Release Of Inorganic Chloride Research Articles

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

Cometabolic degradation of dichloroethenes by Comamonas testosteroni RF2

An environmental isolate Comamonas testosteroni strain RF2, which has been found to cometabolize trichloroethene (TCE) in the presence of phenol and sodium lactate as growth substrates, was tested to investigate its capacity for degrading 1,2-cis-dichloroethene (cDCE), 1,2-trans-dichlorothene (tDCE), and 1,1-dichloroethene (1,1DCE). Degradation assays were performed for single DCEs, as well as for a mixture of DCEs with TCE, which resembled contaminated plume in groundwater. RF2 was capable of efficiently removing all three dichloroethenes (DCEs) at the initial aqueous concentrations of 6.01 mg L−1 for cDCE, 3.80 mg L−1 for tDCE and 0.65 mg L−1 for 1,1DCE, with a removal efficiency of 100% for cDCE, 65.8% for tDCE, and 46.8% for 1,1DCE. Furthermore, complete removal of TCE, cDCE and 1,1DCE (122.5 μg L−1, 84.3 μg L−1 and 51.4 μg L−1, respectively) was observed in a mixture sample that also contained 72.33 μg L−1 of tDCE, which was removed to the amount of 72.3%. Moreover, degradation of cDCE (6.01 mg L−1) led to a 93.8% release of inorganic chloride, and 2,2-dichloroacetaldehyde was determined as the first intermediate of cDCE transformation. The findings of this study suggest that the strain RF2 exhibits the potential to remediate groundwater contaminated with less chlorinated ethenes.

Ozonation, photocatalysis and photocatalytic ozonation of diuron. Intermediates identification

Aqueous 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) has been oxidized by ozonation, photocatalysis and photocatalytic ozonation. Diuron degradation takes place via radical pathway through hydroxyl radicals in those systems involving ozone. Diuron elimination in photocatalytic ozonation is not enhanced if compared to single ozonation; however, TOC removal was significantly improved. Specifically, 80% TOC removal in 2h was reached in photocatalytic ozonation while single ozonation just led to 25% TOC reduction. Photocatalysis required 9h to reach 25% TOC reduction. Ten transformation by-products generated during the application of the three technologies were tentatively identified by liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–QTOF–MS/MS). Single ozonation and photocatalytic ozonation led to the formation and complete elimination of all by-products. Low weight carboxylic acids evolution suggests that high TOC removal in photocatalytic ozonation is linked to its capacity to oxidize small oxygenated compounds and release of inorganic chloride and nitrate. Toxicity evolution to Vibrio fischeri in photocatalytic ozonation displayed an increase in inhibition at the initial stages (>90% of inhibition), followed by a decrease of this parameter as the reaction progressed. The final treated sample shows a lower toxicity than the initial one (55% vs 20%).

A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon.

Biodegradation of vinyl chloride, cis-dichloroethene and 1,2-dichloroethane in the alkene/alkane-oxidising Mycobacterium strain NBB4

Mycobacterium chubuense strain NBB4 can grow on both alkanes and alkenes as carbon sources, and was hypothesised to be an effective bioremediation agent for chlorinated aliphatic pollutants. In this study, the ability of NBB4 to biodegrade vinyl chloride (VC), cis-dichloroethene (cDCE) and 1,2-dichloroethane (DCA) was investigated under pure-culture conditions and in microcosms. Ethene-grown NBB4 cells were capable of biodegrading VC and cDCE, while ethane-grown cells could biodegrade cDCE and DCA. The stoichiometry of inorganic chloride release (1mol/mol in each case) indicated that VC was completely dechlorinated, while cDCE and DCA were only partially dechlorinated, yielding chloroacetate in the case of DCA, and unknown metabolites in the case of cDCE. The apparent maximum specific activities (k) of whole cells against ethene, cDCE, ethane and DCA were 93±4.6, 89±18, 39±5.5, and 4.8±0.9nmol/min/mg protein, respectively, while the substrate affinities (K(S)) of whole cells with the same substrates were 2.0±0.15, 46±11, 11±0.33 and 4.0±3.2μM, respectively. In microcosms containing contaminated aquifer sediments and groundwater, NBB4 cells removed 85-95% of the pollutants (cDCE or DCA at 2mM) within 24h, and the cells remained viable for >1month. Due to its favourable kinetic parameters, and robust survival and biodegradation activities, strain NBB4 is a promising candidate for bioremediation of chlorinated aliphatic pollutants.

