Microbes Environ. Vol. 19, No. 2, 172–177, 2004
http://wwwsoc.nii.ac.jp/jsme2/
Short Communication
Distribution of Dibenzofuran-Degrading Bacteria in Soils Polluted with Different Levels of Polychlorinated Dioxins HIROYUKI FUTAMATA1, TETSUYA UCHIDA1, NAOKO YOSHIDA1, YOSHITOMO YONEMITSU1 and AKIRA HIRAISHI1* 1
Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441–8580, Japan
(Received February 27, 2004—Accepted April 13, 2004) Aerobic chemoorganotrophic bacteria in soils polluted with different levels of polychlorinated dioxins (6.8 to 4,600 pg-TEQ g-1 dry weight) were isolated by the quantitative agar-plating method and tested for their ability to degrade dibenzofuran (DF) using DF-overlaid agar and DF-containing liquid media. For comparison, bacteria isolated from river sediments were also tested. Out of the 5,069 strains thus isolated, 23 strains were found to be able to degrade DF, and the majority produced soluble yellow metabolites during the degradation. A higher isolation frequency for DF degraders was obtained with samples containing higher concentrations of polychlorinated dioxins. Most of the DF-degrading isolates were identified as members of the class Actinobacteria, particularly of the genera Nocardioides and Rhodococcus. These results suggest that particular actinobacterial species constitute the major populations of culturable DF-degrading bacteria in dioxin-polluted environments. Key words: Dibenzofuran degraders, dioxins, Nocardioides, Rhodococcus, bioremediation
The biodegradation of dibenzofuran (DF) as well as of dibenzo-p-dioxin (DD) by microorganisms has been studied extensively in connection with potential utilization in the bioremediation of dioxin-polluted environments. Large numbers of DF-degrading bacteria have been isolated and characterized in terms of physiological, biochemical and genetic attributes [for reviews, refs. 2, 5, 14, 15, 21]. The information obtained to date indicates that the bacterial degradation of DF proceeds via lateral or angular dioxygenation initially and that both modes of degradation are found in a wide variety of bacterial species. From ecological and biotechnological points of view, however, information about the quantitative distribution of DF-degrading bacteria in the environment and its relation to dioxin pollution levels is still lacking despite its importance for bioremediation purpose. In a previous study, an attempt to isolate DFdegrading bacteria from highly dioxin-polluted soils was * Corresponding author; E-mail:
[email protected], Tel: +81– 532–44–6913, Fax: +81–532–44–6929
successful, and the isolates from enrichment cultures were identified as members of the genus Sphingomonas and some Gram-positive genera6). Thus, this study was designed to more thoroughly investigate the distribution of DFdegrading bacteria in polluted soils in terms of quantity and quality. The isolation frequency for DF-degrading bacteria is discussed in relation to dioxin pollution levels. Soil samples were collected from various polluted sites in Japan as described previously6) (see Table 1). Surface soil at a depth of up to 5 cm was collected with a Tosen stainless soil sampler (Tosen Techno Co., Toyonaka, Japan) and taken into polyethylene bottles. In addition, sediment samples were collected from rivers in the Aichi, Kanagawa, and Tokyo Metropolitan Prefectures. All these samples were kept in an insulated cooler during transportation to the laboratory. Samples for dioxin analysis were dried at 110°C for 2 days and then stored at -80°C until analyzed. All of the tetra- and higher chlorinated congeners of DD and DF were analyzed as polychlorinated dibenzo-p-dioxins/ dibenzofurans (PCDD/Fs). Dioxins were extracted from the
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Dibenzofuran-Degrading Bacteria in Polluted Soils Table 1. Sample Soil NSA1 NSA2 NSA3 NSA4 NSA5 NSA6 RS1 RS2 RS3 KS1 KS2 KS3 MS1 MS2 MS3 TS1 TS2 TS3 TS4 SS1 SS2 SS3 Sediment FRS1 HRS1 HRS2 HRS3 TRS1 TRS2 NRS1 NRS2 NRS3 Total a b c d
Isolation of DF-degrading bacteria from soil and river sediment samples polluted with different levels of PCDD/Fs
PCDD/F concn. Total count Plate count No. of colonies tested (pg-TEQ g-1 dry wt) (´109 cells g-1 dry wt or ml-1) (´106 CFU g-1 dry wt or ml-1)a
4,600 3,100 2,700 1,100 890 560 330 210 110 440 290 180 62 44 32 49 33 28 18 9.8 8.0 6.8 540 110 82 78 63 53 32 32 30
1.4 1.9 3.9 4.2 5.2 6.3 7.1 6.2 7.6 11 6.7 10 12 5.8 4.2 12 13 16 17 9.7 8.5 10 0.74 0.67 1.1 1.4 ntd nt 1.7 2.2 1.6
