Bioremediation of halogenated compounds: comparison of dehalogenating bacteria and improvement of catalyst stability

Five bacterial strains were compared for halogenated compounds conversion in aqueous media. Depending on the strain, the optimal temperature for dehalogenase activity of resting cells varied from 30 to 45 degrees C, while optimal pH raised from 8.4 to 9.0. The most effective dehalogenase activity for 1-chlorobutane conversion was detected with Rhodococcus erythropolis NCIMB13064 and Escherichia coli BL21 (DE3) (DhaA). The presence of 2-chlorobutane or propanal in the aqueous media could inhibit the 1-chlorobutane transformation.     

Effects of sensitizers on the photodegradation of the systemic fungicide triadimenol

The photochemical behaviour of triadimenol (1) under various conditions has been examined. Significant degradation is obtained only in the presence of electron-acceptor sensitizers as 9,10-dicyanoanthracene or 2,4,6-triphenylpyrylium tetrafluoroborate, and long irradiation times are required. 1H-1,2,4-Triazole (2), 4-chlorophenyl formate (3), 4-chlorophenol (4), 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one (5), 4-chlorophenyl 2,2-dimethylpropanoate (6) and 4-chlorobenzoic acid (7) were identified as photoproducts by NMR and GC-MS.     

A novel ortho-dehalogenation reaction of 2-chlorocinnamic acid catalyzed by the pink yeast Rhodotorula rubra Y-1529

In the present study, a resting cells suspension of Rhodotorula rubra Y-1529 was shown to have the capacity to perform an ortho-dehalogenation reaction on 2-chlorocinnamic acid. The results from the biodegradation of U-[¹⁴C]benzoic acid, cinnamic acid, 3-chlorocinnamic acid and 4-chlorocinnamic acid suggest that the first step of the ortho-dehalogenation reaction occurred during the oxidation of the unsaturated C3 side chain of 2-chlorocinnamic acid to 2-chlorobenzoic acid. None of the 2-chlorobenzoic acid was found in the biodegradation system, suggesting that this step was a highly regulated step. After the side-chain oxidation reaction, the hydroxylation of the benzene ring was determined to be at the para-position first, followed by the meta-position. The occurrence of 3:4-position ring fission reactions and the production of the final product, CO₂, was proven by the biodegradation of U-[¹⁴C] benzoic acid. This oxidative dehalogenation reaction catalyzed by R. rubra was found to be regiospecific for 2-chlorocinnamic acid; the chloride ion was probably removed after the ring fission reaction. A pathway of the ortho-dehalogenation reaction of 2-chlorocinnamic acid catalyzed by R. rubra was proposed based on these data.     

Biodegradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) by using Serratia marcescens NCIM 2919

A solvent tolerant bacterium Serratia marcescens NCIM 2919 has been evaluated for degradation of DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane). The bacterium was able to degrade up to 42% of initial 50 mg L^?1 of DDT within 10 days of incubation. The highlight of the work was the elucidation of DDT degradation pathway in S. marcescens. A total of four intermediates metabolites viz. 2,2-bis (chlorophenyl)-1,1-dichloroethane (DDD), 2,2-bis (chlorophenyl)-1,1-dichloroethylene (DDE), 2,2-bis (chlorophenyl)-1-chloroethylene (DDMU), and 4-chlorobenzoic acid (4-CBA) were identified by GC-Mass and FTIR. 4-CBA was found to be the stable product of DDT degradation. Metabolites preceding 4-CBA were not toxic to strain as reveled through luxuriant growth in presence of varying concentrations of exogenous DDD and DDE. However, 4-CBA was observed to inhibit the growth of bacterium. The DDT degrading efficiency of S. marcescens NCIM 2919 hence could be used in combination with 4-CBA utilizing strains either as binary culture or consortia for mineralization of DDT. Application of S. marcescens NCIM 2919 to DDT contaminated soil, showed 74.7% reduction of initial 12.0 mg kg^?1 of DDT after 18-days of treatment.     

