Biodegradation of the mixture of 2,4,6-trichlorophenol, 4-chlorophenol,and phenol by a defined mixed culture

Two new strains, Pseudomonas sp. TCP114 degrading 2,4,6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

厌氧微生物菌群XH-1对2,4,6-三氯苯酚的降解特性

有机污染物2,4,6-三氯苯酚(2,4,6-TCP)普遍存在于地下水和河流底泥等厌氧环境中。为了探究厌氧微生物菌群XH-1对2,4,6-TCP的降解能力,本研究以2,4,6-TCP为底物,接种XH-1建立微宇宙培养体系,并以中间产物4-氯苯酚(4-CP)和苯酚为底物分别进行分段富集培养,利用高效液相色谱分析底物的降解转化,同时基于16S rRNA基因高通量测序分析微生物群落结构变化。结果表明: 2,4,6-TCP(122 μmol·L^-1)以0.15 μmol·d^-1的速率在80 d内被完全降解转化,降解中间产物分别为2,4-二氯苯酚(2,4-DCP)、4-氯苯酚和苯酚,所有中间产物最终在325 d被完全降解。高通量测序结果表明,脱卤杆菌和脱卤球菌可能驱动2,4,6-TCP还原脱氯,其中,脱卤球菌可能在4-CP的脱氯转化中发挥重要作用,并与丁酸互营菌和产甲烷菌联合作用彻底降解2,4,6-TCP。     

Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent

The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms.     

Anaerobic treatment of 2,4,6-trichlorophenol in an expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactor at 15 degrees C

Expanded granular sludge bed-anaerobic filter (EGSB-AF) bioreactors were operated at 15 degrees C for the treatment of 2,4,6-trichlorophenol (TCP)-containing volatile fatty acid (VFA)-based wastewaters. The seed sludge used as inoculum for the control (no TCP) and test reactor was unexposed to chlorophenols (CPs) prior to the 425-day trial. TCP supplementation to the feed at 50 mg TCPl(-1) partially inhibited the anaerobic degradation of the VFA feed measured as COD removal efficiency. However, the withdrawal and subsequent application of stepwise increments to the TCP loading resulted in steady COD removal. Terminal restriction fragment length polymorphism analysis showed Methanosaeta-like Archaea in the control reactor over the experimental period. Different methanogenic populations were detected in the test reactor and responded to the changes in feed composition. Bacterial community analyses indicated changes in the community structure over time and suggested the presence of Campylobacter-like, Acidimicrobium-like and Heliophilum-like organisms in the samples. TCP mineralisation was by a reductive dechlorination pathway through 2,4-dichlorophenol (DCP) and 4-chlorophenol (4-CP) or 2-chlorophenol (2-CP). CP degradation rates in sludge granules from the lower chamber of the hybrid EGSB-AF reactor was in the order TCP > DCP > 4-CP > 2-CP. However, a biodegradability order of lower CPs > TCP was observed in fixed-film biomass taken from the upper reactor chamber, thus reflecting the role of this reactor section in the metabolism of residual lower CPs from the lower sludge-bed stage of operation.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

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.     

Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1

A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate.     

Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1.

A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate.     

Removal of chlorophenols from synthetic solutions using Phanerochaete chrysosporium

The potential use of the fungus Phanerochaete chrysosporium to remove chlorophenols (phenol, o-chlorophenol, p-chlorophenol and 2,4,6-trichlorophenol) from aqueous solutions was evaluated. The kinetics of both adsorption and desorption of phenolic compounds was rapid for all adsorbates. The maximum adsorptions of phenol and chlorophenols onto the Phanerochaete chrysosporium were 1.23 mmol/g for phenol, 1.49 mmol/g for o-chlorophenol, 1.78 mmol/g for p-chlorophenol and 2.14 mmol/g for 2,4,6-trichlorophenol. The affinity order was as follows: 2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol. Phenol and chlorophenols binding with Phanerochaete chrysosporium were clearly pH dependent. The adsorption of phenol and chlorophenols increased as pH increased. Desorption of phenol or chlorophenols was achieved using methanol solution (30% (v/v)). Phanerochaete chrysosporium is suitable for reuse for more than ten cycles without noticeable loss of adsorption capacity.     

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.     

Biodegradation kinetics of 2,4,6-Trichlorophenol by an acclimated mixed microbial culture under aerobic conditions

The objective of this study was to achieve a better quantitative understanding of the kinetics of 2,4,6-trichlorophenol (TCP) biodegradation by an acclimated mixed microbial culture. An aerobic mixed microbial culture, obtained from the aeration basin of the wastewater treatment plant, was acclimated in shake flasks utilizing various combinations of 2,4,6-TCP (25–100 mg l⁻¹), phenol (300 mg l⁻¹) and glycerol (2.5 mg l⁻¹) as substrates. Complete primary TCP degradation and a corresponding stoichiometric release of chloride ion were observed by HPLC and IEC analytical techniques, respectively. The acclimated cultures were then used as an inoculum for bench scale experiments in a 4 l stirred-tank reactor (STR) with 2,4,6-TCP as the sole carbon/energy (C/E) source. The phenol acclimated mixed microbial culture consisted of primarily Gram positive and negative rods and was capable of degrading 2,4,6-TCP completely. None of the predicted intermediate compounds were detected by gas chromatography in the cell cytoplasm or supernatant. Based on the disappearance of 2,4,6-TCP, degradation was well modelled by zero-order kinetics which was also consistent with the observed oxygen consumption. Biodegradation rates were compared for four operating conditions including two different initial 2,4,6-TCP concentrations and two different initial biomass concentrations. While the specific rate constant was not dependent on the initial 2,4,6-TCP concentration, it did depend on the initial biomass concentration (X _init). A lower biomass concentration gave a much higher zero-order specific degradation rate. This behaviour was attributed to a lower average biomass age or cell retention time (θ_x) for these cultures. The implications of this investigation are important for determining and predicting the potential risks associated with TCP, its degradation in the natural environment or the engineering implications for ex situ treatment of contaminated ground water or soil.     