No. of colonies positive for DF degradationb
Enrichment with DFc
4.8 4.5 9.3 7.6 11 16 7.1 11 9.2 25 8.9 13 54* 9.2* 8.6* 23 24 49 51 33 12 64
246 117 245 150 153 156 98 160 137 165 187 190 127 186 192 220 81 197 93 124 87 144
3 (3) 1 (1) 4 (3) 0 4 (3) 1 (1) 0 1 2 1 (1) 0 0 0 0 0 0 0 0 0 0 0 0
+Y +Y +Y +Y +Y +Y + -
1.2 1.5 2.2 6.5 2.4*
178 199 186 190 75
3 (3) 1 (1) 0 0 2 (2)
+Y +Y + nt
1.7* 11 14 17
75 252 264 195 5,069
0 0 0 0 23 (17)
nt -
Data obtained with PBYS agar or 1/10´TS agar (asterisked). Data obtained with DF-overlaid agar medium. Figures in parentheses indicate the number of colonies producing soluble yellow pigment. Direct enrichment in DF-containing two-layer liquid medium. +, growth positive without pigment production; +Y, growth positive with yellow pigment production; -, growth negative. nt, not tested.
dried samples by the Soxhlet method, fractionated by multiphase column chromatography and alumina column chromatography, and analyzed by gas-liquid chromatographymass spectrometry as described previously6). PCDD/F concentrations as toxicity equivalent (TEQ) levels were calculated based on the toxicity equivalency factors for
different congeners redefined by van den Berg et al.19). As shown in Table 1, the concentration of PCDD/Fs detected in the soil and sediment samples (31 samples in total) ranged from 6.8 to 4,600 pg-TEQ g-1 dry wt. All samples were subjected to microbiological analyses upon return to the laboratory. For cell counting, 1 g (wet wt)
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of sample was suspended in 9 ml of filter-sterilized phosphate-buffered saline (PBS, 10 mM potassium phosphate and 130 mM sodium chloride, pH 7.0), dispersed for 1 min with a POLYTRON homogenizer (Kinematica Co., Littau/ Luzern, Switzerland) and then settled for 5 min. One milliliter of the upper fraction of the homogenate was diluted serially with the same buffer, and these serial dilutions were used for direct cell counting and plating. Total counts of bacteria were measured by epifluorescence microscopy with ethidium bromide or SYBR Green II (Molecular Probes, Inc., Eugene, USA) staining as reported previously11,13). For the quantitative isolation of aerobic chemoorganotrophic bacteria, 50 ml of sample at the appropriate dilution was smeared onto PBYS agar medium (0.05% Proteose Peptone No. 3 [Difco Laboratories, Detroit, USA], 0.01% beef extract [Difco], 0.01% yeast extract [Difco], 1 mM sodium salicylate and 1.5% agar, pH 7.0) and onto 1/10-diluted (1/10´) Tryptic Soy (Difco) agar medium. Inoculated plates were prepared in triplicate for each dilution step and incubated in an air incubator at 25°C for 3 weeks before the counting of CFU. For the enrichment of DF-degrading bacteria from soil and sediment samples, a DF-containing two-layer liquid medium in screw-capped test tubes was used. Namely, the mineral medium RM2 supplemented with the vitamin solution PV18) (designated BSV medium) was used as the basal medium, to which a filter-sterilized 0.2% DF solution in 2,2’,4,4’,6,8,8’-heptamethylnonane was added at a volume ratio of 10:1 (DF-BSV medium). The resultant solvent-aqueous two-layer medium was inoculated with 100 ml of 1/10-diluted soil and sediment samples in PBS buffer and incubated on a reciprocal shaker at 28–30°C for 3 weeks. Almost all of the single colonies recovered on “countable” plates were picked up and subjected to the standard purification procedure with repeated streaking on PBY agar (0.5% Bact-Peptone [Difco], 0.3% beef extract, 0.1% yeast extract and 1.5% agar, pH 7.0). All isolates were tested for DF degradation using DF-coated 1/10´PBY agar plates which were prepared by spreading 5 ml of 2% DF solution in diethyl ether onto the agar medium and subsequent drying in a clean bench. The resultant DF layer-overlaid plates were inoculated with precultures (grown in PBY medium) and incubated at 28–30°C for one month. DF degradation was determined by monitoring colony formation, yellow metabolite production and cleared “halo” formation around colonies. In addition, the DF-containing two-layer liquid medium supplemented with 0.05% yeast extract (Difco) in place of the vitamin solution PV1 (designated DF-BSY medium) was used for testing DF degradation, which was
