Anaerobic dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol by methanogenic pentachlorophenol-degrading granules

Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized¹⁴C-labeled PCP to¹⁴CH₄ and¹⁴CO₂. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of¹⁴C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of¹⁴CH₄ and¹⁴CO₂ from [¹⁴C]PCP. 2,4,6-trichlorophenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chlorophenol or 2-chlorophenol to phenol at pH 7.0–7.2.     

Kinetics of the sequential dechlorination of 2,4,6-trichlorophenol by an anaerobic microbial population

Summary A mathematical model was developed to describe the sequential dechlorination of 2,4,6-trichlorophenol to 2,4-dichlorophenol, 4-chlorophenol and phenol. Each compound was assumed to be degraded according a Michaelis-Menten expression. Experimental data were used to obtain the model kinetic constants and to test its validity. Good agreement between the model predictions and the experimental data was obtained.     

Enhanced dechlorination of 2,4,6-trichlorophenol by anaerobic microbial populations in the presence of ethanol

Summary A consortium of anaerobic microorganisms was grown on acetate, ethanol, glucose or methanol and dechlorinated 50 umol 2,4,6-trichlorophenol, through 2,4-dichlorophenol, to 4-chlorophenol. The highest rate of dechlorination of 2,4,6-trichlorophenol was observed when ethanol was used as a growth substrate.     

Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium.

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.     

Constitutive dechlorination of chlorinated ethenes by a methanol degrading methanogenic consortium

The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes.     

Reductive dechlorination of chlorophenols by a pentachlorophenol- acclimated methanogenic consortium.

Anaerobic digester sludge fed 5,300 mg of acetate per liter, 3.4 microM pentachlorophenol, and nutrients for 10 days biotransformed pentachlorophenol by sequential ortho dechlorinations to produce 2,3,4,5-tetrachlorophenol and 3,4,5-trichlorophenol. Upon acclimation to 3.4 microM pentachlorophenol for 6 months, the methanogenic consortium removed chlorines from the ortho, meta, and para positions of pentachlorophenol and its reductive dechlorination products. Pentachlorophenol was degraded to produce 2,3,4,5-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and 2,3,5,6-tetrachlorophenol. Dechlorination of 2,3,4,5-tetrachlorophenol produced 3,4,5-trichlorophenol, which was subsequently degraded to produce 3,4-dichlorophenol and 3,5-dichlorophenol. 2,3,4,6-Tetrachlorophenol was dechlorinated at the ortho and meta positions to produce 2,4,6-trichlorophenol and 2,4,5-trichlorophenol. 2,3,5,6-Tetrachlorophenol yielded 2,3,5-trichlorophenol, followed by production of 3,5-dichlorophenol. 2,4,6-Trichlorophenol was degraded to form 2,4-dichlorophenol, and 2,4,5-trichlorophenol was dechlorinated at two positions to form 2,4-dichlorophenol and 3,4-dichlorophenol. Of the three dichlorophenols produced (2,4-dichlorophenol, 3,4-dichlorophenol, and 3,5-dichlorophenol), only 2,4-dichlorophenol was degraded significantly within 3 weeks, to produce 4-chlorophenol.     

Aerobic degradation of 2,4,6-trichlorophenol by a microbial consortium - selection and characterization of microbial consortium

A microbial consortium that efficiently degrades 2,4,6-TCP (2,4,6-trichlorophenol), as the sole source of carbon and energy under aerobic conditions was selected from municipal activated sludge. Six bacterial strains, designated S(1), S(2), S(3), S(4), S(5) and S(6), were isolated from the selected consortium and five were identified as Sphingomonas paucimobilis (S(2), S(3)), Burkholderia cepacia(S(4)), Chryseomonas luteola (S(5)) and Vibrio metschnikovii (S(6)). After prolonged cultivation followed by successive transfers, the consortium's degradation ability was improved and reached a specific degradation rate of 34 mg 2,4,6-TCP g(-1) dry weight h(-1) (about 51 mg 2,4,6-TCP g(-1) cell protein h(-1)). The soluble chemical oxygen demand, chloride and oxygen uptake balance data clearly indicate the complete dechlorination and mineralization of 2,4,6-TCP. The consortium's activity was not inhibited by 2,4,6-TCP concentrations 相似文献   

Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms

Anaerobically digested municipal sewage sludge which had been acclimated to monochlorophenol degradation for more than 2 years was shown to degrade pentachlorophenol (PCP). Di-, tri-, and tetrachlorophenols accumulated when PCP was added to the individual acclimated sludges. When the 2-chlorophenol- (2-CP), 3-CP-, and 4-CP-acclimated sludges were mixed in equal volumes, PCP was completely dechlorinated. The same results were obtained in sludge acclimated to the three monochlorophenol isomers simultaneously. With repeated PCP additions, 3,4,5,-trichlorophenol, 3,5-dichlorophenol, and 3-CP accumulated in less than stoichiometric amounts. All chlorinated compounds disappeared after PCP additions were stopped. All chlorinated compounds disappeared after PCP additions were stopped. Incubations with [14C]PCP resulted in 66% of the added 14C being mineralized to 14CO2 and 14CH4. Technical-grade PCP was found to be degraded initially at a rate very similar to that of reagent-grade PCP, but after repeated additions, the technical PCP was degraded more slowly. Pentabromophenol was also rapidly degraded by the mixture of acclimated sludges. These results clearly show the complete reductive dechlorination of PCP by the combined activities of three chlorophenol-degrading populations.     

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.     

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.     

Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms.

Anaerobically digested municipal sewage sludge which had been acclimated to monochlorophenol degradation for more than 2 years was shown to degrade pentachlorophenol (PCP). Di-, tri-, and tetrachlorophenols accumulated when PCP was added to the individual acclimated sludges. When the 2-chlorophenol- (2-CP), 3-CP-, and 4-CP-acclimated sludges were mixed in equal volumes, PCP was completely dechlorinated. The same results were obtained in sludge acclimated to the three monochlorophenol isomers simultaneously. With repeated PCP additions, 3,4,5,-trichlorophenol, 3,5-dichlorophenol, and 3-CP accumulated in less than stoichiometric amounts. All chlorinated compounds disappeared after PCP additions were stopped. All chlorinated compounds disappeared after PCP additions were stopped. Incubations with [14C]PCP resulted in 66% of the added 14C being mineralized to 14CO2 and 14CH4. Technical-grade PCP was found to be degraded initially at a rate very similar to that of reagent-grade PCP, but after repeated additions, the technical PCP was degraded more slowly. Pentabromophenol was also rapidly degraded by the mixture of acclimated sludges. These results clearly show the complete reductive dechlorination of PCP by the combined activities of three chlorophenol-degrading populations.     

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 μM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 μM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 μM. Lactate and ethanol were also accumulated at 760 μM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 μM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 μM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 μM h(-1) and K(s)=5.8 and 13.1 μM for control and bioaugmented sludges, respectively).     

Rapid dehalogenation of 2,4,6-trichlorophenol at alkaline pH by an anaerobic enrichment culture

An enrichment culture, derived from the anaerobic stage of a two-step sequential anaerobic-aerobic reactor system which mineralized 2,4,6-trichlorophenol, stoichiometrically converted 2,4,6-trichlorophenol to 4-chlorophenol. Dehalogenation occurred only in alkaline media (pH 8–9) at concentrations of substrate up to 1 mmol 1¹. Formate plus acetate or trypticase could serve as electron donors. Neither vitamins nor trace elements were required in a chloride-free defined medium. The dehalogenating organism was oxygen-resistant, but was not active in media which were oxidized with respect to resazurin indicator dye. Most probable number counts of the dehalogenating cultures showed that the dehalogenating organisms were present in very small numbers, yet catalysed dehalogenation at rates considerably faster than other dehalogenating organisms described in the literature.     

Total reductive dechlorination of chlorobenzenes to benzene by a methanogenic mixed culture enriched from Saale river sediment

 A methanogenic mixed culture enriched from Saale river sediment was able to transform all chlorobenzenes by reductive dechlorination via monochlorobenzene to unsubstituted benzene after a short lag phase of only 1 week. The formation of benzene was confirmed by GC/MS in pentane extracts of the sludge samples, as well as in the headspace gas of the batch cultures. The spectra of substrate utilization of the adapted cultures depended mainly on the substrates used for adaptation. Sediment microbes that had been adapted with a mixture of the three isomers of trichlorobenzene offered the widest spectrum for the utilization of chlorobenzenes in all chlorine stages. Reductive dechlorination was stimulated when the mixed cultures were supplemented with pyruvate and methanol. Received: 17 July 1995/Received revision: 11 December 1995/Accepted: 18 December 1995     

Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence that, under methanogenic conditions, mixed cultures are able to completely dechlorinate PCE and TCE to ethylene, a product which is environmentally acceptable. Radiotracer studies with [14C]PCE indicated that [14C]ethylene was the terminal product; significant conversion to 14CO2 or 14CH4 was not observed. The rate-limiting step in the pathway appeared to be conversion of vinyl chloride to ethylene. To sustain reductive dechlorination of PCE and TCE, it was necessary to supply an electron donor; methanol was the most effective, although hydrogen, formate, acetate, and glucose also served. Studies with the inhibitor 2-bromoethanesulfonate suggested that methanogens played a key role in the observed biotransformations of PCE and TCE.     