Induction of plant defence compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum

Bacillus subtilis strain BSCBE4 and Pseudomonas chlororaphis strain PA23 (=P. aureofaciens) were effective biocontrol agents against Pythium aphanidermatum, the causal agent of damping-off of hot pepper (Capsicum annum L.) in greenhouse vegetable production systems. Application of strains BSCBE4 and PA23 at the rate of 20 g kg^-1 of seed significantly increased the growth of hot pepper seedlings. The efficacies of various carriers in sustaining the population of these strains in storage were assessed. Both the antagonists survived up to 180 days of storage in peat and talc-based formulations. The two bacterial strains induced development of plant defence-related enzymes including phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, phenol content, suppressed incidence of damping-off and increased growth of hot pepper seedlings.     

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 degradation of xenobiotics by organisms from municipal solid waste under landfilling conditions

The potential for biological transformation of 23 xenobiotic compounds by microorganisms in municipal solid waste (MSW) samples from a laboratory scale landfill reactor was studied. In addition the influence of these xenobiotic compounds on methanogenesis was investigated. All R11, 1,1 dichloroethylene, 2,4,6 trichlorophenol, dimethyl phthalate, phenol, benzoate and phthalic acid added were completely transformed during the period of incubation (> 100 days). Parts of the initially added perchloroethylene, trichloroethylene, R12, R114, diethyl phthalate, dibutyl phthalate and benzylbutyl phthalate were transformed. Methanogenesis from acetate was completely inhibited in the presence of 2,5 dichlorophenol, whereas 2,4,6 trichlorophenol and R11 showed an initial inhibition, whenafter methane formation recovered. No transformation or effect on the anaerobic microflora occurred for R13, R22, R114, 3 chlorobenzoate, 2,4,6 trichlorobenzoate, bis(2 ethyl)hexyl phthalate, diisodecyl phthalate and dinonyl phthalate. The results indicate a limited potential for degradation, of the compounds tested, by microorganisms developing in a methanogenic landfill environment as compared with other anaerobic habitats such as sewage digestor sludge and sediments.Abbreviations BBP benzylbutylphthalate - DEHP bis(2 ethylhexyl) phthalate - 3 CB 3 chlorobenzoate - R22 chlorodifluoromethane - CFC chlorofluorocarbon - R13 chlorotrifluoromethane - cis1,2 DCE cis 1,2 dichloroethylene - DBP dibutyl phthalate - R12 dichlorodifluoromethane - 1,1 DCE 1,1 dichloroethylenel - R114 dichlorotetrafluoroethane - 2,5 DCP 2,5 dichlorophenol - DEP diethyl phthalate - DiDP diisodecyl phthalate - DMP Dimethyl phthalate - DNP dinonyl phthalate - MSW dunicipal solid waste - PCE perchloroethylene - PA phthalic acid - PAE phthalic acid esters - R11 trichlorofluoromethane - 2,4,6 TCB 2,4,6 trichlorobenzoate - 2,4,6 TCP 2,4,6 trichlorophenol - VC vinylchloride     

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.     

Utilization of d-carnitine by Pseudomonas sp. AK 1

Abstract The degradation of chlorophenols by Alcaligenes eutrophus JMP134 (pJP4) was studied. The strain grew on 2,4,6-trichlorophenol or 2,4,6-tribromophenol as the sole carbon and energy source. Complete degradation of 2,4,6-trichlorophenol was confirmed by chloride release and gas chromatography analysis of supernatants from growth cultures. The 2,3,5-, 2,3,4-, 2,3,6-and 2,4,5-isomers of trichlorophenol did not support growth. However, up to 40% of 2,4,5-trichlorophenol was mineralized during growth of A. eutrophus on chemostats fed with either phenol (0.4 mM) or 2,4,6-trichlorophenol (0.4 mM) plus 2,4,5-trichlorophenol (0.1 mM). Growth on 2,4,6-trihalophenols was also observed in A. Eutrophus JMP222, the strain lacking pJP4, suggesting that this new degradative ability reported for A. eutrophus is not related to pJP4 encoded catabolic functions.     

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.     

Phospholipids of Five Pseudomonad Archetypes for Different Toluene Degradation Pathways

Liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) was used to determine intact phospholipid profiles for five reference pseudomonad strains harboring different (aerobic) toluene catabolic pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. These five strains contained a predominant pool of phosphatidylethanolamines. Other phospholipids identified include phosphatidylglycerol, phosphatidylserine, phosphatidylmethylethanolamine, and phosphatidyldimethylethanolamine. There was a clear separation in phospholipid profiles that allows for the differentiation between the Pseudomonas and Burkholderia genera. Factor analysis of the phospholipid profiles showed that B. cepacia G4, P. putida mt-2, and B. pickettii PKO1 were clearly separated, while P. putida F1 and P. mendocina KR1 were clustered as a group. These results suggest that intact phospholipid profiling could be used to evaluate the relative abundance of specific degraders in bioreactors or in aquifer material. Nevertheless, the usefulness of this technique for taxonomic characterization of such complex samples remains to be demonstrated because of potential confounding effects of overlapping profiles and potential changes in phospholipid composition due to different growth conditions.