TY - JOUR
AU - Lechner,, Ute
AB - Abstract An anaerobic mixed culture enriched over 16 transfers (1/10) from Saale river sediment reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) to di- and monochlorinated congeners in the presence of pyruvate (or lactate) and fumarate as cosubstrates. Besides TrCDD, tetrachloroethene and 1,2,3-trichlorobenzene were dechlorinated. Dioxin dehalogenation was sensitive to pasteurization, but was not remarkably influenced by inhibitors of methanogens, sulfate-reducing bacteria or Gram-positive bacteria. The rate of 1,3-dichlorodibenzo-p-dioxin formation increased with rising initial concentrations of 1,2,4-TrCDD (1–250 μM) from 0.05 to 5.4 μmol l−1 day−1. Two isolates, belonging to Sulfurospirillum and Trichococcus, did not show reductive dehalogenation. 16S rDNA-targeted methods further revealed the presence of Acetobacterium, Desulfitobacterium, Desulfuromonas and Dehalococcoides. Nested polymerase chain reaction (PCR) indicated the presence of Dehalococcoides in highest most probable number (MPN) dilutions that were positive for dioxin dechlorination. Reductive dehalogenation, Chlorinated dibenzo-p-dioxin, Trichlorobenzene, Dehalococcoides, Sulfurospirillum, Trichococcus 1 Introduction Polychlorinated dioxins and furans (PCDD/F) have been released into the environment as unwanted byproducts of many different processes such as the production of phenoxyherbicides, bleaching of paper pulp with chlorine or production of water-free magnesium chloride [1]. This led in some industrialized areas such as the Bitterfeld region (middle Germany) to large-scale dioxin pollution of sediments and soils [2]. Due to the high toxicity of dioxin congeners chlorinated at the lateral 2, 3, 7 and 8 positions, high attention and efforts are necessary to prevent contamination of drinking water and food. Despite their relatively high persistence in the environment, reductive dehalogenation of PCDD by anaerobic bacteria and an aerobic microbial breakdown of low chlorinated congeners has been reported (for a review see [3]). A change of the congener distribution pattern within Lake Ketelmeer sediment over a time period of 20 years indicated anaerobic in situ reductive dechlorination [4]. Using microcosms established from anaerobic sediment and aquifer material, the microbially catalyzed reductive dechlorination of highly chlorinated dioxins (five to seven chlorines per molecule) was demonstrated [5]. Tetrachlorodibenzo-p-dioxin (TCDD) was the main product [6]. Nonetheless, it was demonstrated that sediment-associated 2,3,7,8-TCDD was further converted to 2-monochlorodibenzo-p-dioxin (2-MCDD) [7]. Different dechlorination pathways have been observed, but the bacteria mediating specific dechlorination patterns are not known yet. The peripheral (peri-) dechlorination of octachlorodibenzo-p-dioxin and the ability to dechlorinate trichlorodibenzo-p-dioxins (TrCDDs) was assigned to subpopulations of non-methanogenic, heat-sensitive bacteria, whereas lateral dechlorination was exhibited by pasteurized cells [8]. Dechlorination of 1,2,3,4-TCDD via a lateral and a subsequent peri-dechlorination step was reported for a primary enrichment culture from Saale river sediment [9] and for a methanogenic mixed culture enriched from estuarine sediment [10] and reflected the most favorable route according to thermodynamics [11]. However, using the same model congener, other dechlorination sequences have also been observed. A sediment-free culture enriched with hexachlorobenzene (HCB) from Lake Ketelmeer sediment [12] as well as different primary enrichment cultures from the highly PCDD/F-contaminated sediment of river Spittelwasser [13] dechlorinated spiked 1,2,3,4-TCDD via both 1,2,3- and 1,2,4-TrCDD to a mixture of 1,3- and 2,3-dichlorodibenzo-p-dioxin (DCDD). This observed diversity of dechlorination routes might be due to diverse microorganisms which grew under the selective pressure of specific contaminants or natural chlorinated compounds [14] present in the original habitat and might have been further adapted by the individual enrichment conditions used, e.g. by ‘priming’ with bromophenols [10] or chlorobenzene [12]. Recently, Dehalococcoides sp. strain CBDB1 isolated from a chlorobenzene-dechlorinating mixed culture [15] was shown to dechlorinate a range of chlorinated dioxins [16]. This bacterium had also been detected by polymerase chain reaction (PCR) targeting 16S rRNA genes in dioxin-dechlorinating enrichment cultures from Spittelwasser sediment [16]. In this study we examined factors that influence reductive dechlorination of TrCDD by an enrichment culture from sediment of the river Saale [9]. For the first time an insight into the microbial diversity of a dioxin-dechlorinating anaerobic mixed culture is presented, which demonstrates the presence of Dehalococcoides and other bacteria with known dechlorination capability. 2 Materials and methods 2.1 Medium, organisms and enrichment procedure Anoxic minimal medium M204 was prepared under N2/CO2 (80:20, [vol/vol]) atmosphere, buffered with bicarbonate and contained all components as previously described (medium 1 [17]) modified by the addition of 11 nM Na2SeO3, 12 nM Na2WO4, 63 nM 1,4-naphthoquinone and routinely contained 0.05 g l−1 yeast extract. Electron donors (5–40 mM), non-chlorinated electron acceptors (1–20 mM), titanium (III) citrate (0.2 mM; prepared according to [18]), specific inhibitors (bromoethanesulfonic acid [BES; 5 mM], sodium molybdate [1 mM], vancomycin [5 mg l−1]), humic acids mixture (Aldrich, Steinheim, Germany; 2 g l−1) or 2,6-anthraquinone disulfonic acid (AQSA; 100 μM) [19] were added from separately sterilized aqueous stock solutions. The final pH of the medium was 7.0±0.1 and cultures were incubated in 125-ml serum bottles or 16-ml anaerobic culture tubes (culture volumes of 80 and 2–3 ml, respectively) with agitation (150 rpm) in the dark at 20°C. TrCDDs (AccuStandard Inc., New Haven, CT, USA) were added from acetone stock solutions to empty culture tubes and the acetone was evaporated before the medium was injected [9]. Starting from an 1,2,3,4-TCDD-dechlorinating primary incubation of Saale river sediment [9], dioxin-dechlorinating enrichment cultures were established with 1,2,4- and 1,2,3-TrCDD over 16 and two sequential transfers (5–10% [vol/vol] each), respectively, using 50–100 μM of TrCDD. Table 1 indicates the addition of cosubstrates (potential electron donors and carbon sources) and BES during the course of the enrichment procedure and the sequential transfer, which served as inoculum for the individual experiments. 