Characterization of pyrene-degrading bacterial consortia obtained by a biofilm enrichment strategy in a model trickling filter

Introduction
　Polycyclic aromatic hydrocarbons (PAHs) are pollutant, which consist of two or more fused benzene rings and they are degradable by various bacteria and fungi. Traditionally, the PAH degrading bacteria have been isolated using liquid enrichment culture method. However, only a small proportion of the total community is isolated using this method.
　Cunliffe (2005) developed a biofilm community trickle column system to isolate pyrene-degrading microorganisms. Columns were constructed from glass tubing with insertion of activated carbon impregnated matrix (ACIM) inoculated with contaminated soil, and 180 pyrene-degrading consortia were isolated from the top, middle, and bottom of columns. Most of the consortia were able to grow as biofilms and the colonization of the ACIM was unaffected by location on the column, however, the activity and the structure of the community were affected by location on the column.
　Further analyses on these pyrene-degrading consortia were done in this study.

Material and Methods
　10 consortia from each top, middle, and bottom of the columns were used in this study. Planktonic growth rate, PAH degrading ability, motility, hydrophobicity, and biofilm formation of the consortia from top, middle, and bottom of the column were examined. Moreover, bacterial species constituting the top and bottom consortia were determined by cloning and sequencing the consortia 16SrDNA and the gradient of the concentration of phosphate and ammonium in the column were also measured. From these results, the relationships between the location on the column and the character of the community were evaluated.

Results and Discussion
　The physiological characters of the consortia isolated from the middle of the columns were outstanding compared to the consortia from the top and bottom. They were excellent in PAH degradation, motility, and in forming biofilms. These findings suggest that the middle consortia are excellent PAH-degraders.
　Each consortium contained 5 to 9 species, and Pseudomonads and Stenotrophomonads are the dominant groups constituting the consortia. Interestingly, if we look at the proportion of the genera within the consortia, Stenotrophomonads were dominant in the top consortia while Pseudomonads were dominant in the bottom consortia. From the phosphate and ammonium analysis, there was almost no difference in the concentration of these two substances among the location of the column. Therefore, it can be assumed that the concentration of the carbon source is a possible key factor regulating the consortium structures.

Future works
　It was proved that the culturable bacteria using the trickle column enrichment method were very diverse in the previous study (Cunliffe, 2005) and in this study. Twenty-six different consortia were isolated from the top and the bottom of the column (Cunliffe, 2005) and each consortium contained 5 to 9 species. It is interesting to know how many great a diversity of culturable bacteria this trickle column enrichment method can produce compared to the classical liquid enrichment, and this should be a main target for future research.