Uptake of phenol by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

Phenol was absorbed unspecifically by active and by inactivated cells of a strictly anaerobic, phenol-degrading consortium to reach about twice the concentration of the medium. The absorption was temperature-dependent. A Q₁₀ of 1.7 was determined, indicating that accumulation was due to diffusion or facilitated diffusion and not to an active transport process. At increasing phenol concentration in the medium, concentrated cell suspensions adsorpted phenol proportionally until saturation was reached at about 25 nmol phenol/mg cell dry weight. At a phenol concentration in the medium of 2 mm, the washed cell pellet contained 3.5 mm phenol. Under conditions that allowed phenol metabolism (presence of CO₂), [¹⁴C]4-hydroxybenzoyl-coenzyme A and [¹⁴C]4-hydroxybenzoate were found as early intermediates of [U-¹⁴C]phenol degradation for the first time. [¹⁴C]Benzoate was excreted stoichiometrically if phenol degradation to acetate was prevented by H₂. Absolutely no ¹⁴C-label was found in the phenylphosphate peak after HPLC separation, which excluded phosphorylation of phenol during uptake or during degradation in the cells. Correspondence to: J. Winter     

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP 2,3,5,6-tetrachlorophenol 2,3,5-trichlorophenol 3,5-dichlorophenol 3-chlorophenol phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 mol (1 reactor volume)^–1 day^–1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.     

Dehalogenation and mineralization of 2,4,6-trichlorophenol by the sequential activity of anaerobic and aerobic microbial populations

Summary A sequential anaerobic-aerobic treatment process that can mineralize 2,4,6-trichlorophenol has been developed. The process uses diluted anaerobic digester fluid as a culture medium, and a single microbial population enriched from the digester fluid for both the anaerobic and aerobic steps.     

Biological reductive dechlorination of tetrachloroethylene and trichloroethylene to ethylene under methanogenic conditions.

A biological process for remediation of groundwater contaminated with tetrachloroethylene (PCE) and trichloroethylene (TCE) can only be applied if the transformation products are environmentally acceptable. Studies with enrichment cultures of PCE- and TCE-degrading microorganisms provide evidence that, under methanogenic conditions, mixed cultures are able to completely dechlorinate PCE and TCE to ethylene, a product which is environmentally acceptable. Radiotracer studies with [14C]PCE indicated that [14C]ethylene was the terminal product; significant conversion to 14CO2 or 14CH4 was not observed. The rate-limiting step in the pathway appeared to be conversion of vinyl chloride to ethylene. To sustain reductive dechlorination of PCE and TCE, it was necessary to supply an electron donor; methanol was the most effective, although hydrogen, formate, acetate, and glucose also served. Studies with the inhibitor 2-bromoethanesulfonate suggested that methanogens played a key role in the observed biotransformations of PCE and TCE.     

Reductive dechlorination of halogenated phenols by a sulfate-reducing consortium

A sulfidogenic consortium enriched from an estuarine sediment utilized 4-chlorophenol as a sole source of carbon and energy. Reductive dechlorination as the initial step in chlorophenol degradation by the sulfate-reducing consortium was confirmed with the use of chloro-fluorophenols. Both 4-chloro-2-fluorophenol and 4-chloro-3-fluorophenol were dechlorinated, resulting in stoichiometric accumulation of 2-fluorophenol and 3-fluorophenol, respectively. The fluorophenols were not degraded further. Furthermore, phenol was detected as a transient intermediate during degradation of 4-chlorophenol in the presence of 3-fluorophenol. Reductive dechlorination was inhibited by molybdate and did not occur in the absence of sulfate. These results indicate that 4-chlorophenol is reductively dechlorinated to phenol under sulfate-reducing conditions and mineralization of the phenol ring to CO₂ is coupled to sulfate reduction.