1 Indication of the origin of inoculum from different stages of the enrichment culture and of additions used for individual experiments Experiment Inoculum from transfer Cosubstratesa/BESb Kinetics of TrCDD dechlorination 2 I Influence of temperature, initial TrCDD concentration, pasteurization, AQSA, humic acids on dechlorination 2 I Dechlorination studies with 2,4,6-trichlorophenol (TCP), 3Cl4OHPA and PCEc 2 I Influence of single electron donors on dechlorination 5 –d/BES Influence of molybdate and bromoethane sulfonate on dechlorination 5 I/BES Dechlorination studies with HCB, 3,4,5,6-TeCV, 2,3,5,6-TeCP, 2,4,8-TrCDF 6 I/BES Agar shake dilution series and cultivation of single colonies 7 II/BES Detection of Desulfitobacterium by nested PCR 6, 11 III, see text Community analyses (ARDRA, nested PCR targeting 16S rDNA of different dehalogenating bacteria, MPN technique) 12 III/BES Influence of vancomycin on dechlorination 13 III/BES Dechlorination of 1,2,3-TCB 15 III/BES Detection of Dehalococcoides in the presence/absence of dioxin 15 III/BES Experiment Inoculum from transfer Cosubstratesa/BESb Kinetics of TrCDD dechlorination 2 I Influence of temperature, initial TrCDD concentration, pasteurization, AQSA, humic acids on dechlorination 2 I Dechlorination studies with 2,4,6-trichlorophenol (TCP), 3Cl4OHPA and PCEc 2 I Influence of single electron donors on dechlorination 5 –d/BES Influence of molybdate and bromoethane sulfonate on dechlorination 5 I/BES Dechlorination studies with HCB, 3,4,5,6-TeCV, 2,3,5,6-TeCP, 2,4,8-TrCDF 6 I/BES Agar shake dilution series and cultivation of single colonies 7 II/BES Detection of Desulfitobacterium by nested PCR 6, 11 III, see text Community analyses (ARDRA, nested PCR targeting 16S rDNA of different dehalogenating bacteria, MPN technique) 12 III/BES Influence of vancomycin on dechlorination 13 III/BES Dechlorination of 1,2,3-TCB 15 III/BES Detection of Dehalococcoides in the presence/absence of dioxin 15 III/BES TrCDD: trichlorodibenzo-p-dioxin, AQSA: 2,6-anthraquinone disulfonic acid, 3Cl4OHPA: 3-chloro-4-hydroxyphenylacetate, PCE: tetrachloroethene, HCB: hexachlorobenzene, TeCV: tetrachloroveratrole, TeCP: tetrachlorophenol, TrCDF: trichlorodibenzofuran, ARDRA: amplified ribosomal DNA restriction analysis, TCB: trichlorobenzene. aThe following cosubstrates were routinely added to the medium: I, acetate, L-lactate (or pyruvate), fumarate and benzoate (5 mM each) and 0.05 g l−1 of yeast extract (up to the 11th transfer); II, lactate (10 mM), fumarate (20 mM) and yeast extract (see text); III, pyruvate (10 mM), fumarate (10 mM) and 0.2 g l−1 of yeast extract (beginning with the 12th transfer). bBES (5 mM) was routinely added starting with the sixth transfer. cFor PCE dechlorination studies, inoculum was used from transfers 2, 3, 5, 6, 7 and 13 (stored at 20°C). L-lactate (40 mM) was added as the electron donor. dSingle electron donors were added as indicated in the text. Open in new tab 1 Indication of the origin of inoculum from different stages of the enrichment culture and of additions used for individual experiments Experiment Inoculum from transfer Cosubstratesa/BESb Kinetics of TrCDD dechlorination 2 I Influence of temperature, initial TrCDD concentration, pasteurization, AQSA, humic acids on dechlorination 2 I Dechlorination studies with 2,4,6-trichlorophenol (TCP), 3Cl4OHPA and PCEc 2 I Influence of single electron donors on dechlorination 5 –d/BES Influence of molybdate and bromoethane sulfonate on dechlorination 5 I/BES Dechlorination studies with HCB, 3,4,5,6-TeCV, 2,3,5,6-TeCP, 2,4,8-TrCDF 6 I/BES Agar shake dilution series and cultivation of single colonies 7 II/BES Detection of Desulfitobacterium by nested PCR 6, 11 III, see text Community analyses (ARDRA, nested PCR targeting 16S rDNA of different dehalogenating bacteria, MPN technique) 12 III/BES Influence of vancomycin on dechlorination 13 III/BES Dechlorination of 1,2,3-TCB 15 III/BES Detection of Dehalococcoides in the presence/absence of dioxin 15 III/BES Experiment Inoculum from transfer Cosubstratesa/BESb Kinetics of TrCDD dechlorination 2 I Influence of temperature, initial TrCDD concentration, pasteurization, AQSA, humic acids on dechlorination 2 I Dechlorination studies with 2,4,6-trichlorophenol (TCP), 3Cl4OHPA and PCEc 2 I Influence of single electron donors on dechlorination 5 –d/BES Influence of molybdate and bromoethane sulfonate on dechlorination 5 I/BES Dechlorination studies with HCB, 3,4,5,6-TeCV, 2,3,5,6-TeCP, 2,4,8-TrCDF 6 I/BES Agar shake dilution series and cultivation of single colonies 7 II/BES Detection of Desulfitobacterium by nested PCR 6, 11 III, see text Community analyses (ARDRA, nested PCR targeting 16S rDNA of different dehalogenating bacteria, MPN technique) 12 III/BES Influence of vancomycin on dechlorination 13 III/BES Dechlorination of 1,2,3-TCB 15 III/BES Detection of Dehalococcoides in the presence/absence of dioxin 15 III/BES TrCDD: trichlorodibenzo-p-dioxin, AQSA: 2,6-anthraquinone disulfonic acid, 3Cl4OHPA: 3-chloro-4-hydroxyphenylacetate, PCE: tetrachloroethene, HCB: hexachlorobenzene, TeCV: tetrachloroveratrole, TeCP: tetrachlorophenol, TrCDF: trichlorodibenzofuran, ARDRA: amplified ribosomal DNA restriction analysis, TCB: trichlorobenzene. aThe following cosubstrates were routinely added to the medium: I, acetate, L-lactate (or pyruvate), fumarate and benzoate (5 mM each) and 0.05 g l−1 of yeast extract (up to the 11th transfer); II, lactate (10 mM), fumarate (20 mM) and yeast extract (see text); III, pyruvate (10 mM), fumarate (10 mM) and 0.2 g l−1 of yeast extract (beginning with the 12th transfer). bBES (5 mM) was routinely added starting with the sixth transfer. cFor PCE dechlorination studies, inoculum was used from transfers 2, 3, 5, 6, 7 and 13 (stored at 20°C). L-lactate (40 mM) was added as the electron donor. dSingle electron donors were added as indicated in the text. Open in new tab 2.2 Dioxin dechlorination under various conditions To study the rate of TrCDD transformation and the influence of temperature, substrate concentration and pasteurization (75, 80 or 85°C for 15 min) several replicates of 2- to 3-ml cultures or of 1.5- and 6-ml cultures, if high (250 μM) and low (1 μM) dioxin concentrations were tested, respectively, were incubated. Duplicate or triplicate samples were sacrificed for analysis in intervals of 10–14 days. The influence of individual electron donors was studied in 125-ml serum bottles (30 ml culture volume). Samples (2 ml) were withdrawn in duplicate and frozen until analysis. Electron donors were generally added at 20 mM. Hydrogen (100% in headspace or 80:20 [vol/vol] H2:CO2) was supplied in combination with 4 mM acetate as carbon source. A mixture of fatty acids (acetate, propionate, butyrate, isobutyrate, isovalerate, valerate, β-hydroxybutyrate and ethanol; at 3 mM each), a mixture of amino acids (L-alanine, L-asparagine, L-threonine, L-cysteine; at 0.7 mM each) or yeast extract (at 1 g l−1) were also added as electron donors. 2.3 Dechlorination of various chlorinated compounds 2,4,6-Trichlorophenol (200 μM) or 3-chloro-4-hydroxyphenylacetate (3Cl4OHPA; 1 mM) were added to 20-ml batch cultures from aqueous stock solutions, 1,2,3- or 1,2,4-trichlorobenzene (TCB; 60 μM) from 1 M stock solutions in acetone. HCB, 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP), 3,4,5,6-tetrachloroveratrole (3,4,5,6-TeCV) or 2,4,8-trichlorodibenzofuran (2,4,8-TrCDF) (each 200 μM) were added from stock solutions in acetone to empty serum bottles or Hungate tubes before medium addition as already described [9]. Samples (2 ml) were taken over a period of 80 days and appropriately analyzed for the chlorinated compounds. Tetrachloroethene (PCE) dechlorination was studied using a hexadecane phase containing 200 mM PCE to give an initial 40 μM concentration in the water phase [20], while 40 mM L-lactate served as electron donor. 2.4 Most probable number (MPN) A dilution series was prepared in serum bottles, gassed with N2/CO2 (80:20, vol/vol), filled with 18 ml of the above described medium M204 supplemented with 10 mM pyruvate, 10 mM fumarate, 0.02% yeast extract and 5 mM BES, inoculated with 2 ml of the mixed culture and further diluted up to 10−8. The content of each inoculated bottle was dispensed in 3-ml portions into six replicate Hungate tubes containing 50 μM 1,2,4-TrCDD. The tubes were incubated for 8 weeks at 20°C and 150 rpm in the dark. Subsequently, three replicate Hungate tubes of each dilution step were sacrificed for dioxin analysis. Dechlorination was regarded as positive if at least 0.5 μM of 1,3-DCDD (detection limit 0.2 μM) had been formed. Three replicate tubes of each dilution step were analyzed for fatty acids and hydrogen and used for DNA extraction. 