Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Bacterial degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal-EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg2+, Ca2+, Mn2+, Pb2+, Co2+, Cd2+, Zn2+, Cu2+, or Fe3+. The metal-EDTA complexes (Me-EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10(16) (lg K < 16), such as Mg-EDTA, Ca-EDTA, and Mn-EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5-10 h of incubation. Me-EDTA complexes with lg K above 16 (Zn-EDTA, Co-EDTA, Pb-EDTA, and Cu-EDTA) were not completely degraded during a 24-hour incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd-EDTA or Fe(III)-EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Bacterial Strain Characterizing by EDTA Requirement

A novel strain of bacteria (LPM-4) characterized by a unique EDTA requirement for cell growth was isolated. Suspensions of washed cells of strain LPM-4 degraded EDTA complexes with Ba²⁺, Mg²⁺, Ca²⁺, and Mn²⁺ at constant rates ( 0.310 ± 0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 ± 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrading bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095^{? 1}) and lower mass cell yield (0.219 g cells/g EDTA), which is promising for its practical applications for EDTA removal in wastewater treatment plants.     

Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Bacterial strain characterizing by EDTA requirement

A novel strain of bacteria (LPM-4) was isolated that is characterized by a unique EDTA requirement for cell growth. Suspensions of washed cells of strain LPM-4 degrated EDTA complexes with Ba2+, Mg 2+, Ca2+, and Mn2+ at constant rates (0.310-0.486 mmol EDTA/(g h)) and Zn-EDTA at an initial rate of 0.137 +/- 0.016 mmol EDTA/(g h). The temperature optima for cell growth and EDTA degradation were determined under pH-auxostat cultivation. As compared with the known EDTA-degrating bacteria, strain LPM-4 exhibited a higher specific growth rate (0.095 h(-1)) and lower mass cell yield (0.219 g cells/g EDTA) that is promising for its practical applications for EDTA removal in wastewater treatment plants.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Evidence that bacterial ABC-type transporter imports free EDTA for metabolism

EDTA, a common chelating agent, is becoming a major organic pollutant in the form of metal-EDTA complexes in surface waters, partly due to its recalcitrance to biodegradation. Even an EDTA-degrading bacterium, BNC1, does not degrade stable metal-EDTA complexes. In the present study, an ABC-type transporter was identified for possible uptake of EDTA because the transporter genes and the EDTA monooxygenase gene were expressed from a single operon in BNC1. The ABC-type transporter had a periplasmic-binding protein (EppA) that should confer the substrate specificity for the transporter; therefore, EppA was produced in Escherichia coli, purified, and characterized. EppA was shown to bind free EDTA with a dissociation constant as low as 25 nM by using isothermal titration calorimetry. When unstable metal-EDTA complexes, e.g., (Mg-EDTA)²⁻, were added to the EppA solution, binding was also observed. However, experimental data and theoretical analysis supported EppA binding only of free EDTA. When stable metal-EDTA complexes, e.g., (Cu-EDTA)²⁻, were titrated into the EppA solution, no binding was observed. Since EDTA monooxygenase in the cytoplasm uses some of the stable metal-EDTA complexes as substrates, we suggest that the lack of EppA binding and EDTA uptake are responsible for the failure of BNC1 cells to degrade the stable complexes.     

Biodegradation of EDTA

The chelating agent ethylenediaminetetraacetate (EDTA) is not degraded by conventional biological and physicochemical methods for the treatment of wastewater and the purification of drinking water. Of the measurable organic compounds it is the one present at the highest concentration in many surface and drinking waters. In recent years, however, studies have demonstrated that EDTA can be degraded by specially enriched bacterial cultures and in wastewater treatment plants receiving EDTA-containing effluents. The amounts of EDTA released into the aquatic environment could thus be reduced by establishing appropriate biological wastewater treatment plants. This article describes the degradation of EDTA and its metal chelates by different bacterial cultures, catabolic steps in EDTA degradation, and biological methods for the removal of this chelating agent from wastewaters. Received: 14 September 1998 / Received revision: 9 December 1998 / Accepted: 11 December 1998     

EDTA degradation by cells of Chelativorans oligotrophicus immobilized on a biofilter

A biofilter based on light expanded clay aggregate (LECA) and cells of the obligate ethylenediamine tetraacetate (EDTA) destructor Chelativorans oligotrophicus LPM-4 has been developed. The culture steadily maintained a high level of EDTA monooxygenase activity of 180–200 nmol/min/mg of protein during three months. EDTA was converted completely or by 80% at initial concentrations of 0.5–0.7 or 2.0 g/l, respectively, in a 2-dm² biofilter at a flow rate of 20 ml/h.     

