Biodegradation of nitrilotriacetate (NTA) by bacteria—I. Isolation of bacteria able to grow anaerobically with NTA as a sole carbon source

Bacterial strains with the ability to utilize nitrilotriacetate (NTA) under aerobic and anaerobic conditions have been isolated from natural waters exposed to NTA. One of these strains (NTA-A2) is a facultative anaerobe which grows under anaerobic conditions on NTA if nitrate is available in the medium. (Under aerobic conditions this strain can utilize acetate, glucose and some other sugars as well as NTA but not EDTA (ethylenediaminotetraacetate) or lactose as the sole carbon source.) The properties and characteristics of the strain NTA-A2 are described.     

The bacterial degradation of synthetic anionic detergents

Experimental work carried out during the period from October 1966 to April 1968 has been concerned with the assessment of the biodegradability of synthetic anionic detergents, with particular reference to the “slope culture” technique. Solutions of the detergents known as JNX, JNQ, sodium tetrapropylene benzene sulphonate, a “difficult” alkyl benzene sulphonate and Dobane 055 were inoculated with bacteria from various sewage works effluents which had undergone prior acclimatization to a particular detergent on an agar slope; unacclimatized bacteria were also investigated. Further tests were carried out using a single, standard effluent and a microbial slime from an experimental recirculating filter together with a more stringent testing procedure. The results of all tests showed that the detergents had a low biodegradability value when compared with other methods, and they were inconsistent. Bacteria which degraded JNX were isolated from a sewage works effluent and from the microbial slime which developed on an experimental recirculating filter matured on JNX. Inoculation of the bacteria into a detergent solution was found to be ineffective in assessing their detergent degrading ability. A recirculating filter was developed for this purpose and proved to be successful. The active bacteria were members of the genera Klebsiella, Achromobacter, Flavobacterium and Micrococcus. The environmental conditions controlling detergent degradation by a pure culture of bacteria were also investigated. With the prior knowledge that the recirculating filter provided a favourable environment, each of its predominant physical characteristics was simulated individually. Degradation occurred in the presence of inert materials such as gravel, asbestos, or dried activated sludge provided that the detergent solution was vigorously agitated. The presence of an additional nutrient such as peptone did not appear to interfere with the normal degradation of the detergent. With the knowledge of the essential conditions for degradation, attempts were made to induce the bacteria E. coli and Serratia marcescens to degrade JNX in a recirculating filter; these attempts were unsuccessful.     

Microbial degradation of nitrilotriacetate (NTA) during river water/groundwater infiltration: Laboratory column studies

In laboratory column experiments with aquifer material collected from a natural river water/groundwater infiltration site, the effects of changes in NTA concentration (0.06–3.40 μM), temperature (5–20°C), and redox conditions on the microbial degradation of NTA during infiltration have been investigated. Under both aerobic and denitrifying conditions, NTA was rapidly mineralized and supported microbial growth as a sole carbon and energy source. The presence of other degradable organic compounds and of trace metals had no significant effect on the total rate of NTA elimination after a 21.8 cm flow distance. At concentrations between 0.02 and 0.05 μM, NTA degradation was still rapid (apparent pseudo first-order rate constants of up to 15 d⁻¹). From the results of the column experiments it may be concluded that under environmental conditions typical for Switzerland, very low residual NTA concentrations (< 0.01 μM) should be present at all times of the year in the groundwater after only a few meters of infiltration, even when concentrations of NTA in river water reach 3–4 μM. This conclusion is corroborated by results of field measurements.     

Environmental aspects of the use of NTA as a detergent builder

The behaviour, fate and significance of the detergent builder nitrilotriacetic acid (NTA) has been reviewed with particular reference to the removal of NTA during wastewater treatment and the effects of NTA on heavy metal solubility both during treatment and in the receiving environment. It is concluded that NTA removal during secondary biological treatment is subject to considerable variation, both temporal and between works as a result of changes in NTA load, temperature, water hardness and treatment process parameters. As a result of such variability, effluent NTA concentrations may be sufficient to mobilise heavy metals resulting in metal contamination of receiving waters and potable waters, particularly in areas of low effluent dilution and high water re-use. Removal of NTA during primary sedimentation and septic tank treatment is concluded to be predominantly due to adsorption to the sludge solids while removal in anaerobic sludge digestion is subject to operational characteristics of the treatment works. Disposal of NTA contaminated sludge to land may contaminate groundwaters and affect heavy metal speciation, while the disposal of contaminated sludge or sewage to sea may result in toxic algal blooms, in addition to effects on metal speciation.     