2.5 Procedures for the isolation and characterization of pure cultures Agar shake dilution series were prepared in sterilized medium M204 supplemented with 0.9% agar, 0.06% yeast extract, cosubstrates and BES (Table 1). The agar was kept molten at 50°C and dispensed in 1.8-ml aliquots into 5-ml culture tubes containing sterilized 1,2,4-TrCDD crystals (nominal concentration 10 μM). The agar shakes were inoculated with 0.2 ml of a 10−1 to 10−9 dilution series of the enrichment culture. After 2 weeks, colonies of different shape, density and color (yellowish to light pink) were picked from the 10−5 and 10−6 dilution steps and transferred into 5-ml tubes containing 2 ml medium M204 with 10 μM 1,2,4-TrCDD, 0.01% (w/v) yeast extract, cosubstrates and BES (Table 1) and 10% (vol/vol) filter-sterilized culture fluid of the sixth transfer. Two uninoculated tubes served as controls. After 8 weeks a volume of 1.5 ml was used for appropriate analyses and 0.5 ml was transferred as 10% (vol/vol) inoculum into fresh medium. The pure cultures EK7 and Coc4 were obtained by consecutive agar shake dilution series using medium M204 with 20 mM fumarate and 10 mM L-lactate (EK7) and 10 mM fumarate and 20 mM pyruvate (Coc4). Strain Coc4 fermentation products from pyruvate were determined in the presence of 0.01% yeast extract (products released from 0.01% yeast extract were used for correction). Determination of the guanine-plus-cytosine (G+C) content and of menaquinones was done as described [21]. DNA preparation and determination of DNA–DNA reassociation was carried out according to [22]. For microscopy, cells were concentrated by centrifugation. Gram staining was done using the Difco detection kit (Difco, Detroit, MI, USA). Transmission electron micrographs were obtained by negative staining with uranyl acetate [20] 2.6 Chemical determinations and chemicals Organic acids were determined on a FFAP capillary column (25 m×0.25 mm internal diameter [i.d.], film thickness 0.25 μm) [23]. Methane production was monitored as described previously [9]. Hydrogen was analyzed in 0.5 or 2.0 ml headspace samples on a RGA3 model E-001 reduction gas analyzer (Trace Analytical, Menlo Park, CA, USA) equipped with 60/80 unibeads 1S precolumn and a 60/80 molesieve 5A column (Supelco, Taufkirchen, Germany). Separation occurred isothermally at 105°C oven temperature with nitrogen as carrier gas (flow rate 20 ml min−1) and 265°C detector temperature. Concentrations of sulfide and formate were determined colorimetrically [24,25]. Chlorinated ethenes were analyzed as previously described [26]. Chlorophenols, 3Cl4OHPA and 3,4,5,6-TeCV were analyzed by high-performance liquid chromatography (HPLC) or gas chromatography (GC) [27]. Prior to analysis by GC-electron capture detection (ECD), chlorophenols and 3,4,5,6-TeCV were acetylated and extracted with n-hexane [28]. HCB was extracted twice with n-hexane and the combined extracts were analyzed by GC-ECD. Mono- to trichlorinated benzenes were determined from 1-ml samples of the culture fluid extracted by hexane and analyzed by GC-flame ionization detection (FID) using 2,4-dichlorotoluene as recovery standard and 1,3,5-tribromobenzene as internal standard [29,30]. The extraction, clean-up procedure and quantification of the PCDD congeners and 2,4,8-TrCDF by GC-ECD and GC-mass spectroscopy-selected ion monitoring (MS-SIM) followed the procedures previously described [9]. Due to the low signal intensity in GC-ECD, mono- and non-chlorinated dioxins were quantified by additional measurements using GC-FID (Shimadzu GC 14A) equipped with a DB608 megabore capillary column (30 m, 0.33 mm i.d., film thickness 0.5 μm; J&W Scientific, Folsom, CA, USA; detector: 320°C; injector: 260°C; split 1:10). The compounds (injection volume 4 μl; four-fold concentrated) were separated by initially holding the oven temperature 3 min at 170°C, then raising the temperature to 175°C with 1°C min−1, to 290°C with 5°C min−1 held finally constant for 5 min. The congeners were identified comparing the retention times with authentic standards and quantified according to a six-point calibration curve using a linear fit of the data ranging from 3 to 50 μM per congener. Chemicals were obtained from commercial sources, and in each case the highest purity available was used. Dioxin congeners were from Amchro, Hattersheim, Germany, except 1,3-DCDD, which was a kind gift from John R. Parsons, University of Amsterdam, The Netherlands. 2.7 Isolation of DNA and amplification, restriction and sequence determination of 16S rRNA genes Total community DNA was isolated from a 1.5-ml sample according to a combination of freeze-thawing [27] and ultrasonication (35 kHz, 10 min). MPN samples were treated by combined freeze-thawing and subsequent bead-beater homogenization. Freeze-thawed cell suspensions were mixed with an equal volume of 2×TENS buffer [31] and transferred onto autoclaved glass beads (diameter 0.25 mm) in an 1.5-ml reaction tube. Samples were homogenized in a bead-beater (Retsch, Haan, Germany) for 15 min at highest speed. After centrifugation (15 min, 13 000×g) the supernatant was stored and the glass beads were washed twice with sterile water (600 μl). After subsequent centrifugation, the supernatants were combined and total DNA was concentrated by ethanol precipitation. The resulting extracts contained 200–600 ng total DNA μl−1. PCR amplification of 16S rRNA genes was performed using 50-μl reaction mixtures containing 0.5 U Taq-polymerase, 5 μl 10×PCR buffer, 1.6 mM MgCl2, 0.21 μM of each primer (Table 2) and 200 μM of each deoxynucleoside triphosphate (Roche Diagnostics, Mannheim, Germany). All amplifications were carried out in a thermocycler (PCR Sprint, Hybaid, Heidelberg, Germany) using the following temperature program: 94°C for 3 min, 10 cycles 94°C for 15 s, 50–58°C for 45 s and 72°C for 1.5 min; 20 similar cycles with a time increment of 10 s added to the elongation phase of each cycle; final extension 72°C for 5 min. Almost complete 16S rRNA genes of Bacteria and fragments of archaeal 16S rRNA genes were amplified from 1-μl aliquots of total DNA with a bacterial primer mixture (fD1/fD2 and rP1/rP2) and Archaea-specific primers Archaea F/R, respectively (Table 2). The resulting bulk 16S rDNA was purified with the PCR purification kit (Qiagen, Hilden, Germany) and used as template (1 μl) for a subsequent nested PCR (for primers see Table 2) targeting Dehalobacter restrictus (DRE445/858N), Desulfomonile tiedjei (DTI178/858N), Dehalococcoides ethenogenes (DET730/DET1350), Trichococcus species (LPA208/858N) and Desulfuromonas chloroethenica (DCH205/DCH1033). The latter primer pair was shown to be specific for D. chloroethenica and Desulfuromonas sp. strain BB1, but gave also a product from Desulfuromonas acetexigens[32]. 16S rRNA gene fragments of the Sulfurospirillum group were amplified with primers SUG016/796. SUG016 perfectly matched the respective sequence stretches of the members of the Sulfurospirillum clade [33,34] (Sulfurospirillum deleyianum, Sulfurospirillum barnesii, Sulfurospirillum multivorans, Sulfurospirillum halorespirans, Sulfurospirillum arsenophilum; except Sulfurospirillum archachonense [one mismatch]). 