Influence of physiological conditions on EDTA degradation

Aerobic biodegradation of the chelating agent EDTA by a mixed bacterial culture was investigated. Bacterial growth and degradation of the substrate required the presence of sufficient metal ions in the culture fluid. Uncomplexed EDTA interacted negatively with the cell walls of the bacteria and completely inhibited bacterial growth, whereas Mg(II)/Ca(II)-EDTA was degraded up to an initial concentration of 4.7 g/l. Therefore, concentrations of metal ions must be stoichiometric to that of EDTA or higher. Specific degradation rates ranged between 120 mg EDTA g^–1 (cell dry weight) h^–1 and 285 mg EDTA g^–1 h^–1. In contrast, complexes with high thermodynamic stability constants such as Fe(III)-EDTA remained as inert compounds in the solution. Specific growth rates of the mixed culture were found to vary between 0.03 h^–1 and 0.07 h^–1, which could be explained by population dynamics within the synergistic mixed community. Growth was significantly accelerated by the addition of vitamins.     

Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O₂ were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH₂ and O₂. The FMN reductase provided EDTA monooxygenase with FMNH₂ by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C. K_ms were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA²⁻; this difference in K_m indicates that the enzyme has greater affinity for MgEDTA²⁻. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH₂-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Genetic toxicology of ethylenediaminetetraacetic acid (EDTA)

EDTA and its salts have a number of applications in medicine and pharmacy. EDTA is used to remove calcium from the human body, and serves as an anticoagulant and as a detoxicant after poisoning by heavy metals. It is often used in analytical chemistry for complexometric titrations and many other purposes. Because the compound is of rather low toxicity, it is used as a food additive to bind metal ions. EDTA affects the inhibition of DNA synthesis in primary cultures of mammalian cells. This may be due to impairment of enzymes involved in DNA replication. Some early studies have shown that EDTA leads to morphological changes of chromatin and chromosome structure in plant and animal cells. These alterations consist of dispersion or swelling of chromosomes or a loss of interphase chromatin structure. For several test systems, a low chromosome-breaking activity of EDTA has been reported. A weak activity in the induction of gene mutations has also been observed. It is well established that EDTA influences chromosome breakage by mutagenic agents. In particular, when applied in combination with chemical mutagens, EDTA enhances mutagen-induced aberration frequencies. Furthermore, the chelating agent is able to increase the incidence of meiotic crossing-over. This has been demonstrated for many gene loci in Drosophila melanogaster, Chlamydomonas reinhardi, Neurospora crassa and Zea mays. EDTA interferes with DNA repair processes that take place after exposure to mutagens. In E. coli or Micrococcus radiodurans as well as in Chinese hamster cells, the fast repair component detectable after treatment with ionizing radiation or bleomycin is inhibited by EDTA. In plant cells exposed to gamma-rays, EDTA inhibits unscheduled DNA synthesis. The mechanism by which EDTA causes these effects remains poorly understood. The sequestering of metal ions by the chelating agent is obviously responsible for functional and structural alterations of the genetic material. Although EDTA produces a whole set of genetic effects it seems to be a harmless compound to man as far as genotoxicity is concerned. The data presently at hand, however, are not sufficient for a reliable risk assessment.     

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA

Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins.     

A new approach for Fe(III)EDTA reduction in NOx scrubber solution using bio-electro reactor

A new process for the removal of NO_x by a combined Fe(II)EDTA absorption and microbial reduction has been demonstrated, in which part of the Fe(II)EDTA will be oxidized by oxygen in the flue gas to form Fe(III)EDTA. In former studies, strain FR-2 has been found to reduce Fe(III)EDTA efficiently. Otherwise, it has been reported that bio-electro reactor could efficiently provide a chance for simultaneous denitrification and metal ion removal. Therefore, a use of bio-electro reactor is suggested to promote the reduction of Fe(III)EDTA by strain FR-2 in this paper. The results showed that the concentration of Fe(III)EDTA decreased rapidly when electric current was applied, and that as the current density rose, the Fe(III)EDTA reduction rate increased while followed by a decrease afterward. The formation of the biofilm on the electrode was observed by ESEM (Environmental Scan Electro-Microscope). In addition, the Fe(III)EDTA reduction rate obviously decreased with the existence of NaNO₂.     

In vitro inhibitory activity of EDTA against planktonic and adherent cells ofCandida sp.