The bacterial metabolism of 4,4′-dichlorobiphenyl,and its suppression by alternative carbon sources

The metabolism of 4,4′-dichlorobiphenyl by mixed cultures of bacteria, isolated from activated sludge, was studied in shake cultures and in soil, both in presence and absence of alternative carbon sources. When 4,4′-dichlorobiphenyl was used as sole carbon source, 4-chlorobenzoic acid and 4,4′-dichloro-2,3-biphenyldiol could be isolated from the culture medium. Polar metabolites however, could not be detected in soil and in media in which alternative carbon sources such as glucose, glycerol, peptone, yeast-extract, humic acid or activated sludge were present. No hydroxylated or carboxylic acid derivatives could be isolated when 2,4′, 5-tri-, 2,2′,5,5′,-textra-, 2,2′,3,4,5′-penta-, 2,2′,3,4,5,5′-hexa- and decachlorobiphenyl were used as the sole carbon sources for incubation with bacteria in shake culture.     

Biodegradation of aromatic compounds by white rot and ectomycorrhizal fungal species and the accumulation of chlorinated benzoic acid in ectomycorrhizal pine seedlings

The capability of different white rot (WR, Heterobasidion annosum, Phanerochaete chrysosporium, Trametes versicolor) and ectomycorrhizal (ECM, Paxillus involutus, Suillus bovinus) fungal species to degrade different aromatic compounds and the absorption of 3-chlorobenzoic acid (3-CBA) by ECM pine seedlings was examined. The effect of aromatic compounds on the fungal biomass development varied considerably and depended on (a) the compound, (b) the external concentration, and (c) the fungal species. The highest effect on the fungal biomass development was observed for 3-CBA. Generally the tolerance of WR fungi against aromatic compounds was higher than that of the biotrophic fungal species. The capability of different fungi to degrade aromatic substances varied between the species but not generally between biotrophic and saprotrophic fungi. The highest degradation capability for aromatic compounds was detected for T. versicolor and H. annosum, whereas for Phanerochaete chrysosporium and the ECM fungi lower degradation rates were found. However, Paxillus involutus and S. bovinus showed comparable degradation rates at low concentrations of benzoic acid and 4-hydroxybenzoic acid. In contrast to liquid cultures, where no biodegradation of 3-CBA by S. bovinus was observed, mycorrhizal pines inoculated with S. bovinus showed a low capability to remove 3-CBA from soil substrates. Additional X-ray microanalytical investigations showed, that 3-CBA supplied to mycorrhizal plants was accumulated in the root cell cytoplasm and is translocated across the endodermis to the shoot of mycorrhizal pine seedlings.     

Growth of Dehalococcoides spp. and increased abundance of reductive dehalogenase genes in anaerobic PCB-contaminated sediment microcosms

Polychlorinated biphenyls (PCBs) contaminate 19% of US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs.

However, functional genes that act as biomarkers for PCB dechlorination processes (i.e., reductive dehalogenase genes) are poorly understood. Here, we developed anaerobic sediment microcosms that harbor an OHRB community dominated by the genus Dehalococcoides. During the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 10⁷ copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70?+?74?+?76, 95, 90?+?101, and PCB110 without exogenous electron donor. We quantified candidate reductive dehalogenase (RDase) genes over a 430-day incubation period and found rd14, a reductive dehalogenase that belongs to Dehalococcoides mccartyi strain CG5, was enriched to 10⁷ copies/g of sediment. At the same time, pcbA5 was enriched to only 10⁵ copies/g of sediment. A survey for additional RDase genes revealed sequences similar to strain CG5’s rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, our results suggest candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.