Degradation and mineralization of 2-chloro-, 3-chloro-and 4-chlorobiphenylby a newly characterized natural bacterial strain isolated froman electrical transformer fluid-contaminated soil

A bacterium classified as Achromobacter xylosoxidans strain IR08 by phenotypic typing coupled with 16S rRNA gene analysis was isolated from a soil contaminated with electrical transformer fluid for over sixty years using Aroclor 1221 as an enrichment substrate. The substrate utilization profiles revealed that IR08 could grow on all three monochlorobiphenyls (CBs), 2,4'- and 4,4'-dichlorobiphenyl as well as 2-chlorobenzoate (2-CBA), 3-CBA, 4-CBA, and 2,3-dichlorobenzoate. Unusually, growth was poorly sustained on biphenyl and benzoate. In growth experiments, IR08 degraded all CBs (0.27 mmol/L) in less than 96 h with concomitant stoichiometric release of inorganic chloride and growth yields were 2-3 times higher than those observed on biphenyl. In contrast to most of the chlorobiphenyl-degrading strains described in the literature, which are reported to form CBA, no metabolite was identified in the culture broth by HPLC analysis. When co-incubated with respective CBs and biphenyl, strain IR08 preferentially utilized the chlorinated analogues in less than 96 h while it took another 264 h before 90% of the initially supplied biphenyl could be degraded. The promotion of co-metabolic transformation of halogenated substrates by the inclusion of their non-halogenated derivatives may not therefore, result in universal benefits.

Trichloroethylene degradation in a coupled anaerobic/aerobic reactor oxygenated using hydrogen peroxide.

In this work, trichloroethylene (TCE) degradation under combined anaerobic-aerobic conditions was studied in an ethanol-fed biofilm reactor oxygenated using hydrogen peroxide. The reactor was inoculated with a biomass originating from an anaerobic digestor. Granulated peat was added to the reactor as a substratum for biofilm development. Extensive characterization of reactor populations using activity tests and PCR analysis revealed the development of a mutualistic consortium, particularly methanotrophic and methanogenic microorganisms. This consortium was shown to degrade TCE by a combination of reductive and oxidative pathways. A near complete degradation of TCE at a load of 18 mg L(R)(-1) day(-1) was evidenced by a stoichiometric release of inorganic chloride.

Degradation of 2,3,4,6-tetrachlorophenol at low temperature and low dioxygen concentrations by phylogenetically different groundwater and bioreactor bacteria.

Effects of low temperature and low oxygen partial pressure on the occurrence and activity of 2,3,4,6-tetrachlorophenol degrading bacteria in a boreal chlorophenol contaminated groundwater and a full-scale fluidized-bed bioreactor were studied using four polychlorophenol degrading bacterial isolates of different phylogenetic backgrounds. These included an alpha-proteobacterial Sphingomonas sp. strain MT1 isolated from the full-scale bioreactor and three isolates from the contaminated groundwater which were identified as beta-proteobacterial Herbaspirillum sp. K1, a Gram-positive bacterium with high G + C content Nocardioides sp. K44 and an alpha-proteobacterial Sphingomonas sp. K74. The Sphingomonas strains K74 and MT1 and Nocardioides sp. K44 degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol as the sole carbon and energy sources. Close to stoichiometric inorganic chloride release with the 2,3,4,6-tetrachlorophenol removal and the absence of methylation products indicated mineralization. Tetrachlorophenol degradation by the Herbaspirillum sp. K1 was enhanced by yeast extract, malate, glutamate, pyruvate, peptone and casitone. At 8 degrees C, Sphingomonas sp. K74 had the highest specific degradation rate (mu(max) = 4.9 x 10(-2) mg h(-1) cell(-1)) for 2,3,4,6-tetrachlorophenol. The Nocardioides strain K44 had the highest affinity (K(s) = 0.46 mg l(-1)) fortetrachlorophenol. K1 and MT1 grew microaerophilically in semisolid glucose medium. Furthermore, the growth of MT1 was inhibited in liquid glucose medium at high oxygen partial pressure indicating sensitivity to accumulating toxic oxygen species. On the other hand, trichlorophenol degradation was not affected by oxygen concentration (2-21%). The isolates K44, K74 and MT1, with optimum growth temperatures between 23 and 25 degrees C, degraded tetrachlorophenol faster at 8 degrees C than at room temperature indicating distinctly different temperature optima for chlorophenol degradation and growth on complex media. These results show efficient polychlorophenol degradation by the isolates at the boreal groundwater conditions, i.e., at low temperature and low oxygen concentrations. Differences in chlorophenol degradation and sensitivities to chlorophenols and oxygen among the isolates indicate that the phylogenetically different chlorophenol degraders have found different niches in the contaminated groundwater and thus potential for contaminant degradation under a variety of saturated subsurface conditions.