16S rRNA gene fragments of Desulfitobacterium hafniense were amplified directly from total DNA or by nested PCR using primers PCP2G/PCP4G. 2 Sequences of primers used in this work for the amplification of 16S rDNA fragments Primer Sequence (5′→3′) E. coli position [39] Organism(s) with complementary target sequences fD1a AGA GTT TGA TCC TGG CTC AG 8–27 most Bacteria fD2a AGA GTT TGA TCA TGG CTC AG 8–27 enterics and relatives, many Bacteria rP1a ACG GTT ACC TTG TTA CGA CTT 1512–1492 most Bacteria rP2a ACG GCT ACC TTG TTA CGA CTT 1512–1492 most Bacteria Archaea Fb TTC CGG TTG ATC CTG CCG GA 7–27 Archaea Archaea Rb CCC GCC AAT TCC TTT AAG TTT C 927–905 Archaea 796c GGG TTG CGC TCG TTG 1114–1100 most Bacteria 858Nc GGG TTG CGC TCG TTG C 1114–1099 most Bacteria DTI178d ATG AGA CCA CAT GAG CTC 179–197 D. tiedjei LPA208d TTG TGC TGT CGC TTA TGG 209–227 Trichococcus species DRE445d GGA AGA ACG GCA TCT GTG 446–464 D. restrictus DET730d GCG GTT TTC TAG GTT GTC 731–748 Dehalococcoides species DET1350d CAC CTT GCT GAT ATG CGG 1351–1333 D. ethenogenes PCP2Ge AGA TGG CCT CTG AAA ATG 205–220 D. hafniense PCP4De AGG TAC CGT CAT GTA AGT AC 490–471 D. hafniense SUG016d GCT AAA GGA TGG GGC T 220–235 Sulfurospirillum species DCH205f AAC CTT CGG GTC CTA CTG TC 205–222 D. chloroethenica DCH1015f GCC GAA CTG ACC CCT ATG TT 1033–1015 D. chloroethenica PG1fg TGG CGG CCG CGG GAA TTC PG2rg GGC CGC GAA TTC ACT AGT G Primer Sequence (5′→3′) E. coli position [39] Organism(s) with complementary target sequences fD1a AGA GTT TGA TCC TGG CTC AG 8–27 most Bacteria fD2a AGA GTT TGA TCA TGG CTC AG 8–27 enterics and relatives, many Bacteria rP1a ACG GTT ACC TTG TTA CGA CTT 1512–1492 most Bacteria rP2a ACG GCT ACC TTG TTA CGA CTT 1512–1492 most Bacteria Archaea Fb TTC CGG TTG ATC CTG CCG GA 7–27 Archaea Archaea Rb CCC GCC AAT TCC TTT AAG TTT C 927–905 Archaea 796c GGG TTG CGC TCG TTG 1114–1100 most Bacteria 858Nc GGG TTG CGC TCG TTG C 1114–1099 most Bacteria DTI178d ATG AGA CCA CAT GAG CTC 179–197 D. tiedjei LPA208d TTG TGC TGT CGC TTA TGG 209–227 Trichococcus species DRE445d GGA AGA ACG GCA TCT GTG 446–464 D. restrictus DET730d GCG GTT TTC TAG GTT GTC 731–748 Dehalococcoides species DET1350d CAC CTT GCT GAT ATG CGG 1351–1333 D. ethenogenes PCP2Ge AGA TGG CCT CTG AAA ATG 205–220 D. hafniense PCP4De AGG TAC CGT CAT GTA AGT AC 490–471 D. hafniense SUG016d GCT AAA GGA TGG GGC T 220–235 Sulfurospirillum species DCH205f AAC CTT CGG GTC CTA CTG TC 205–222 D. chloroethenica DCH1015f GCC GAA CTG ACC CCT ATG TT 1033–1015 D. chloroethenica PG1fg TGG CGG CCG CGG GAA TTC PG2rg GGC CGC GAA TTC ACT AGT G aPrimer was taken from [61]. bPrimer was taken from [62]. cPrimers are complementary to highly conserved regions within the bacterial 16S rDNA. dPrimers were designed during this work using ARB software [63]. ePrimer was taken from [42]. fPrimer was taken from [32]. gPrimer sequences are complementary to the 5′ and 3′ sequences in immediate vicinity of the insertion site of the pGEM-T-Easy vector (Promega Corp., Madison, WI, USA). Open in new tab 2 Sequences of primers used in this work for the amplification of 16S rDNA fragments Primer Sequence (5′→3′) E. coli position [39] Organism(s) with complementary target sequences fD1a AGA GTT TGA TCC TGG CTC AG 8–27 most Bacteria fD2a AGA GTT TGA TCA TGG CTC AG 8–27 enterics and relatives, many Bacteria rP1a ACG GTT ACC TTG TTA CGA CTT 1512–1492 most Bacteria rP2a ACG GCT ACC TTG TTA CGA CTT 1512–1492 most Bacteria Archaea Fb TTC CGG TTG ATC CTG CCG GA 7–27 Archaea Archaea Rb CCC GCC AAT TCC TTT AAG TTT C 927–905 Archaea 796c GGG TTG CGC TCG TTG 1114–1100 most Bacteria 858Nc GGG TTG CGC TCG TTG C 1114–1099 most Bacteria DTI178d ATG AGA CCA CAT GAG CTC 179–197 D. tiedjei LPA208d TTG TGC TGT CGC TTA TGG 209–227 Trichococcus species DRE445d GGA AGA ACG GCA TCT GTG 446–464 D. restrictus DET730d GCG GTT TTC TAG GTT GTC 731–748 Dehalococcoides species DET1350d CAC CTT GCT GAT ATG CGG 1351–1333 D. ethenogenes PCP2Ge AGA TGG CCT CTG AAA ATG 205–220 D. hafniense PCP4De AGG TAC CGT CAT GTA AGT AC 490–471 D. hafniense SUG016d GCT AAA GGA TGG GGC T 220–235 Sulfurospirillum species DCH205f AAC CTT CGG GTC CTA CTG TC 205–222 D. chloroethenica DCH1015f GCC GAA CTG ACC CCT ATG TT 1033–1015 D. chloroethenica PG1fg TGG CGG CCG CGG GAA TTC PG2rg GGC CGC GAA TTC ACT AGT G Primer Sequence (5′→3′) E. coli position [39] Organism(s) with complementary target sequences fD1a AGA GTT TGA TCC TGG CTC AG 8–27 most Bacteria fD2a AGA GTT TGA TCA TGG CTC AG 8–27 enterics and relatives, many Bacteria rP1a ACG GTT ACC TTG TTA CGA CTT 1512–1492 most Bacteria rP2a ACG GCT ACC TTG TTA CGA CTT 1512–1492 most Bacteria Archaea Fb TTC CGG TTG ATC CTG CCG GA 7–27 Archaea Archaea Rb CCC GCC AAT TCC TTT AAG TTT C 927–905 Archaea 796c GGG TTG CGC TCG TTG 1114–1100 most Bacteria 858Nc GGG TTG CGC TCG TTG C 1114–1099 most Bacteria DTI178d ATG AGA CCA CAT GAG CTC 179–197 D. tiedjei LPA208d TTG TGC TGT CGC TTA TGG 209–227 Trichococcus species DRE445d GGA AGA ACG GCA TCT GTG 446–464 D. restrictus DET730d GCG GTT TTC TAG GTT GTC 731–748 Dehalococcoides species DET1350d CAC CTT GCT GAT ATG CGG 1351–1333 D. ethenogenes PCP2Ge AGA TGG CCT CTG AAA ATG 205–220 D. hafniense PCP4De AGG TAC CGT CAT GTA AGT AC 490–471 D. hafniense SUG016d GCT AAA GGA TGG GGC T 220–235 Sulfurospirillum species DCH205f AAC CTT CGG GTC CTA CTG TC 205–222 D. chloroethenica DCH1015f GCC GAA CTG ACC CCT ATG TT 1033–1015 D. chloroethenica PG1fg TGG CGG CCG CGG GAA TTC PG2rg GGC CGC GAA TTC ACT AGT G aPrimer was taken from [61]. bPrimer was taken from [62]. cPrimers are complementary to highly conserved regions within the bacterial 16S rDNA. dPrimers were designed during this work using ARB software [63]. ePrimer was taken from [42]. fPrimer was taken from [32]. gPrimer sequences are complementary to the 5′ and 3′ sequences in immediate vicinity of the insertion site of the pGEM-T-Easy vector (Promega Corp., Madison, WI, USA). Open in new tab The sensitivity of the nested PCR assay to detect Dehalococcoides was determined using 1:5 dilution series (6×1010 to 0 copies) of a plasmid containing the 16S rRNA gene amplified and cloned from a recently described dioxin-dechlorinating mixed culture [16]. The determined sequence (1421 bp) was almost identical (one mismatch at Escherichia coli position 689) with the sequence of strain CBDB1 [15]. The first step of the nested PCR with universal primers fD1/fD2 and rP1/rP2 was performed with 1 μl of the respective plasmid dilution. Additionally, 1 μl (500 ng) of total DNA extracted from a pure culture of D. hafniense strain PCP-1 was added to mimic the presence of total DNA in extracts from mixed cultures. Following the nested PCR step with primers DET730/DET1350, 2-μl aliquots were separated on 1% agarose gels. For visualization of a PCR product on gel, at least 250 plasmid copies in the primary assay were necessary. Assuming one 16S rRNA gene copy per Dehalococcoides cell and considering a concentration factor of 15 during DNA extraction, this would account for a detection limit of about 103Dehalococcoides cells ml−1. 