Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37

AIMS: To analyse the possible effect of poly-beta-hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. METHODS AND RESULTS: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25-400 microm). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 microm of 2,4,6-TCP and PHA in cells investigated by spectrofluorimetric and flow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the first 24 h. CONCLUSION: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a significant role in the degradation of toxic compounds.     

Biodegradation of 2,4,6-Tribromophenol by Ochrobactrum sp. Strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 μM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Biosynthesis of poly-β-hydroxyalkanoates by Sphingopyxis chilensis S37 and Wautersia sp. PZK cultured in cellulose pulp mill effluents containing 2,4,6-trichlorophenol

Poly-β-hydroxyalkanoates (PHA) polymer is synthesized by different bacterial species. There has been considerable interest in the development and production of biodegradable polymers; however, the high cost of PHA production has restricted its applications. Kraft cellulose industry effluents containing 2,4,6-trichlorophenol (10 or 20 μg ml⁻¹) were used by the bacteria Sphingopyxis chilensis S37 and Wautersia sp. PZK to synthesize PHA. In this condition, S. chilensis S37 was able to grow and degrade 2,4,6-trichlorophenol (ca. 60%) and 80% of these cells accumulated PHA. Wautersia PZK completely degraded 2,4,6-TCP and more than 90% of the cells accumulated PHA in 72 h. The PHA detection was performed by flow cytometry and polyester composition was characterized by gas chromatography-mass spectroscopy (GC-MS), indicating that these polymers are made by 3-hydroxybutyric acid and 3-hydroxyhexadecanoic acid for S37 and PZK strains, respectively. Results demonstrated that strains’ growth and PHA production and composition are not modified in cellulose effluents with or without 2,4,6-TCP (10–20 μg ml⁻¹). Therefore, our results indicate that S. chilensis S37 and Wautersia sp. PZK are able to degrade a toxic compound such as a 2,4,6-TCP and simultaneously produce a valuable biopolymer using low-value substrates.     

A Previously Unexposed Forest Soil Microbial Community Degrades High Levels of the Pollutant 2,4,6-Trichlorophenol

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant that is efficiently degraded by some aerobic soil bacterial isolates under laboratory conditions. The degradation of this pollutant in soils and its effect on the soil microbial community are poorly understood. We report here the ability of a previously unexposed forest soil microbiota to degrade high levels of 2,4,6-TCP and describe the changes in the soil microbial community found by terminal restriction fragment length polymorphism (T-RFLP) analysis. After 30 days of incubation, about 50% degradation of this pollutant was observed in soils amended with 50 to 5,000 ppm of 2,4,6-TCP. The T-RFLP analysis showed that the soil bacterial community was essentially unchanged after exposure to up to 500 ppm of 2,4,6-TCP. However, a significant decrease in richness was found with 2,000 and 5,000 ppm of 2,4,6-TCP, even though the removal of this pollutant remained high. The introduction of Ralstonia eutropha JMP134 or R. eutropha MS1, two efficient 2,4,6-TCP degraders, to this soil did not improve degradation of this pollutant, supporting the significant bioremediation potential of this previously unexposed, endogenous forest soil microbial community.     

Transformation of 2,4,6-trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor

The toxicity of thirteen isomers of mono-, di-, tri- and pentachlorophenols was tested in potato-dextrose agar cultures of the white rot fungi Panus tigrinus and Coriolus versicolor. 2,4,6-Trichlorophenol (2,4,6-TCP) was chosen for further study of its toxicity and transformation in liquid cultures of these fungi. Two schemes of 2,4,6-TCP addition were tested to minimize its toxic effect to fungal cultures: stepwise addition from the moment of inoculation and single addition after five days of growth. In both cases the ligninolytic enzyme systems of both fungi were found to be responsible for 2,4,6-TCP transformation. 2,6-Dichloro-1,4-hydroquinol and 2,6-dichloro-1,4-benzoquinone were found as products of primary oxidation of 2,4,6-TCP by intact fungal cultures and purified ligninolytic enzymes, Mn-peroxidases and laccases of both fungi. However, primary attack of 2,4,6-TCP in P. tigrinus culture was conducted mainly by Mn-peroxidase, while in C. versicolor it was catalyzed predominantly by laccase, suggesting a different mode of regulation of these enzymes in the two fungi.     

