Transformation of microflora during degradation of gaseous toluene in a biofilter detected using PCR-DGGE

Abstract

A laboratory-scale biofiltration system, the rotatory-switching biofilter (RSB), was operated for 199 days using toluene as a model pollutant. The target gaseous pollutant for the biofiltration experiment was ∼300 ppmv of toluene. Toluene removal efficiency (RE, %) was initially approximately 20% with a 247-ppmv concentration (0.9 g m⁻³) of toluene during the first 10 days. Although the RE decreased several times whenever nitrogen was consumed, it again reached almost 100% when the nitrogen source was in sufficient supply. Denaturing gradient gel electrophoresis (DGGE) analysis was employed to assess the transformation of microflora during operation of the biofilter. The results based on a 16S rRNA gene profile showed that the microbial community structure changed with operation time. Although the microflora changed during the initial period (before day 40), transformation of the bacterial component was hardly observed after day 51. Statistical analyses of the DGGE profiles indicated that the bacterial community was almost unaffected by the environmental factors, such as adding ozone, high-level nitrogen supply, increase of loading toluene, and the shutdown of the RSB. The DGGE profile using tmoA-like genes, which encode proteins belonging to the hydroxylase component mono-oxygenases involved in the initial attack of aerobic benzene, toluene, ethylbenzene, and xylene degradation, confirmed the existence of toluene-degrading bacteria. There were at least four kinds of toluene-degradable bacteria having tmoA-like genes up to day 36, which decreased to two species after day 40. Sequence analysis after DGGE profiling revealed that Burkholderia cepacia, Sphingobacterium multivorum, and Pseudomonas putida were present in the biofilter. Only Alicycliphilus denitrificans was present throughout the whole operation period. In the initial stage of operating the RSB, many types of bacteria may have tried to adapt to the conditions, and subsequently, only selected bacteria were able to grow and to degrade toluene.

Implications:

Biofiltration technology, which is more cost-effective and environmentally friendly than any other physicochemical method, has been applied to the treatment of volatile organic compounds (VOCs). It was difficult to maintain a high level of activity because the performance of the biofilter depends on microbial degradation of pollutants. However, there is little knowledge about the transformation of bacteria during long-term biofiltration operations. This study revealed the transformation of microflora in a biofilter using a molecular biological method, PCR-DGGE.

Acknowledgments

This research was supported by the Environment Research and Technology Development Fund (ERTDF) of the Ministry of the Environment in 2006–2007. Kimura Chemical Plants Co., Ltd., collaborated throughout this study, especially in designing and manufacturing the RSB biofilter. We deeply appreciate the help of Dr. Yuki Kobayashi (Kyoto University) in the NMDS analyses. We are especially grateful to Dr. S. Rand (Antwerp University Hospital) for checking the English language of the paper and for his valuable comments.