16S rRNA genes of the mixed culture were cloned into E. coli JM 109 using the pGEM-T-Easy vector system (Promega, Madison, WI, USA). Amplified ribosomal DNA restriction analysis (ARDRA) was performed as described [27]. For reamplification of the cloned inserts vector-specific primers (PG1f and PG2r, Table 2) were used. For sequence analysis, 16S rDNA amplicons or plasmids were purified using the PCR purification kit or the plasmid miniprep kit (Qiagen, Hilden, Germany), respectively. Cycle sequencing was performed using the automated laser fluorescence DNA sequencer ABI Prism 377, version 4.0 (PE Applied Biosystems, Langen, Germany), universal bacterial primers binding to conserved regions of 16S rRNA genes [35] or the vector-specific primers PG1f and PG2r. Bidirectional sequencing of the partial Dehalococcoides sequence was performed using primers Det730 and Det1350 (Table 2). 2.8 Nucleotide sequence accession numbers The partial sequences of 16S rRNA genes of strains Sulfurospirillum sp. EK7 and Trichococcus sp. Coc4, and the 16S rDNA sequence of clone A4, similar to the sequence of Acetobacterium malicum, were deposited in the EMBL, GenBank, and DDBJ nucleotide sequence databases under accession numbers AJ535704–AJ535706, respectively. 3 Results 3.1 Reductive dechlorination of 1,2,4- and 1,2,3-TrCDD Two previously described 1,2,4- and 1,2,3-TrCDD-dechlorinating second sequential transfer cultures supplied with 50% (vol/vol) of gamma ray-sterilized sediment [9] served as inoculum for the establishment of dioxin-dechlorinating enrichment cultures (Table 1). A 10% transfer of the 1,2,3-TrCDD-grown subculture was used to study reductive dechlorination of both 1,2,3- and 1,2,4-TrCDD without further addition of gamma ray-sterilized sediment (Fig. 1). 1,2,4-TrCDD was transformed at a rate of 0.2 μmol l−1 day−1 (as taken for the period of 0–32 days) to equimolar amounts of 1,3-DCDD plus 2-MCDD (Fig. 1A). In comparison, reductive dechlorination of 1,2,3-TrCDD and formation of the products 1,3-DCDD, 2,3-DCDD and 2-MCDD was somewhat slower (0.12 μmol l−1 day−1) (Fig. 1B). Interestingly, the ratio of both intermediary DCDDs became slightly changed to an increased amount of 1,3-DCDD compared to results with the previously described 1,2,3-TrCDD-dechlorinating culture [9]. This suggests that consecutive subcultivation with 1,2,3-TrCDD may select for enhanced removal of chlorine atoms from a lateral position (Fig. 1C). However, the observed rate and products were in accordance with those of the sediment-supplied precultures. Thus, further transfers were performed without addition of sterilized sediment. Due to the higher dechlorination rate, 1,2,4-TrCDD was chosen as the electron acceptor for further transfers. The 1,2,4-TrCDD-dechlorinating second sequential transfer [9] has further been successfully transferred (10% [vol/vol]) 16 times. In no case was reductive dechlorination observed in uninoculated or autoclaved controls. 1 Open in new tabDownload slide Reductive dechlorination of 1,2,4-TrCDD (A) and 1,2,3-TrCDD (B) in the absence of added sterilized sediment and comparison with setups pasteurized at 80°C. Mean values and standard deviations of three replicates are shown. The dechlorination pathways were proposed on the basis of the products formed. The sites of chlorine removal from peri(pheral) and lateral positions are indicated at the arrows. 1 Open in new tabDownload slide Reductive dechlorination of 1,2,4-TrCDD (A) and 1,2,3-TrCDD (B) in the absence of added sterilized sediment and comparison with setups pasteurized at 80°C. Mean values and standard deviations of three replicates are shown. The dechlorination pathways were proposed on the basis of the products formed. The sites of chlorine removal from peri(pheral) and lateral positions are indicated at the arrows. Dechlorination took place with four different initial concentrations of 1,2,4-TrCDD (1, 10, 40 and 250 μM) (Fig. 2). Within 10 days 1 μM 1,2,4-TrCDD was converted to 80 mol% of 1,3-DCDD (not shown in Fig. 2). The rate of product formation increased with increasing substrate concentration from 0.05 to 5.4 μmol l−1 day−1. All concentrations tested exceeded the water solubility of 1,2,4-TrCDD (30 nM) [36], suggesting that its availability might not depend just on its water solubility but rather on the direct contact of cells (or their lipids) to dioxin crystals. Some electron acceptors with similar low water solubility such as ferric iron can be faster reduced by anaerobic bacteria by addition of humic acids or the model compound AQSA as electron shuttle [19]. However, neither humic acids nor AQSA stimulated microbial reductive dechlorination of 1,2,4-TrCDD compared to controls (data not shown). 2 Open in new tabDownload slide Formation of 1,3-DCDD from 10, 40 and 250 μM 1,2,4-TrCDD. Data represent mean values of duplicate or triplicate cultures sacrificed for analysis. 2 Open in new tabDownload slide Formation of 1,3-DCDD from 10, 40 and 250 μM 1,2,4-TrCDD. Data represent mean values of duplicate or triplicate cultures sacrificed for analysis. The extent of 1,3-DCDD formation from 10 μM 1,2,4-TrCDD was highest at 20°C (36 mol% at day 90), the usual incubation temperature of the culture. However, reductive dechlorination was also observed at 10 and 30°C (23 and 28 mol%, respectively, at day 90). No dechlorination occurred at 37°C within 90 days. 3.2 Reductive dechlorination of various chlorinated compounds Of other chlorinated aromatic compounds tested (see Section 2) only 1,2,3-TCB (60 μM) was reductively dechlorinated to 60 mol% of 1,3-dichlorobenzene (1,3-DCB) within 56 days. Dechlorination started after a lag phase of 14 days and reached a rate of 1.4 μmol l−1 day−1 between days 42 and 56. The enrichment culture was also able to dechlorinate chlorinated ethenes. Inoculum (10% [vol/vol]) was taken from different stages of the enrichment culture (Table 1) and incubated with a PCE-containing hexadecane phase. After 157 days, only the culture inoculated with the second transfer showed an almost complete conversion of the nominal amount of 10 mM PCE to cis-dichloroethene (DCE), traces of trans-DCE and trichloroethene (TCE). In all the other cultures only partial dechlorination of PCE to mainly TCE occurred. 3.3 Influence of different potential electron donors on the dechlorination process The single constituents of the cosubstrate mixtures (acetate, fumarate, pyruvate, lactate, benzoate, and yeast extract ([0.05 and 0.1%]) and some other potential electron donors (a fatty acid and an amino acid mixture, formate and hydrogen) were separately added to parallel inoculated cultures (Table 1). Within 23 days, 1,3-DCDD was formed in amounts of 2, 3, 5, 20 or 41 mol% only in cultures, which were supplied with lactate, acetate, pyruvate, fumarate or 0.1% yeast extract, respectively. After a longer period (74 days) 1,2,4-TrCDD was almost completely converted. In addition, a similar conversion was observed after 74 days in cultures incubated with the fatty acid or the amino acid mixture, 0.005% yeast extract and even without an added electron donor, suggesting that a carryover of residual yeast extract or lysed cell material from the preculture might be sufficient to maintain dechlorination. Dechlorination did not occur, when benzoate, formate or hydrogen was added as an electron donor. Although benzoate was utilized by the fourth sequential transfer (data not shown), no conversion was observed later, indicating a loss of benzoate-oxidizing bacteria during the enrichment procedure. In the presence of hydrogen, 40–50 mM acetate was formed indicating activity of acetogenic bacteria. In general, the fermentation of the electron donating cosubstrates was almost complete within 16 days. Acetate was the main organic acid formed from pyruvate, formate, yeast extract and the fatty acid mixture, whereas acetate plus propionate were formed from lactate. Fumarate was first reduced to succinate and then converted to acetate plus propionate. Fig. 3 demonstrates the metabolism of the mixture of electron donors usually added up to the 11th transfer, which reflects the fermentation processes observed with the single cosubstrates. However, the formation of 1,3-DCDD from 1,2,4-TrCDD seemed not to depend directly on one of the individual processes because it continued after the metabolism of the organic acids had ceased. 