     

Chloromuconolactone dehalogenase ClcF of actinobacteria

This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram‐positive bacteria, Rhodococcus opacus 1CP, adapted to 2‐chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R. opacus 1CP. CMLDs isolated from the biomass of strains Rhodococcus spp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of the clcF gene from the ClcF of strain R. opacus 1CP. Matrix‐assisted laser dissociation/ionization time‐of‐flight mass spectrometry showed the presence of a peak with m/z 11,194–11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats.     

Isolation and characterization of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. SR1 degrading two β-triketone herbicides

In this study, a bacterial strain able to use sulcotrione, a β-triketone herbicide, as sole source of carbon and energy was isolated from soil samples previously treated with this herbicide. Phylogenetic study based on16S rRNA gene sequence showed that the isolate has 100 % of similarity with several Bradyrhizobium and was accordingly designated as Bradyrhizobium sp. SR1. Plasmid profiling revealed the presence of a large plasmid (>50 kb) in SR1 not cured under nonselective conditions. Its transfer to Escherichia coli by electroporation failed to induce β-triketone degrading capacity, suggesting that degrading genes possibly located on this plasmid cannot be expressed in E. coli or that they are not plasmid borne. The evaluation of the SR1 ability to degrade various synthetic (mesotrione and tembotrione) and natural (leptospermone) triketones showed that this strain was also able to degrade mesotrione. Although SR1 was able to entirely dissipate both herbicides, degradation rate of sulcotrione was ten times higher than that of mesotrione, showing a greater affinity of degrading-enzyme system to sulcotrione. Degradation pathway of sulcotrione involved the formation of 2-chloro-4-mesylbenzoic acid (CMBA), previously identified in sulcotrione degradation, and of a new metabolite identified as hydroxy-sulcotrione. Mesotrione degradation pathway leads to the accumulation of 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4 methylsulfonylbenzoic acid (AMBA), two well-known metabolites of this herbicide. Along with the dissipation of β-triketones, one could observe the decrease in 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition, indicating that toxicity was due to parent molecules, and not to the formed metabolites. This is the first report of the isolation of bacterial strain able to transform two β-triketones.     

Study of MCPA and MCPP herbicides mobility in soils from North-West Croatia as affected by presence of fertilizers

The mobility of acid herbicide (4-chloro-2-methylphenoxy)acetic acid [MCPA] and 2-(4-chloro-2-methylphenoxy)propionic acid [MCPA] in soils of North-West Croatia has been studied by soil thin-layer chromatography (STLC). Mobility of MPCA and MCPP was influenced by the change in concentration of soluble salts and the effect of mineral composition of the system studied, i.e. content of kaolin and sand in soil thin layer. The objective of this work was also to investigate how the mobility of phenoxy herbicides MCPA and MCPP is altered by the presence of fertilizers when both coexist in soil as a result of human activity. It has been found that mobility of acidic herbicides increases with application of fertilizers especially on soil with low clay and low organic matter content.     

Biodegradation capability of Absidia fusca Linnemann towards environmental pollutants

The purpose of this work was to study the bioremediation capability of Absidia fusca Linnemann (Zygomycete) towards different classes of xenobiotics (lignin-derived compounds, chloroaromatic compounds, polycyclic aromatic hydrocarbons) the presence of which in contaminated soils, water and sediments poses a significant risk to the environment and human health. Two strains from different origins were compared. One was from an official collection and grown in non-inducing conditions, while the other was isolated during the course of the survey of fungal flora in a polluted soil from Annaba (Algeria). All data were analyzed and results validated via a statistical treatment. We showed the effect of the factors studied (origin of the strain, xenobiotic) but also the interactions between these factors. The strain of A. fusca isolated from a polluted soil was able to efficiently degrade most of the xenobiotics tested, particularly: pentachlorophenol, phenol, catechol, guaiacol and ferulic acid. This property also existed in the other strain but at a very low level.     