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determined by monitoring growth, yellow metabolite production and the concentration of DF remaining in the liquid cultures. The concentration of DF was determined by HPLC as described previously8). As shown in Table 1, the soil and sediment samples yielded total bacterial populations in the order of 108 to 1010 cells g-1 (dry wt) or ml-1. Plate counts accounted for 0.1 to 1.1% of the total count. No significant differences were noted in plate counts in each sample between PBYS and 1/10 ´TS agar media. A total of 5,069 strains of aerobic chemoorganotrophic bacteria were isolated and tested for their ability to degrade DF. Out of the strains thus tested, 23 exhibited growth with cleared halo formation on DF-overlaid agar plates. HPLC experiments revealed that all of these 23 isolates degraded DF completely after 3 to 10 days of incubation when grown in DF-BSY medium (data not shown). There seemed no correlation between the velocity of DF degradation by the isolates and pollution levels of their sources. Attempts to concentrate DF-degrading bacteria using DF-BSV medium gave positive results in 10 of the test samples. In view of the results shown in Table 1, it seemed evident that a higher isolation frequency for DF-degrading bacteria could be obtained with samples polluted with higher levels of PCDD/Fs. Most of the DF-degrading isolates produced a soluble yellow pigment on the agar medium and in the liquid medium. This pigment was produced only when the isolates were exposed to DF, indicating that it was an intermediate product during the degradation of DF. Spectrophotometric experiments revealed that the supernatant from the yellowcolored cultures had an absorption maximum at 465 nm. A pH-dependent shift (pH 3 to pH 7–10) in the absorption maximum from 400 to 465 nm was observed, suggesting the keto-enol tautomerism of the meta cleavage product, 2-hydroxy-4-[3’-oxo-3’H-benzofuran-2’-yliden]but-2-enoic acid (HOBB), found in the lateral degradation pathway5,21). There was an exceptional case in some strains identified as members of the genus Nocardioides (see below), which produced yellow metabolite(s) showing an atypical pHdependent absorption change. Namely, the absorption spectra of the supernatants from the Nocardioides cultures at pH 3, 7, and 10 showed an absorption maximum at around 400, 466, and 507 nm, respectively. This interesting phenomenon should be elucidated by further study. All of the DF-degrading isolates thus obtained were identified phylogenetically based on 16S rDNA sequence comparisons. 16S rDNA fragments (positions 8 to 1543 of Escherichia coli 16S rRNA3)) from the DF-degrading isolates were amplified by PCR as described7), sequenced directly
Dibenzofuran-Degrading Bacteria in Polluted Soils
with a SequiTherm Long Read sequencing kit (Epicentre Technologies, Madison, USA) and analyzed with an Amersham-Pharmacia DNA sequencer. Sequence data were compiled with the GENETYX-MAC program (Software Developing Co., Tokyo, Japan) and compared with those available from the DDBJ/EMBL/GenBank DNA databanks using the BLAST homology search program1). By using CLUSTAL W program18), a multiple alignment of the 16S rDNA sequences of the isolates and those retrieved from the databases was performed, and a neighbor-joining16) phylogenetic tree was constructed based on the distance matrix
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data. As shown in Fig. 1, 78% of the isolates were clustered with members of the class Actinobacteria, particularly those of the genera Nocardioides and Rhodococcus. Isolates assigned to the genera Arthrobacter and Janibacter were also found. All other isolates were identified as members of the family Sphingomonadaceae, a major phylogenetic group belonging to the class Alphaproteobacteria. Similar results of isolation and identification of DF degraders were obtained in previous studies, most of which employed enrichment techniques for the isolation. For example, the DF-degrading actinobacterial strains reported to date in-
Fig. 1. A neighbor-joining distance matrix tree showing the phylogenetic positions of DF-degrading isolates based on 16S rDNA sequences. The 16S rDNA sequence of Bacillus subtilis (X60646) was used as the outgroup to root the tree. Bootstrap values (100 resamplings)4) of more than 80% are given at branching points. Scale=2% nucleotide substitution (Knuc). The isolate names are shaded; the symbols and abbreviations in the brackets behind the isolate names show yellow metabolite production (+, positive; -, negative) and the sample names from which they were isolated (see Table 1).
