the request that the soil remediation method must be low in cost in order to commercialize it. Using self-propelled soil property enriching equipment in order to effectively mix soil enriching agents or microbes, this technique can degrade TCE to less than the standard value in a few days and backfill it (Fig. 3). The cost of remediation is around 10,000 yen per ton of contaminated soil except excavation and backfilling, depending on the degree of contamination.

Bio-augmentation using safety-confirmed microbes degrades harmful substances into harmless residue. Additionally, the technology is low cost and has no secondary contamination, and the organization of administrative guidelines also supports it. The future development of bio-augmentation is expected to be not only a technology with low environmental impacts but also a profitable business method.