Sublethal detergent concentrations increase metabolization of recalcitrant polyphosphonates by the cyanobacterium Spirulina platensis

As a consequence of increasing industrial applications, thousand tons of polyphosphonates are introduced every year into the environment. The inherent stability of the C–P bond results in a prolonged half-life. Moreover, low uptake rates limit further their microbial metabolization. To assess whether low detergent concentrations were able to increase polyphosphonate utilization by the cyanobacterium Spirulina platensis, tolerance limits to the exposure to various detergents were determined by measuring the growth rate in the presence of graded levels below the critical micellar concentration. Then, the amount of hexamethylenediamine-N,N,N′,N′-tetrakis(methylphosphonic acid) that is metabolized in the absence or in the presence of sublethal detergent concentrations was quantified by ³¹P NMR analysis on either P-starved or P-fed cyanobacterial cultures. The strain tolerated the presence of detergents in the order: nonionic > anionic > cationic. When added to the culture medium at the highest concentrations showing no detrimental effects upon cell viability, detergents either improved or decreased polyphosphonate utilization, the anionic sodium dodecyl sulfate being the most beneficial. Metabolization was not lower in P-fed cells—a result that strengthens the possibility of using, in the future, this strain for bioremediation purposes.     

Biodegradation of phthalate esters by two bacteria strains

In this study two aerobic phthalic acid ester (PAE) degrading bacteria strains, DK4 and O18, were isolated from river sediment and petrochemical sludge, respectively. The two strains were found to rapidly degrade PAE with shorter alkyl-chains such diethyl phthalate (DEP), dipropyl phthalate (DPrP), di-n-butyl phthalate (DBP), benzylbutyl phthalate (BBP) and diphenyl phthalate (DPP) are very easily biodegraded, while PAE with longer alkyl-chains such as dicyclohexyl phthalate (DCP) and dihexyl phthalate (DHP) and di-(2-ethylhexyl) phthalate (DEHP) are poorly degraded. The degradation rates of the eight PAEs were higher for strain DK4 than for strain O18. In the simultaneous presence of strains DK4 and O18, the degradation rates of the eight PAEs examined were enhanced. When the eight PAEs were present simultaneously, degradation rates were also enhanced. We also found that PAE degradation was delayed by the addition of nonylphenol or selected polycyclic aromatic hydrocarbons (PAHs) at a concentration of 1 microg/g in the sediment. The bacteria strains isolated, DK4 and O18, were identified as Sphigomonas sp. and Corynebacterium sp., respectively.     

Influences of metals on kinetics of methyl tert-butyl ether biodegradation by Ochrobactrum cytisi

The influence of zinc, manganese, and nickel on the degradation of MTBE (methyl tert-butyl ether), by an aerobic MTBE-degrading strain, Ochrobactrum cytisi, were investigated. The result showed that unlike previous findings, O. cytisi was able to degrade MTBE through direct metabolism when MTBE was present as the only carbon source. The degradation rate of MTBE was rapid, completed within 80 h. MTBE biodegradation by this strain was stimulated at low concentrations of Zn(2+) (1-5 mg l(-1)) and Mn(2+) (1-5 mg l(-1)) but inhibited at high concentrations of Zn(2+) (20 mg l(-1)) and Mn(2+) (20 mg l(-1)), and at low concentration of Ni(2+) (1-4 mg l(-1)). Kinetic parameters for MTBE degradation in the presence or absence of metals were obtained through nonlinear regression and a least-square minimization procedure. In all cases, a good agreement was achieved between kinetic simulations and experimental results.     

Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process

The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L^?1. The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200–250 mg L^?1) and high (4 g L^?1) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.     