Candida sp. can cause infections of indwelling medical devices associated with biofilm formation, which are difficult to treat due to insensitivity of adherent microorganisms to host defence mechanisms and standard antimicrobial therapy. The aim of this paper was to determine the effect of EDTA (disodium salt) on the adhesion ofCandida sp. to some catheters and also on biofilm formation by the yeasts and its eradication in relation to cytotoxicity of this chelating agent to the cell cultures. The adhesion process and biofilm formation, and also EDTA cytotoxicity to green monkey kidney (GMK) cell culture were determined using MTT tetrazolium salt [3-(4,5-dimethylthiazol-2-yl) ?2,5-diphenyltetrazolium bromide)] reduction assay. EDTA inhibited the growth of free-floating forms ofCandida sp. strains with minimal inhibitory concentration (MIC) from 0.06 to 0.25 mM; the minimal fungicidal concentration (MFC) values ranged from 64 to 128 mM. The prevention ofCandida sp. adhesion on the catheters used or eradication of the adherent cells was achieved at 0.5 to 4.0 mM EDTA. Also biofilm formation was prevented by 0.5 to 4.0 mM EDTA. Much higher concentration of EDTA (32 to 128 mM) was needed to eradicate the mature biofilm. EDTA at concentration up to 1 mM was not toxic for GMK cells. At higher concentration, toxicity of EDTA to GMK cells was correlated with the concentration of this agent and the time of exposure. Summing up, EDTA may be regarded as a useful agent rather in prophylaxis of candidal infections of medical devices.     

Cloning, sequencing, and characterization of a gene cluster involved in EDTA degradation from the bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Slow complexation kinetics for ferric iron and EDTA complexes make EDTA non-biodegradable

Published experimental data on ethylenediaminetetraacetic acid (EDTA) biodegradation in the presence of ferric iron (Fe(III)) showed that rapid biodegradation of EDTA suddenly stopped, leaving a residual of unbiodegraded EDTA that was equal to the concentration of dissolved Fe(III). We hypothesize that slow kinetics for the dissociation of two iron-EDTA complexes - FeEDTA^- and FeOHEDTA^2- – sequestered the EDTA in a form that is biologically unavailable. To evaluate this hypothesis, we added to the biogeochemical model CCBATCH a new sub-model for kinetically controlled complexation. CCBATCH simulations with kinetically controlled complexation for FeEDTA^- and FeOHEDTA^2- and the observed concentration of total dissolved Fe(III) accurately predicted the sudden cessation of EDTA biodegradation at the exact time shown experimentally. Our simulations also correctly predicted the observed residual EDTA concentration and the amounts of biomass and NH₄ ⁺. Alternate explanations for the experimental results – strong equilibrium complexation of ferric iron and EDTA and precipitation of calcium and magnesium solids – could not capture the observed trends. This analysis using CCBATCH's new sub-model for kinetically controlled complexation shows that EDTA, once it becomes complexed with Fe(III), becomes biologically unavailable.     

The Ultrastructure of Free Intestinal Cells Isolated eith EDTA

A brief electron microscopic study of free rat intestinal cells, isolated with the calcium chelator EDTA is given. The cells appeared morphologically intact, as was shown by nigrosin-staining. The junctional complexes were modified or lost.     

Synergistic wood preservatives involving EDTA,irganox 1076 and 2-hydroxypyridine-N-oxide

The efficiency of 2-hydroxypyridine-N-oxide (2-HPNO) against wood degradation by the white-rot fungus Coriolus versicolor was demonstrated by monitoring weight loss of treated and untreated wood blocks. The fungistatic properties of 2-HPNO are related to the presence of the hydroxamic acid function as shown using several analogs. Using response surface methodology, strongly significant synergy was observed between either the chelator ethylenediaminetetraacetic acid (EDTA) and 2-HPNO or the hindered phenolic antioxidant Irganox 1076 and 2-HPNO. 2-HPNO is subjected to oxidation by peroxidase explaining the synergy observed with the antioxidant. The chelating properties of 2-HPNO may also explain the synergy observed with EDTA. The implications of the observed synergy for the design of new wood preservation strategies are also discussed.     

柠檬酸和EDTA对铜污染土壤环境中吊兰生长的影响

通过盆栽试验研究了在铜污染条件下,柠檬酸和EDTA作为活化剂对铜污染土壤中吊兰生长状况的影响.结果表明,柠檬酸和EDTA对吊兰富集量的影响与其对土壤中铜的活化能力呈显著性正相关.柠檬酸对土壤铜有较强的活化作用,能够有效提高吊兰对铜的吸收,且在浓度为5mmol/L时效果最为明显,而较高的铜富集量又抑制了吊兰的生长;EDTA对吊兰富集能力的影响相对较弱,对吊兰的生长也无显著影响.相比而言,柠檬酸对铜污染土壤中吊兰生长状况的影响比EDTA大.