Degradation of Chlorophenols Using Pentachlorophenol-Degrading Bacteria Sphingomonas chlorophenolica in a Batch Reactor

Chlorophenols are common environmental contaminants that have been used as the major component in wide-spectrum biocides in industry and agriculture. Many chlorophenols tend to persist in the environment and may become public health hazards. This research studied the ability of the pentachlorophenol (PCP)-degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. In addition, the characteristics of S. chlorophenolica were also investigated. When S. chlorophenolica cells were preincubated with PCP, the lag phase PCP degradation periods became shorter and the PCP concentrations that could be removed became higher. S. chlorophenolica was able to completely degrade 2,3,6-trichlorophenol (2,3,6-TCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), and PCP within 38.1, 15.1, 11.8, and 11.8 h, and to release concentrations of 50.1, 60.9, 63.7, and 58.5 mg/L chloride at the same period of time. In the presence of supplementary carbon sources, the PCP removal efficiency increased with the presence of glucose or pyruvate. However, the removal efficiency of 75 mg/L 2,4-dichlorophenol did not increase with supplemental carbon sources.     

Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.     

Efficient Degradation of 2,4,6-Trichlorophenol Requires a Set of Catabolic Genes Related to tcp Genes from Ralstonia eutropha JMP134(pJP4)

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and β-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.     

Effects of glucose and phenylalanine upon 2,4,6-trichlorophenol degradation by Pseudomonas paucimobilis S37 cells in a no-growth state

Pseudomonas paucimobilis S37, a strain able to degrade 2,4,6-trichlorophenol (246-TCP), was isolated from an aquatic environment polluted with this compound. The effect of two natural organic compounds on the degradation of 246-TCP by this strain, in a no-growth state, was studied. Bacterial cultures were exposed to 0.1 mM and 0.5 mM of 246-TCP, alone, or in the presence of similar concentrations of glucose, a growth supporting substrate, or phenylalanine, a no-growth supporting compound. The effects on viable counts and 246-TCP degradation were measured. The bacterial culture died with 0.5 mM 246-TCP. This effect was overcome by the presence of glucose or phenylalanine, although no degradation of 246-TCP was detected. At 0.1 mM 246-TCP, the viability was not altered, and cells were able to degrade this compound. Glucose at 0.1 mM increased the degradative activity, but higher levels were inhibitory. Phenylalanine at 0.67 mM or higher concentration was also inhibitory of the 246-TCP degradation.     

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 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration

A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6-TCP with stoichiometric release of Cl⁻. In cultures of Alc. eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation. Experiments conducted with soil microcosms gave evidence that : the degradative process had a biotic nature and was accompanied by microbial growth ; the soil used presented an intrinsic degradative capacity versus 2,4,6-TCP ; the specialized organism used as inoculum was effective in degrading 2,4,6-TCP in a short time. These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil.     

Degradation of 2,4,6-trinitrotoluene by Klebsiella sp. isolated from activated sludge

Klebsiella sp. strain C1 isolated from activated sludge metabolized 2,4,6-trinitrotoluene (TNT) by two different pathways. The typical metabolites in the nitro group reduction pathway of TNT, such as hydroxylamino-dinitrotoluenes and amino-dinitrotoluenes, were detected. Dinitrotoluenes and nitrite were also detected, possibly produced by a denitration pathway. After incubation of [U-¹⁴C]TNT for 28 and 77 d, 2.4 and 6.24%, respectively, were released as ¹⁴CO₂. This mineralization rate was higher than those reported by any other TNT degrading bacteria and might be due to the dual pathways of degradation in this bacterium.     