Biacore biosensor immunoassay for 4-nonylphenols: assay optimization and applicability for shellfish analysis

A rapid Biacore biosensor immunoassay of 4-nonylphenols was developed. Two types of antibodies were used in the study: polyclonal antibodies with high cross-reactivity towards technical 4-nonylphenol and a monoclonal antibody very specific to 4-n-nonylphenol. 9-(p-Hydroxyphenyl)nonanoic acid was immobilized onto surface of a sensor chip. The best assay sensitivity was achieved using a flow rate of 50 microl min(-1) and injection time of 2 min. For the assay incorporating monoclonal antibodies a limit of detection 2 ng ml(-1) for 4-n-nonylphenol was achieved. With polyclonal antibodies one order lower sensitivity was observed for 4-nonylphenols. High background level of calibration curve for technical 4-nonylphenol was decreased by using IgG fraction of polyclonal antibodies in combination with lower amount of immobilised 9-(p-hydroxyphenyl)nonanoic acid. Sensitivity of the assay was improved by using a chip with a new derivative on a surface-N-aminobutyl [2-(4-hydroxyphenyl)ethylamine] (limit of detection--5 ng ml(-1)). Applicability of the developed assays to ecological monitoring was checked in experiments using shellfish samples. 4-n-Nonylphenol from spiked samples was extracted into hexane followed by clean-up on NH2 SPE columns. Calibration curves generated for cockles, mussels and oyster samples were identical (limit of detection about 10 ng g(-1)) whereas for scallop samples a slight decrease (about 5-10%) of absolute response was observed. In the assay using the monoclonal antibody specific to 4-n-nonylphenol 31 shellfish samples were found to be negative. Results obtained with polyclonal antibodies indicated that two scallop samples contained a quantity of 4-nonylphenols. The developed biosensor assay could be applied for shellfish analysis as a preliminary screening method.     

Photochemical degradation of methylparathion in the presence of humic acid

Photochemical degradation of methylparathion (O,O,-dimethyl O-4 nitrophenylphosphorothioate) in the presence of humic acid between pH 2 and 7 was monitored by differential pulse polarography. Humic acid was not electro-active under the experimental conditions used in this study. Only the pesticide and its main degradation product at pH 2 exhibited polarographic signals. Photolysis of methylparathion in acid media was sensitized by humic acid since the pesticide did not degrade in the absence of this compound. Methylparathion degradation in the presence of humic acid was observed at each of the studied pHs. The reaction was first-order with rate constant values ranging from 2 x 10(-3) to 6.3 x 10(-3) min(-1).     

PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01–0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6–14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.     

Complete degradation of butyl benzyl phthalate by a defined bacterial consortium: role of individual isolates in the assimilation pathway

Two bacterial strains, in consortium, were isolated by enrichment techniques from municipal waste-contaminated soil, which utilized butyl benzyl phthalate (BBP) as the sole carbon source. One of the isolates was identified as Arthrobacter sp. strain WY and the other one as Acinetobacter sp. strain FW based on the morphological, nutritional and biochemical characteristics and 16S rRNA sequence analysis. Various metabolites of BBP engendered by Arthrobacter sp. strain WY were isolated and identified by a combination of chromatographic and spectrophotometric analyses, which revealed a pathway involving monobutylphthalate (MBuP), monobenzyl phthalate (MBzP), phthalic acid and protocatechuic acid. The protocatechuic acid in turn was processed by ortho-cleavage dioxygenase to form beta-carboxy-cis,cis-muconate, ultimately leading to the TCA cycle. The Arthrobacter sp. strain WY could not utilize the hydrolyzed alcohols of BBP. On the other hand, the Acinetobacter sp. strain FW, which by itself could not utilize BBP as the sole carbon source, is capable of utilizing the hydrolyzed alcohols of BBP. Benzyl alcohol was found to be metabolized by the Acinetobacter sp. strain FW via benzaldehyde, benzoic acid and catechol. Catechol was further degraded by ortho-cleavage dioxygenase to cis,cis-muconic acid and subsequently to muconolactone leading to beta-ketoadipate pathway. Moreover, the Acinetobacter sp. strain FW metabolized 1-butanol through butyraldehyde and butyric acid leading to the tricarboxylic acid cycle via beta-oxidation pathway. This is the first report on the complete degradation of BBP by a defined consortium describing the role of its individual constituents in the BBP assimilation pathway.