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

Figures & data

Table 1. Operating conditions of toluene removal experiment using RSB biofilter

	Term	Flow rate	Toluene concentration	Toluene loading
Run	(day)	(m^3h^−1)	(ppmv) ^a	(g m^−3h^−1) ^a	Sampling for DGGE ^b
Run1	1–10	3.0	247	96	day 2 (1), day 9 (2)
Run2	11–35	2.0	294	76	day 16 (3), day 31 (4)
Run3 ^c	36–80	2.0	322	83	day 36 (5), day 40 (6), day 51 (7), day 68 (8)
Run4	81–96	2.0	276	72
Run5 ^d	97–124	2.0	270	70	day 105 (9), day 118 (10)
Run6 ^d	125–155	4.0	275	142	day 132 (11), day 145 (12)
Run7 ^e	156–182	-	-	-	day 163 (13)
Run8 ^f	183–199	2.0	213	110	day 183 (14)
Notes: ^aaverage value during the run; ^bsample for PCR-DGGE was taken from the nutrient tank. Numbers in parentheses indicates the sample number in figure 3; ^cOzone treatment was carried out in the nutrient tank for an hour after nutrientsupply; ^d(NH4)2SO4 was added ca. 2 times higher than other term (800–1000mg-N L^−1); ^eRSB was stopped completely during Run7; ^fOzone treatment was carried out for 5 minutes before nutrient supply.

Figure 1. Schematic diagram of the experimental setup.

Figure 2. Change of parameters during the operation period of the RSB: (a) Toluene removal efficiency (%) and the toluene inlet loading (g m⁻³ hr⁻¹). (b) Concentration of NH⁴-N (mg L⁻¹) and SS (mg L⁻¹) in the nutrient tank. Toluene removal efficiency (%) was calculated as follows: (1−Co/Ci) × 100, where Co is the toluene outlet concentration (ppmv); Ci, the toluene inlet concentration (ppmv).

Figure 3. DGGE profiles of universal 16S rRNA gene amplicons collected from the nutrient reservoir tank (no.1–14) and biofilm on the packing material (no.15) of RSB. Open circles with numbers indicate excised bands. These bands were analyzed by sequencing. A phylogenetic tree based on the sequence data is shown in Figure 6. Lanes: 1; day 2, 2; day 9, 3; day 16, 4; day 31, 5; day 36, 6; day 40, 7; day 51, 8; day 68, 9; day 105, 10; day 118, 11; day 132, 12; day 145, 13; day 163, 14; day 183, M; marker, 15; day 68.

Figure 4. Nonmetric multidimensional scaling analysis of the DGGE fingerprint (stress value is 0.04). Numbers 1–15 indicate sample numbers from the DGGE analysis (see Table 1 and Figure 3). Numbers 1–14 (solid circles) were samples of the nutrient solution, and number 15 (open circle) was a sample of the biofilm on the packing material.

Figure 5. DGGE profile of tmoA-like gene amplicons collected from the nutrient reservoir tank of RSB. Arrows indicate the excised band.

Figure 6. Neighbor-joining tree of partial 16S rRNA gene sequence (∼550 bp) recovered by denaturing gradient gel electrophoresis bands. Accession numbers appear after the genus name. The neighbor-joining tree was constructed as described in the text. Each sequence, except for those from this study, was obtained from the DDBJ (DNA Data Bank of Japan). The scale bar indicates 0.05 changes per nucleotide. The numbers on the branches refer to bootstrap values for 1000 replicates; only those above 600 are shown. Solid circles mean the reported bacteria that can utilize or tolerate to the toluene in the papers.

Table 2. Nearest match strain for tmoA-like gene based on DGGE analysis by using TMOA primer set

band ^a	nearest match in BLAST analysis	similarity (%)	Accession no.
1	uncultured bacterium (AY450333)	376/429 (87.6%)	AB644256
2	uncultured bacterium (AY450333)	385/428 (87.9%)	AB644257
3	uncultured bacterium (GU225790)	385/428 (90.0%)	AB644258
4	Dechloromonas aromatica (CP000089)	401/434 (92.4%)	AB644259
Note: ^aNumber indicates the arrow in Figure 5.