Effect of specific inhibitors on the anaerobic reductive dechlorination of 2,4,6-trichlorophenol by a stable methanogenic consortium

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20 mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.     

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.     

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.     

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.     

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.     

Integrated photocatalytic-biological reactor for accelerated 2,4,6-trichlorophenol degradation and mineralization

An integrated photocatalytic-biological reactor (IPBR) was used for accelerated degradation and mineralization of 2,4,6-trichlorophenol (TCP) through simultaneous, intimate coupling of photocatalysis and biodegradation in one reactor. Intimate coupling was realized by circulating the IPBR’s liquid contents between a TiO₂ film on mat glass illuminated by UV light and honeycomb ceramics as biofilm carriers. Three protocols—photocatalysis alone (P), biodegradation alone (B), and integrated photocatalysis and biodegradation (photobiodegradation, P&B)—were used for degradation of different initial TCP concentrations. Intimately coupled P&B also was compared with sequential P and B. TCP removal by intimately coupled P&B was faster than that by P and B alone or sequentially coupled P and B. Because photocatalysis relieved TCP inhibition to biodegradation by decreasing its concentration, TCP biodegradation could become more important over the full batch P&B experiments. When phenol, an easy biodegradable compounds, was added to TCP in order to promote TCP mineralization by means of secondary utilization, P&B was superior to P and B in terms of mineralization of TCP, giving 95% removal of chemical oxygen demand. Cl⁻ was only partially released during P experiments (24%), and this corresponded to its poor mineralization in P experiments (32%). Thus, intimately coupled P&B in the IPBR made it possible obtain the best features of each: rapid photocatalytic transformation in parallel with mineralization of photocatalytic products.     

Anaerobic mineralization of pentachlorophenol (PCP) by combining PCP-dechlorinating and phenol-degrading cultures

The dechlorination and mineralization of pentachlorophenol (PCP) was investigated by simultaneously or sequentially combining two different anaerobic microbial populations, a PCP-dechlorinating culture capable of the reductive dechlorination of PCP to phenol and phenol- degrading cultures able to mineralize phenol under sulfate- or iron-reducing conditions. In the simultaneously combined mixture, PCP (about 35 microM) was mostly dechlorinated to phenol after incubation for 17 days under sulfate-reducing conditions or for 22 days under iron-reducing conditions. Thereafter, the complete removal of phenol occurred within 40 days under both conditions. In the sequentially combined mixture, most of the phenol, the end product of PCP dechlorination, was degraded within 12 days of inoculation with the phenol degrader, without a lag phase, under both sulfate- and iron-reducing conditions. In a radioactivity experiment, [14C-U]-PCP was mineralized to 14CO2 and 14CH4 by the combined anaerobic microbial activities. Analysis of electron donor and acceptor utilization and of the production and consumption of H2, CO2, and CH4 suggested that the dechlorinating and degrading microorganisms compete with other microorganisms to perform PCP dechlorination and part of the phenol degradation in complex anoxic environments in the presence of electron donors and acceptors. The presence of a small amount of autoclaved soil slurry in the medium was possibly another advantageous factor in the successful dechlorination and mineralization of PCP by the combined mixtures. This anaerobic-anaerobic combination technology holds great promise as a cost-effective strategy for complete PCP bioremediation in situ.     

The role of exogenous electron donors for accelerating 2,4,6-trichlorophenol biotransformation and mineralization

2,4,6-Trichlorophenol (TCP) is a biologically recalcitrant compound, but its biodegradation via reductive dechlorination can be accelerated by adding an exogenous electron donor. In this work, acetate and formate were evaluated for their ability to accelerate TCP reductive dechlorination, as well to accelerate mono-oxygenation of TCP’s reduction product, phenol. Acetate and formate accelerated TCP reductive dechlorination, and the impact was proportional to the number of electron equivalents released by oxidation of the donor: 8 e^? equivalents per mol for acetate, compared to 2 e^? eq per mol for formate. The acceleration phenomenon was similar for phenol mono-oxygenation, and this increased the rate of TCP mineralization. Compared to endogenous electron equivalents generated by phenol mineralization, the impact of exogenous electron donor was stronger on a per-equivalent basis.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

Isolation of Pseudomonas pickettii strains that degrade 2,4,6-trichlorophenol and their dechlorination of chlorophenols.

Three strains of Pseudomonas pickettii that can grow with 2,4,6-trichlorophenol (2,4,6-TCP) as the sole source of carbon and energy were isolated from different mixed cultures of soil bacterial populations that had been acclimatized to 2,4,6-TCP. These strains released 3 mol of chloride ion from 1 mol of 2,4,6-TCP during the complete degradation of the TCP. Of these strains, P. pickettii DTP0602 in high-cell-density suspension cultures dechlorinated various chlorophenols (CPs). Cells that were preincubated with 2,4,6-TCP converted isomers of 4-CP to the corresponding chloro-p-hydroquinones, but those preincubated with 4-CP converted CPs lacking a chlorine atom(s) at the o position to isomers of chlorocatechol. The ability of DTP0602 to dechlorinate 2,4,6-TCP was induced by 2,6-dichlorophenol, 2,3,6- and 2,4,6-TCP, and 2,3,4,6-tetrachlorophenol and was repressed in the presence of succinate or glucose.     

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.     