Efficient degradation of 2,4,6-Trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4)

2,4,6-Trichlorophenol (2,4,6-TCP) is a hazardous pollutant. Several aerobic bacteria are known to degrade this compound. One of these, Ralstonia eutropha JMP134(pJP4), a well-known, versatile chloroaromatic compound degrader, is able to grow in 2,4,6-TCP by converting it to 2,6-dichlorohydroquinone, 6-chlorohydroxyquinol, 2-chloromaleylacetate, maleylacetate, and beta-ketoadipate. Three enzyme activities encoded by tcp genes, 2,4,6-TCP monooxygenase (tcpA), 6-chlorohydroxyquinol 1,2-dioxygenase (tcpC), and maleylacetate reductase (tcpD), are involved in this catabolic pathway. Here we provide evidence that all these tcp genes are clustered in the R. eutropha JMP134(pJP4) chromosome, forming the putative catabolic operon tcpRXABCYD. We studied the presence of tcp-like gene sequences in several other 2,4,6-TCP-degrading bacterial strains and found two types of strains. One type includes strains belonging to the Ralstonia genus and possessing a set of tcp-like genes, which efficiently degrade 2,4,6-TCP and therefore grow in liquid cultures containing this chlorophenol as a sole carbon source. The other type includes strains belonging to the genera Pseudomonas, Sphingomonas, or Sphingopixis, which do not have tcp-like gene sequences and degrade this pollutant less efficiently and which therefore grow only as small colonies on plates with 2,4,6-TCP. Other than strain JMP134, none of the bacterial strains whose genomes have been sequenced possesses a full set of tcp-like gene sequences.     

Biodegradation of 2,4,6-tribromophenol by Ochrobactrum sp. strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 microM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Degradation of 2,4,6-TCP and a mixture of isomeric chlorophenols by immobilized Streptomyces rochei 303

We investigated the degradation of 2,4,6-trichlorophenol (2,4,6-TCP) by cells of Streptomyces rochei 303 immobilized on various carriers. Polycaproamide fibre was chosen as the optimal carrier for immobilization. The cells immobilized on this carrier degraded high-concentrations of individual chlorophenols and their mixtures: from mono- to pentachlorophenol including the most persistent meta-substituted derivatives. During continuous fermentation in a column with continuous substrate and air flow at a maximal degraded concentration of 2,4,6-TCP of 1 g/l and the specific flow rate of 0.08 h^–1, the efficiency of degradation was 720 mg 2,4,6-TCP/day (36 mg 2,4,6-TCP/day per gram of carrier). The above system of immobilized cells was operated continuously without any loss of activity for 2.5 months, the amount of degraded 2,4,6-TCP being 54 g. At a lower concentration of the reagent (150 mg/l) the system was operated without any decrease in its degradability and without any additional carbon source for 11 months. Correspondence to: L. A. Golovleva     

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     

Isolation and characterization of a novel Dehalobacter species strain TCP1 that reductively dechlorinates 2,4,6-trichlorophenol

Chlorophenols are widely used as biocides, leading them to being prevalent environmental contaminants that pose toxic threats to ecosystems. In this study, a Dehalobacter species strain TCP1 was isolated from a digester sludge sample, which is able to dechlorinate 2,4,6-trichlorophenol (2,4,6-TCP) to 4-monochlorophenol (4-MCP) with H₂ as the sole electron donor and acetate as the carbon source. Strain TCP1 also distinguishes itself from other Dehalobacter species with its capability to dechlorinate tetrachloroethene or trichloroethene (TCE) to both cis- and trans-dichloroethenes in a ratio of 5.6 (±0.2):1. The growth yields of strain TCP1 on TCE and 2,4,6-TCP were 4.14 × 10¹³ and 5.77 × 10¹³ cells mol^?1 of Cl^? released, respectively. Strain TCP1 contains five unusually long 16S rRNA gene copies per genome, and the extra length is due to the ~110 bp insertion sequences at their 5′-ends. This suggests that strain TCP1 may represent a novel Dehalobacter species. A putative chlorophenol reductive dehalogenase gene—debcprA—was identified to catalyze the ortho-chlorine removal from 2,4,6-TCP. Both the culture-dependent and housekeeping rpoB gene-based approaches indicate the purity of the culture. Strain TCP1 can serve as a promising candidate for the bioremediation of 2,4,6-TCP contaminated sites, and its discovery expands our understanding of metabolic capabilities of Dehalobacter species.     

Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134

Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.