Enhanced dechlorination of aroclor 1242 in an anaerobic continuous bioreactor

Disappearance of Aroclor 1242 was observed in a continuously operated upflow anaerobic sludge bed (UASB) reactor inoculated with granular anaerobic sludge. To distinguish between sorption and dechlorination processes, extraction of Aroclor from the sludge was performed along with the measurements of chloride and Aroclor in the reactor effluent. A release of inorganic chloride combined with the enrichment in lightly chlorinated congeners was observed. Based on the chloride balance, the dechlorination process was found to be Aroclor concentration-limited at an Aroclor load of 0.4 mg/(g VSS day) due to a low solubility of Aroclor in water. While an addition of Tween 80 to the influent stream improved solubility of Aroclor, it led to self-inhibition of the dechlorination process at a load of 1.3 mg/(g VSS day). The maximal dechlorination rate observed in this study was 0.6 mg Aroclor/(g VSS day).

Simulation of in situ subsurface biodegradation of polychlorophenols in air-lift percolators.

Air-lift percolator experiments simulated in situ subsurface degradation of 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol, in mixtures and individually, by indigenous microorganisms from a chlorophenol-contaminated aquifer. Inoculation with a chlorophenol(CP)-degrading gram-positive isolate from the CP-contaminated groundwater did not significantly increase CP degradation rates. Feed concentrations of up to 55 mg CP l-1 were degraded. Stable CP degradation was maintained for over 6 months. CP degradation rates up to 54.3 mg l-1 day-1 and effluent concentrations below 40 micrograms CP l-1 were achieved. CP were mineralized as shown by CP reduction, dissolved organic carbon removal and release of inorganic chloride.

Bioremediation of chlorophenol contaminated ground water

Abstract Two aerobic laboratory‐scale continuous‐flow fluidized‐bed reactors were used to study biological ground water decontamination from chlorophenols. Biomass in both reactors was enriched using synthetic chlorophenol feed. The chlorophenol degradation was monitored by inorganic chloride release (ICl), organic carbon removal and gas Chromatographic analyses. The chlorophenol concentration in Karkola aquifer, Finland remained stable through a two year period. Fluidized‐bed treatment of ground water contaminated with 44–55 mgl‐1 of chlorophenols resulted in less than 1 mgl‐1 effluent concentrations and corresponding ICl releases. 99% chlorophenol degradation was maintained even at 45 min hydraulic retention time (HRT) and 1500 gCPm‐3 d‐1 loading rate. During gradual temperature decrease from 25–30°C to 10°C within 4 months, ICl release remained stable. At 10°C, over 99 % chlorophenol removal, and effluent concentrations of less than 0.1 mgl‐1, were maintained at 5 h HRT and 240 gCPm‐3d‐1 loading rate. ...

Aerobic and Anaerobic Biotransformations and Treatment of Chlorinated Pulp Bleach Waste Constituents

Biotransformations and treatment of several chlorinated, hydroxylated and methoxylated monoaromatic compounds were studied in batch bottle bioassays and continuous-flow fluidized-bed reactors. With the aerobic enrichment, polychlorinated phenols were biodegraded with simultaneous release of inorganic chloride in amounts equal to mineralization stoichiometrics. Aerobic removal of 4,5-dichlorocatechol and 4,5- dichlorovanillin were associated with the release of inorganic chlorine. The aerobic enrichment showed no activity against mono- or di-O-methylated phenols. With the anaerobic enrichment, reductive transformations of these compounds were observed. These transformations included reductive dechlorinations, de-O-methylations and dehydroxylations. High-rate operation of an aerobic fluidized-bed reactor resulted in over 99.7% biodégradation of polychlorophenols. In the anaerobic fluidized-bed reactor, over 95% removal of chlorophenols with no apparent accumulation of lower chlorinated phenols indicated complete dechlorination.

Degradation of 4‐chlorophenol in denitrifying fluidized‐bed process

Abstract Chlorophenol degradation by dissimilatory nitrate reduction was studied in an anoxic fluidized‐bed reactor. 4‐Chlorophenol (4‐CP) served as a carbon source for denitrification although both 4‐CP and nitrate removals remained incomplete (23–82 and 9–60%, respectively) at 5 h hydraulic retention time. Under varying experimental conditions, 1.9–2.5 mg 4‐CP was degraded and dechlorinated per 1 mg NO3‐N reduced. This is higher than the theoretical 4‐CP/NO3‐N ratio of 1.8 and the difference is likely due to aerobic degradation in the presence of trace concentrations of oxygen. The mass balance based on inorganic chloride release and disappearance of the parent compound gave further evidence of chlorophenol degradation in the system.