3 Open in new tabDownload slide Formation of 1,3-DCDD from 50 μM 1,2,4-TrCDD and of fermentation products (succinate and propionate) from the cosubstrates added (10 mM L-lactate, 5 mM fumarate, 5 mM acetate and 5 mM benzoate [the latter was not converted; data not shown]). 3 Open in new tabDownload slide Formation of 1,3-DCDD from 50 μM 1,2,4-TrCDD and of fermentation products (succinate and propionate) from the cosubstrates added (10 mM L-lactate, 5 mM fumarate, 5 mM acetate and 5 mM benzoate [the latter was not converted; data not shown]). 3.4 Contribution of heat-resistant, sulfate-reducing and vancomycin-resistant bacteria, and methanogens to the dehalogenation process Subcultures treated at 75, 80 (Fig. 1) or 85°C for 15 min lost completely their 1,2,4- and 1,2,3-TrCDD dechlorination activity. Thus, heat-sensitive, non-spore-forming bacteria appeared essential for the dechlorination of 1,2,4-TrCDD. A possible role of sulfate-reducing bacteria and methanogens in dechlorination was examined using the competitive inhibitors molybdate and BES, respectively [37,38]. BES completely inhibited methane formation, but dechlorination activity of 1,2,4-TrCDD was not affected compared to a control. Likewise, the addition of 1 mM molybdate had no significant influence on dechlorination. When vancomycin was added, the dechlorination of 1,2,4-TrCDD was somewhat delayed but the same amount of 1,3-DCDD (95 mol%) was finally formed compared to a control after 98 days. 3.5 Isolation and characterization of members of the dechlorinating community Colonies from agar shake dilution series were isolated and their potential to dechlorinate 1,2,4-TrCDD was investigated. The colony releasing the highest amount of 1,3-DCDD (16 mol%) after 8 weeks of incubation consisted of cocci, short rods and vibrio-shaped cells. The coccoid bacterium Coc4 was isolated from further consecutive agar shake dilution series. Cells stained Gram-positive, were 1.8–2.5 μm in diameter and non-motile. The G+C content was 47.5 mol%. Coc4 is regarded as a member of the genus Trichococcus according to the 99% identity of the 16S rRNA gene sequence (29–1491 bp E. coli numbering, [39]) with that of Trichococcus collinsii and Trichococcus pasteurii[40,41]. Growth of strain Coc4 was tested on fumarate and pyruvate, the two carbon sources usually added to the mixed cultures. Fumarate did not serve as substrate for growth, but pyruvate (20 mM) was converted to 17 mM acetate and 4.6 mM formate. Strain Coc4 was not able to dechlorinate 1,2,4-TrCDD. Another colony formed more than 10 mol% 1,3-DCDD and consisted of vibrioid cells (diameter 0.4 μm; length, 1.2–2 μm) motile by a polar flagellum. Strain EK7 was isolated from subsequent agar shake dilutions. Colonies formed in agar shakes were lens shaped and yellowish brown, whereas colonies grown on agar surfaces appeared smooth and pale-white. The temperature for growth ranged from 4 to 37°C with an optimum at 20–24°C. The G+C content was 38 mol%, and menaquinone MK-6 and methylmenachinone MMK-6 were present in a ratio of 2:1. Highest sequence identities of 16S rRNA genes (93–1366 bp E. coli numbering, [39]) were found to the ε-Proteobacteria S. deleyianumT (98.8%), S. barnesii (98.1%) and S. multivoransT (97.9%). DNA–DNA reassociation revealed a similarity of 60% with S. deleyianum (DSM 6946T) and 31% with S. multivorans (DSM 12446), suggesting strain EK7 as a species within the genus Sulfurospirillum somewhat different from the type species S. deleyianum. Using formate as electron donor and acetate as carbon source, strain EK7 reduced oxygen (2–7% air in the headspace), nitrate (reduced to ammonium), thiosulfate, elemental sulfur (both reduced to sulfide), Fe(III), selenite and arsenate. Using nitrate as electron acceptor, growth occurred with formate, hydrogen, pyruvate, fumarate and succinate as electron donors. Sulfide was oxidized to sulfur in the presence of fumarate or nitrate. Selenate, sulfate, sulfite, PCE, HCB, γ-hexachlorocyclohexane, 1,2,3,4-TCDD and different TrCDDs (1,2,4-, 1,2,3-, 1,7,8-, 2,3,7-TrCDD; additionally tested in combination with various electron donors) were not reduced. Fermentative growth occurred with fumarate or malate (20 mM), which were both converted to succinate (13.5 mM) and acetate (5.5 mM). 3.6 Identification of members of the dechlorinating community by amplification, sequencing, and restriction analysis of 16S rDNA Two 1,2,4-TrCDD-dechlorinating transfers, and the cultures tested for PCE dechlorination (Table 1), were investigated for the presence of D. hafniense (formerly Desulfitobacterium frappieri), a bacterium isolated previously from the same sampling location able to dechlorinate chlorophenols [27]. To specifically select for Desulfitobacterium, subcultures were inoculated and supplied with thiosulfate, pyruvate, fumarate (10 mM each) and 0.2% yeast extract. After 4 days of incubation, DNA was extracted and subjected to PCR with specific primers PCP2G/PCP4D [42] targeting D. hafniense 16S rRNA genes. PCR products of the expected size (295 bp) were obtained in all cases. Sequence analysis of the 16S rRNA gene fragment revealed identity with the respective sequences of D. hafniense strains [27,43–45] and one to 10 mismatches to other species of Desulfitobacterium. The community structure of a late sequential transfer (Table 1) incubated with 50 μM 1,2,4-TrCDD was studied in detail using a PCR approach. 16S rRNA gene fragments of Bacteria and Archaea were attempted to amplify from the culture at day 55, when 50 μM 1,2,4-TrCDD were almost completely converted to 1,3-DCDD. The archaeal primers Archaea F/R yielded no product probably due to the routinely added BES. The primer combination targeting the domain Bacteria yielded a PCR product, which was subsequently used in a nested PCR with specific primers targeting distinct bacteria. PCR products were obtained with Sulfurospirillum group-targeted and Trichococcus-targeted primers suggesting that strains EK7 and Coc4 were still present in this late sequential transfer. No PCR products were obtained with primers specific for D. restrictus and D. tiedjei. However, products of the expected sizes were produced with primers targeting D. chloroethenica and D. ethenogenes. The product of the latter PCR was bidirectionally sequenced and revealed high identity with the sequences of the chlorobenzene-dehalorespiring Dehalococcoides sp. strain CBDB1 [15] and the PCE-dehalogenating D. ethenogenes strain 195 [46] (Table 3). 