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clude Janibacter sp. strain YY-124), Rhodococcus erythropolis strain SBUG 27117), Rhodococcus opacus strain SAO10112), Rhodococcus sp. strain YK29) and Terrabacter (Janibacter) sp. strain DBF6310). A representative of DFdegrading alphaproteobacteria is Sphingomonas wittichii strain RW1T 20,22,23). 16S rDNA fragments were also PCR-amplified from the enrichment cultures of samples NSA1, NSA3, NSA5, KS1, MS2, FRS1 and HRS2, subcloned using a pTBlue Perfectly Blunt cloning kit (Novagen, Madison, USA) as previously described11), and sequenced as noted above. As a result, 16S rDNA clones corresponding to the isolates shown on the phylogenetic tree were obtained, except for samples MR2 and HR2, in which clones belonging to the genera Burkholderia, Variovorax and Wautersia were mainly detected (data not shown). The results of this study provide circumstantial evidence that the isolation frequency for DF-degrading bacteria from soils is related to their pollution levels. A previous study has suggested that biological reductive dehalogenation of PCDD/Fs takes place in highly polluted soils, resulting in the formation of lower chlorinated congeners6). In the present study, we detected small but significant amounts of DD, DF and their monochlorinated congeners (220 to 1,400 pg g-1 dry wt in total) in the samples of the NSA series, although it is unclear whether the DD and DF were actually derived from the PCDD/Fs in situ. Probably, the availability of the nonchlorinated and lower chlorinated congeners as substrates is one of the most important factors affecting the proliferation of DF-degrading bacteria in situ. Since the isolates belonging to the class Actinobacteria, particularly those of the genera Nocardioides and Rhodococcus, were most abundantly obtained from different geographical locations, it is our conclusion at this time that these Grampositive taxa are the major constituents of DF-degrading bacterial populations in dioxin-polluted soils. In addition, it is suggested that DF-degrading bacteria having the lateral dioxygenation pathway producing HOBB as a metabolite are more widely distributed in polluted environments than DF degraders having only the angular-type pathway. One major problem to be considered in the present study is a possible culture bias. In light of the fact that the plate count accounted for only 0.1 to 1.1% of the total count in the samples tested, the reliability of the culture-dependent community analysis is not necessarily high. Therefore, we cannot exclude the possibility that bacteria not detectable by the method used in this study were the major constituents of the DF-degrading populations in the polluted soils and sediments. Another problem with this study relates to the sam-
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pling size. Although we tested more than 5,000 isolates for DF degradation, we could obtain only 23 positive strains. This number of test isolates seems insufficient to describe the DF-degrading community structure only based on culture-dependent data. A promising approach to solving these problems is the use of culture-independent molecular markers, such as genes coding for aromatic carbon dioxygenases, in the community analysis. In order to design consensus PCR primers applicable to a wide variety of DF-degrading bacteria, the structural analysis of genes encoding DF dioxygenases of the isolates is now in progress. The 16S rDNA sequences determined in this study have been deposited under DDBJ accession numbers AB087721, AB087725 and AB177880 to AB177889.
Acknowledgments We are grateful to T. Kondo and T. Takanashi for their technical assistance. This study was performed as a part of “The Project for Development of Technologies for Analyzing and Controlling the Mechanism of Biodegrading and Processing” entrusted by the New Energy and Industrial Technology Development Organization (NEDO) and supported in part by grants from the Ministry of the Environment, Japan. This work was also carried out as a part of the 21st Century COE Program “Ecological Engineering and Homeostatic Human Activities” founded by the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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