Chlorophenol toxicity removal and monitoring in aerobic treatment: recovery from process upsets

Bioremediation of simulated ground water containing 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), and pentachlorophenol (PCP) was studied in a laboratory-scale aerobic fluidized-bed reactor. Chlorophenols were the sole source of carbon and energy in the enrichment culture. At a hydraulic retention time of 5 h and chlorophenol loading rate of 445 mg L -1 d -1 , stable chlorophenol removal of over 99.7% and adsorbable organic halogen removal of 99.4% were maintained with the mean inorganic chloride release (ICl) of 94%

Dechlorination of high-molecular-mass compounds in spent sulphite bleach effluents by free and immobilized cells of streptomycetes

Streptomyces chromofuscus and S. rochei dechlorinated high-molecular-mass compounds (HMM) from industrial bleach effluents. Compounds of the effluents from the first chlorination [(C + D)red] and the subsequent alkaline extraction stage (E1O) of a sulphite cellulose pulp mill were used as substrates for the microbial transformations. HMM bleach effluent fractions obtained by ultrafiltration were treated by free and immobilized cells in two types of bioreactors. The dechlorination was followed by measuring the reduction of activated-carbon-adsorbable organic-bound halogen (AOX) and the release of inorganic chloride. While 38–45% of the organic bound chlorine was released from a mixture of (C + D)red and E1O stage effluents within 20 days of incubation with S. chromofuscus, only 11–16% were liberated from E1O-stage HMM bleach-effluent compounds by S. chromofuscus and S. rochei. Immobilized cells of both strains remained active for over 100 days in successive batches, each releasing 12–24% of chloride from HMM bleach-effluent compounds.

Dechlorination of 2,4,6-trichlorophenol by a nitrifying biofilm

To study the possibility of removing nitrogen and organic halogens from bleached kraft pulp mill effluent, we investigated nitrification and dechlorination in a laboratory scale fluidized-bed reactor. The nitrifying reactor was fed with ammonia and exposed to 2,4,6-trichlorophenol as a model substrate. The reactor responded with a decrease in the production of nitrate and acid. Nitrification recovered after six days of continuous 2,4,6-trichlorophenol feed and inorganic chloride began to accumulate into the effluent. When the application of 2,4,6-trichlorophenol was repeated, no inhibition of nitrification was observed and dechlorination began with no lag phase. Inorganic chloride release and total organic carbon removal pointed out that 2,4,6-trichlorophenol was dechlorinated and mineralized in the reactor. Batch experiments showed that enzyme synthesis was necessary for the adaptation of the nitrifying biofilm to 2,4,6-trichlorophenol dechlorination. 2,6-dichlorohydroquinone formed as an intermediate product from 2,4,6-trichlorophenol. The electron micrographs of the biomass revealed bacteria morphologically similar to known ammonia oxidizing species; mainly Nitrosomonas and Nitrosospira.

Aerobic fluidized-bed treatment of polychlorinated phenolic wood preservative constituents

Degradation of polychlorinated phenols was studied in continuous-flow fluidized-bed reactors using pure oxygen for aeration and celite carrier for cell immobilization. High dilution rates and chlorophenols as the only source of carbon and energy were used for maintenance of the mixed biofilm cultures. The chlorophenol degradation performance was monitored as release of inorganic chloride and removal of total organic carbon and by direct gas chromatographic analyses. Continuous polychlorophenol biodegradation activity was maintained in a fluidized-bed reactor for 315 days. Chloride release and chlorophenol removal efficiencies of over 99% were achieved at substrate loading rates of up to 430 g 2,4,6-trichlorophenol/m1/d and 400 g 2,3,4,6-tetrachlorophenol/m1/d at 3–5 h hydraulic retention times, respectively. Immobilized mixed cultures biodegraded 2,3,4,6-tetrachlorophenol with partial efficiency at feed concentrations as high as 157 mg/l as indicated by inorganic chloride release. Inorganic chloride release in experiments with practical grade 2,3,4,6-tetrachlorophenol exceeded that which is possible by dechlorination of tetrachlorophenol alone suggesting dechlorination of unidentified chlorinated impurities of the preparation as well. Fluidized-bed treatment of simulated wood preservative contaminated groundwater at a chlorophenol loading rate of 217 g/m1/d and 5 h hydraulic retention time resulted in the following removal efficiencies: 99.5% for 2,4,6-trichlorophenol, 99.6% for 2,3,4,6-tetrachlorophenol and 92.5% for pentachlorophenol.