3 Sequence analysis of 16S rRNA gene fragments amplified from the 13th sequential transfer culture Source of the fragment Sequenced region of 16S rRNA genea Closest relative (accession number) Identity (%) D. ethenogenes-targeted nested PCR 749–1350 Dehalococcoides sp. strain CBDB1 (AF230641) 100 D. ethenogenes strain 195T (AF004928) 98.5 Profile A3 (84% frequency) 512–1491 Trichococcus strain Coc4 (this work) 100 T. pasteuriiT (X87150) 100 Profile A32 (4.5% frequency) 702–1491 Sulfurospirillum sp. strain EK7 (this work) 98.7 S. deleyianumT (Y13671) 98.6 S. multivoransT (X82931) 98.3 Profile A4 (2% frequency) 31–1488 Acetobacterium malicumT (X96957) 99.0 Acetobacterium wieringaeT (X96955) 99.0 Source of the fragment Sequenced region of 16S rRNA genea Closest relative (accession number) Identity (%) D. ethenogenes-targeted nested PCR 749–1350 Dehalococcoides sp. strain CBDB1 (AF230641) 100 D. ethenogenes strain 195T (AF004928) 98.5 Profile A3 (84% frequency) 512–1491 Trichococcus strain Coc4 (this work) 100 T. pasteuriiT (X87150) 100 Profile A32 (4.5% frequency) 702–1491 Sulfurospirillum sp. strain EK7 (this work) 98.7 S. deleyianumT (Y13671) 98.6 S. multivoransT (X82931) 98.3 Profile A4 (2% frequency) 31–1488 Acetobacterium malicumT (X96957) 99.0 Acetobacterium wieringaeT (X96955) 99.0 aAccording to E. coli 16S rDNA [39]. Open in new tab 3 Sequence analysis of 16S rRNA gene fragments amplified from the 13th sequential transfer culture Source of the fragment Sequenced region of 16S rRNA genea Closest relative (accession number) Identity (%) D. ethenogenes-targeted nested PCR 749–1350 Dehalococcoides sp. strain CBDB1 (AF230641) 100 D. ethenogenes strain 195T (AF004928) 98.5 Profile A3 (84% frequency) 512–1491 Trichococcus strain Coc4 (this work) 100 T. pasteuriiT (X87150) 100 Profile A32 (4.5% frequency) 702–1491 Sulfurospirillum sp. strain EK7 (this work) 98.7 S. deleyianumT (Y13671) 98.6 S. multivoransT (X82931) 98.3 Profile A4 (2% frequency) 31–1488 Acetobacterium malicumT (X96957) 99.0 Acetobacterium wieringaeT (X96955) 99.0 Source of the fragment Sequenced region of 16S rRNA genea Closest relative (accession number) Identity (%) D. ethenogenes-targeted nested PCR 749–1350 Dehalococcoides sp. strain CBDB1 (AF230641) 100 D. ethenogenes strain 195T (AF004928) 98.5 Profile A3 (84% frequency) 512–1491 Trichococcus strain Coc4 (this work) 100 T. pasteuriiT (X87150) 100 Profile A32 (4.5% frequency) 702–1491 Sulfurospirillum sp. strain EK7 (this work) 98.7 S. deleyianumT (Y13671) 98.6 S. multivoransT (X82931) 98.3 Profile A4 (2% frequency) 31–1488 Acetobacterium malicumT (X96957) 99.0 Acetobacterium wieringaeT (X96955) 99.0 aAccording to E. coli 16S rDNA [39]. Open in new tab Inoculum from the 15th transfer was subcultured two times (10%, vol/vol) in duplicate with 1,2,4-TrCDD (60 and 25 μM, respectively) or without dioxin. A distinct Dehalococcoides 16S rRNA gene fragment was detected in all transfers incubated with dioxins, but only in the first transfer cultivated without dioxin, suggesting the loss of Dehalococcoides during further subcultivation in the absence of dioxins. 16S rRNA genes amplified and cloned from the 13th sequential transfer was subjected to restriction analysis following the ARDRA strategy described earlier [27]. Seven different profiles (A2, A3, A4, A10, A21, A29, A32) were obtained. Representative inserts (except A21 and A29) have been sequenced. The most frequent pattern A3 (37 of 44 clones) represented 16S rRNA genes of Trichococcus (Table 3). Another profile (A32) corresponded to 16S rRNA genes of Sulfurospirillum with high similarity to the sequence of strain EK7 (Table 3). All other restriction patterns occurred only once. Novel was the identification of Acetobacterium (profile A4), a Gram-positive obligate anaerobe able to use hydrogen and carbon dioxide to form acetate via the acetyl-coenzyme A (CoA) pathway (Table 3). The profiles A2 and A10 were analyzed as fragments of chimeric 16S rDNA from Trichococcus and S. deleyianum. The remaining two profiles (A21 and A29) were also suspected to represent chimeras as the fragment pattern showed a high similarity to the most frequent profile A3. 3.7 Estimation of cell numbers and metabolic properties of dioxin-dehalogenating and other members of the community using the MPN technique The sequential transfer culture also studied for the community composition was additionally subjected to MPN analysis at day 59. 1,2,4-TrCDD dechlorination occurred until the 10−3 dilution step in all three replicates and in no replicate of higher dilution steps resulting in an estimated number of 2.5×103 dioxin-dechlorinating bacteria per ml. The total bacterial number was >108 cells per ml as indicated by metabolic activities and amplification of 16S rRNA genes from higher dilution steps (see below). The formation of hydrogen, methane, volatile and non-volatile fatty acids was also studied in these MPN series. Methane was not detectable. Hydrogen concentrations calculated for the water phase were between 3 and 45 nM in dilution steps 10−1 and 10−2 and higher than 50 nM in all other dilution steps up to 10−8. Other products of fermentative and/or acetogenic activities were acetate (4–11 mM) and propionate (2–6.4 mM) found in all dilution steps. Traces (<0.2 mM) of isobutyrate, butyrate and isovalerate were detected in most cases probably due to the 0.02% yeast extract added. 16S rRNA genes were amplified from one replicate of each dilution step and subjected to nested PCR directed against 16S rRNA genes of Trichococcus, Sulfurospirillum, Dehalococcoides, D. chloroethenica and D. hafniense (Table 4). In accordance with the results of ARDRA, the high abundance of Trichococcus and the Sulfurospirillum group both present up to the 108 and 107 dilution, respectively, was shown. Interestingly, D. hafniense was also found in numbers of at least 108 per ml, although it was not represented in the 16S rRNA genes comprising clone library of this culture, perhaps due to inefficient cell lysis or other pitfalls in PCR and cloning procedures [47]. The D. chloroethenica- and Dehalococcoides-targeted PCR yielded products up to the dilutions 10−5 and 10−4, respectively (Table 4). 4 Occurrence of selected bacteria in MPN dilution tubes of the 13th sequential transfer culture as indicated by nested PCR amplification of specific 16S rDNA fragments after 8 weeks of incubation in the presence of 50 μM 1,2,4-TrCDD Target organisma Dilution step 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8 Trichococcus + + + + + + + + Sulfurospirillum group + + + + + + + − D. ethenogenes + + +/±b ±/−b − − − − D. chloroethenica + + + − + − − − D. hafniense + + + + + + + + Target organisma Dilution step 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8 Trichococcus + + + + + + + + Sulfurospirillum group + + + + + + + − D. ethenogenes + + +/±b ±/−b − − − − D. chloroethenica + + + − + − − − D. hafniense + + + + + + + + aPrimer pairs are given in Section 2. bResults of three replicate tubes are shown. Open in new tab 4 Occurrence of selected bacteria in MPN dilution tubes of the 13th sequential transfer culture as indicated by nested PCR amplification of specific 16S rDNA fragments after 8 weeks of incubation in the presence of 50 μM 1,2,4-TrCDD Target organisma Dilution step 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8 Trichococcus + + + + + + + + Sulfurospirillum group + + + + + + + − D. ethenogenes + + +/±b ±/−b − − − − D. chloroethenica + + + − + − − − D. hafniense + + + + + + + + Target organisma Dilution step 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8 Trichococcus + + + + + + + + Sulfurospirillum group + + + + + + + − D. ethenogenes + + +/±b ±/−b − − − − D. chloroethenica + + + − + − − − D. hafniense + + + + + + + + aPrimer pairs are given in Section 2. bResults of three replicate tubes are shown. Open in new tab 4 Discussion This is the first description of an anaerobic dioxin-dechlorinating enrichment culture, which could be successfully transferred without additions of sterilized sediment. 