Degradation pathway, toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR

The present study deals with cultivation of 2,4,6-trichlorophenol (TCP) degrading aerobic granules in two SBR systems based on glucose and acetate as co-substrate. Biodegradation of TCP containing wastewater starting from 10 to 360 mg L⁻¹ with more than 90% efficiency was achieved. Sludge volume index decreases as the operation proceeds to stabilize at 35 and 30 mL g⁻¹ while MLVSS increases from 4 to 6.5 and 6.2 g L⁻¹ for R1 (with glucose as co-substrate) and R2 (with sodium acetate as co-substrate), respectively. FTIR, GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocatechol pathway and the cleavage may be at ortho-position. Haldane model for inhibitory substrate was applied to the system and it was observed that glucose fed granules have a high specific degradation rate and efficiency than acetate fed granules. Genotoxicity studies shows that effluent coming from SBRs was non-toxic.     

Anaerobic dechlorination of pentachlorophenol in fixed-film and upflow anaerobic sludge blanket reactors using different inocula

Longterm performance and stability of two upflow anaerobic sludge blanket (UASB) reactors inoculated with granular sludge and treating a synthetic waste water containing pentachlorophenol (PCP) and phenol were studied. A similar system consisting of two fixed-film reactors inoculated with anaerobic digested sewage sludge were further studied. One reactor in each series received glucose in addition to the phenols. Dechlorination of PCP proceeded via two different dominating pathways in the respective reactor systems, suggesting that two distinct microbial populations were present, probably originating from the different inocula. Dechlorinating activity was maintained for more than 18 months in the UASB reactors and was generally higher than in the fixed-film reactors. In the fixed-film reactors, dechlorination of PCP suddenly decreased after 15.5 months of operation compared to earlier performance. Since no operational parameters had been changed, this indicated that the enriched culture was unstable on a longterm basis. Addition of yeast extract to the medium restored activity. General process stability in both reactor systems was clearly enhanced by the addition of glucose and was superior in the UASB/granular sludge system. The better performance and the higher stability in the UASB/granular sludge reactor highlights the importance of thorough screening of inocular prior to start-up of processes treating waste waters containing xenobiotic compounds.Abbreviations PCP pentachlorophenol - TeCP tetrachlorophenol - TCP trichlorophenol - DCP dichlorophenol - UASB upflow anaerobic sludge blanket - HRT hydraulic retention time     

Gratuitous dechlorination of chloroethanes by methanogenic granular sludge

The dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B₁₂ and F₄₃₀. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG ^0′). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG ^0′ value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge. Received: 8 June 1998 / Received revision: 7 September 1998 / Accepted: 13 September 1998     

Detoxification of 2,4,6-trichlorophenol by an indigenous bacterial community

An indigenous bacterial community capable of degrading 2,4,6-trichlorophenol (TCP) as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. The community consists of three gram-negative bacterial, non-fermentative strains, two of them was identified as belonging to genus Pseudomonas and the other to genus Stenotrophomonas. None of the individual strains were capable of degrading TCP as the sole carbon source. Aerobic biodegradation assays were performed using a 2-l microfermentor at 28 °C with agitation. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna as test organisms. The indigenous bacterial community degrades 100 mg l^?1 of TCP in 27 h. The absence of metabolites and toxicity was proved at the end of the process. The influence of the initial concentration of compound, pH, cell inoculum, presence of other substrates and toxic related compounds in the biodegradation process was assayed. Also the application for polluted water was studied. The promising behavior of the bacterial community under the different conditions assayed allows us to suggest its possible use in remediation processes.     

Degradation of prochloraz and 2,4,6-trichlorophenol by environmental bacterial strains

 Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the β subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests. Received: 3 July 1995/Received revision: 27 July 1995/Accepted: 31 July 1995     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Impact of long-term partial aeration on the removal of 2,4,6-trichlorophenol in an initially methanogenic fluidized bed bioreactor

A fluidized bed bioreactor (FBBR) was operated for more than 1000 days under two regimes, Methanogenic (M) and Methanogenic-Aerobic (M-A), to remove 2,4,6-trichlorophenol (TCP) and phenol (Phe) from a synthetic wastewater, containing different amounts of TCP and Phe, using different aeration flow-rates (0, 2.13, and 1.06 NL O(2)/L.day). M conditions (80:20 mg/L of TCP:Phe, 0 NL O(2)/L.day) showed similar TCP and Phe removal (>95%). Nevertheless accumulation of 4-chlorophenol (4CP) up to 16 mg/L and Phe up to 4 mg/L was observed, while in M-A conditions (80:20 mg/L of TCP:Phe, 2.13 NL O(2)/L.day) TCP and Phe removal achieved 99.9(+)% and after 70 days no accumulation of intermediates were detected. The increase of TCP and Phe in the influent under M-A conditions from 80:20 to 120:30 mg/L of TCP:Phe did not negatively affect the removal of TCP, intermediates and Phe; in fact, they were similar to those in previous M-A conditions. The decrease in the oxygen flow rate from 2.13 to 1.06 NL O(2)/L.day had no negative effect on pollutant removals, which were as high as in previous two M-A conditions. The specific methanogenic activity of bioparticles of the fluidized bed decreased with long-term partial aeration, starting from 1.097 mmol CH(4)/h.g(TKN) in the M regime (day 60) to <0.02 mmolCH(4)/h.g(TKN) at day 1050, suggesting aerobic regime in the bioreactor rather than an M-A regime. In conclusion, complete removal of TCP and less chlorinated intermediates could be achieved in an initially methanogenic FBBR under conditions of partial aeration, although long-term operation seemed to negatively affect the methanogenic activity of biomass. It is also likely that after extended aeration the microbial community was finally enriched with strains with the ability to attack 2,4,6-TCP under aerobic conditions. This report represents the first evidence of a long exposure to oxygen of an anaerobic microbial consortium that efficiently remove TCP.