1,2,4-TrCDD was consistently dechlorinated after 16 transfers via 1,3-DCDD to 2-MCDD. Dechlorination proceeded via two subsequent steps of chlorine removal from the peri-positions 1 and 4 (Fig. 1). The dechlorination products from 1,2,3-TrCDD reflected chlorine removal from a lateral and peripheral position leading to 1,3-DCDD and 2,3-DCDD, respectively. The final product of both pathways seemed to be 2-MCDD, although it cannot be excluded that only 1,3- or 2,3-DCDD was the precursor. Similar dechlorination products from both TrCDDs were observed in a methanogenic enrichment culture from river Rhine (except for the additional observation of 1,2- and 1,4-DCDDs from 1,2,4-TrCDD) [48] and two enrichment cultures from river Spittelwasser [13]. In contrast, other enrichment cultures exhibited a strong lateral dechlorination activity which yielded only 1,3-DCDD from 1,2,3-TrCDD [13]. This indicates that different dioxin-dechlorinating microbial populations exist that mediate chlorine removal from distinct positions. A diversity of organisms was identified in the mixed culture by a combination of microbiological and molecular methods. The most frequent bacteria, as identified by 16S rDNA targeting methods in the MPN experiment, belonged to Trichococcus, Sulfurospirillum and Desulfitobacterium. Members of Trichococcus and Desulfitobacterium ferment pyruvate [20,41], whereas Sulfurospirillum species ferment fumarate [33]. Sulfurospirillum sp. strain EK7 and members of Desulfitobacterium[20] are also able to grow by fumarate respiration. These properties might explain the high abundance of these organisms in the mixed culture supplied with fumarate and pyruvate as cosubstrates. The Trichococcus and Sulfurospirillum isolates Coc4 and EK7 did not dechlorinate TrCDDs, although the latter is distantly related to PCE-dechlorinating species S. multivorans[49] and S. halorespirans[34]. No dechlorination of TrCDDs and 1,2,3,4-TCDD was found for Desulfitobacterium strains PCE1 (DSM 10344), D. hafniense DCB-2T (DSM 10664) and Desulfitobacterium dehalogenans JW/IU-DC1T (DSM 9161) [50]. Fumarate, pyruvate, acetate or lactate as sole primary energy and carbon donors supported reductive dechlorination of 1,2,4-TrCDD. However, no correlation between consumption of the individual cosubstrates and reductive dechlorination was observed (Fig. 3). Acetate was the only fermentation product, constantly formed in the mixed culture, e.g. as a result of fumarate fermentation by strain EK7 or of pyruvate fermentation by strain Coc4. Thus, acetate might be a putative primary electron donor for reductive dechlorination. For example, the PCE-dechlorinating D. chloroethenica[51] and the recently described ortho-PCB-dechlorinating bacterium o-17 [52] are known to use acetate as electron donor for reductive dechlorination. However, an indirect role of acetate is also possible, because it can be fermented to CO2 and hydrogen in syntrophic interaction with hydrogenotrophic bacteria. Hydrogen was measured in a concentration reasonable for the stimulation of dehalorespiration [53]. The MPN experiment demonstrated a high abundance of hydrogen-producing bacteria within the mixed culture. Despite the fact that an excess of externally added hydrogen inhibited dechlorination (similar to the observation with an ortho-PCB-dechlorinating mixed culture [52]), hydrogen might be electron donor, if directly supplied by the mixed culture at suitable (low) concentrations [54]. Reductive dechlorination of dioxins might be a cometabolic process, however, the observed increase of the dechlorination rate with increasing dioxin concentrations and the stable maintenance of the dechlorination activity over many sequential transfers (up to a final dilution factor of 1016) argue for a dioxin-stimulated growth of the dechlorinator. However, due to the discrepancy in the concentrations of the potential electron acceptor 1,2,4-TrCDD and the cosubstrates added, its number was expected to be orders of magnitude lower compared to fermentatively growing bacteria. The low MPN value of 2.5×103 dioxin-dechlorinating bacteria per ml corresponded to this assumption. Using nested PCR it was demonstrated that the highest MPN dilutions that were positive for dioxin dechlorination contained Dehalococcoides cells. Although the sensitivities of the dechlorination assay and the nested PCR assay might differ, it seems reasonable that during the 8 weeks of incubation of the MPN tubes the amount of dioxin formed and the Dehalococcoides cells grown were both in the detection range. Taking into account a dechlorination rate of 0.1 pmol bacterium−1 day−1[55] ca. 100 continuously dechlorinating bacteria ml−1 were sufficient to produce 0.5 μM 1,3-DCDD (taken as MPN positive) within this time. Considering growth of dechlorinating cells, their number would have been lower in the beginning and higher than 100 ml−1 at the end, thus cells possibly have been recognized by nested PCR if they belonged to Dehalococcoides. Desulfuromonas was indicated in MPN dilutions two orders of magnitude above those positive for dioxin dechlorination. D. chloroethenica can grow by respiration with PCE, but reductive dechlorination of chloroaromatic compounds was not observed so far [51]. A common property of the genus Desulfuromonas is fumarate reduction with acetate as an electron donor [56], which possibly explains its presence in our mixed culture. In contrast, the three known cultivated members of the genus Dehalococcoides are regarded to be physiological restricted to dehalorespiration as a lifestyle [15,46,57]. Dehalococcoides sp. strain CBDB1 is the only known pure culture capable to grow by dehalorespiration with chlorobenzenes using hydrogen as electron donor [15]. Recently, its capability to reductively dechlorinate dioxins was described [16]. D. ethenogenes strain 195 grows by dehalorespiration with PCE, TCE and cis-DCE [46]. Two Dehalococcoides strains were identified in mixed cultures that use vinyl chloride as electron acceptor [58,59] and strain BAV1 was isolated as described recently [57]. Most interestingly, two bacteria responsible for dechlorination of 2,3,5,6- and 2,3,4,5-tetrachlorobiphenyl, also belong phylogenetically to the green non-sulfur bacteria with a distant relatedness to Dehalococcoides[52,60]. Dehalococcoides species might be involved in dioxin dechlorination as (i) Dehalococcoides was not detectable by the nested PCR assay after two sequential transfers in the absence of chlorinated dioxins, and (ii) physiological properties of the dioxin-dechlorinating mixed culture like the ability to additionally dechlorinate TCB, the sensitivity to heat, and the resistance to vancomycin were similar to those of other Dehalococcoides strains [15,46]. However, a role of other bacteria in dioxin dechlorination by the mixed culture cannot be completely ruled out. Acknowledgements The authors acknowledge financial support from grants of FEMS (to H.B.), of Land Sachsen-Anhalt (7621/03/98H) (to M.B.) and Fonds der Chemischen Industrie (to J.R.A.). 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TI - Properties of a trichlorodibenzo-p-dioxin-dechlorinating mixed culture with a Dehalococcoides as putative dechlorinating species
JO - FEMS Microbiology Ecology
DO - 10.1016/S0168-6496(03)00282-4
DA - 2004-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/properties-of-a-trichlorodibenzo-p-dioxin-dechlorinating-mixed-culture-BEi4i9nSuR
SP - 223
EP - 234
VL - 47
IS - 2
DP - DeepDyve
ER -