Biodegradation of nitrilotriacetic acid (NTA) and ferric-NTA complex by aerobic microbial granules

Development of mixed-culture microbial granules under aerobic conditions in a sequencing batch reactor (SBR), capable of completely degrading a recalcitrant metal chelating agent is reported. In laboratory-scale reactor studies, the microbial granules degraded 2mM of free nitrilotriacetic acid (NTA) and Fe(III)-NTA completely in 14 and 40 h, respectively. Free NTA was degraded at a specific rate of 0.7 mM (gMLSS)(-1)h(-1), while Fe(III)-NTA was degraded at a specific rate of 0.37 mM (gMLSS)(-1)h(-1). Achievement of significant degradation rates of NTA and ferric-NTA in double-distilled water suggests that the microbial metabolism is not constrained by lack of essential elements. Efficient degradation of recalcitrant synthetic chelating agents by aerobic microbial granules suggests their potential application in a variety of situations where heavy metals or radionuclides are to be co-disposed with metal chelating agents.     

Mobilization of some metals in water and animal tissue by NTA, EDTA and TPP

Parallel experiments were conducted under stagnant and flowing conditions to determine whether sodium tripolyphosphate (TPP), the conventional detergent builder, or two potential replacements, nitrilotriacetic acid (NTA) and ethylene—diaminetetraacetic acid (EDTA) significantly influenced the mobilization of major cations (Ca, Mg, Na, K) and some heavy metals (Fe, Mn, Zn, Cu, Pb, Cr). Water sediments and tissue of chironomid adults (Chironomus tentans Fabricius), crayfish (Orconectes virilis Hagen) and rainbow trout (Salmo gairdnerii Richardson) were examined. The results showed that the chelating agents applied in concentrations of 0.2–5.0 mg 1⁻¹ often increased the concentration of iron, manganese, lead and zinc in water above contaminated lake sediments. Other heavy metals were not affected significantly. EDTA was the most active mobilizing agent while NTA and TPP both had a less significant effect. The chelating agents generally had little effect on the rate of uptake of most metals by chironomids in aquaria. The role of chelating agents in flowing systems was even less pronounced. Zinc concentrations in water were significantly elevated by the treatment with 1 mg 1⁻¹ EDTA and manganese concentrations were significantly higher in trout from the pond treated with an intermediate level (1 mg 1⁻¹) of NTA. All other treatments yielded levels of major ions and heavy metals that were not significantly different from those in the control ponds.     

异养硝化菌强化饱和基质材料降解水体氨氮的研究

将优势菌技术运用于饱和基质材料中,考察基质材料对水中氨氮的吸附特性以及微生物原位强化饱和基质材料后对氨氮的降解效果。试验结果表明,沸石对氨氮的吸附量高于活性炭。对氨氮含量为110mg／L的模拟富营养化水体进行360h动态吸附后,沸石和活性炭吸附后出水中氨氮平均含量分别为73．3l和89．18mg／L,沸石显示出作为基质材料的优越性。对饱和基质材料进行异养硝化茵强化96h后,沸石柱和活性炭柱出水氨氮平均含量分别降低8．58和17．31mg／L,并且活性炭和沸石表面形成稳定的生物膜。因此,对富营养水体在基质吸附基础上进行微生物降解的方法是可行的。     

Assessment of in situ degradation of chlorinated ethenes and bacterial community structure in a complex contaminated groundwater system

The occurrence of in situ degradation of chlorinated ethenes was investigated using an integrated approach in a complex groundwater system consisting of several geological units. The assessment of hydrogeochemistry and chlorinated ethenes distribution using principal component analysis (PCA) in combination with carbon stable isotope analysis revealed that chlorinated ethenes were subjected to substantial biodegradation. Shifts in isotopic values up to 20.4 per thousand, 13.9 per thousand, 20.1 per thousand and 31.4 per thousand were observed between geological units for tetrachloroethene (PCE), trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC), respectively. The use of specific biomarkers (16S rRNA gene) indicated the presence of Dehalococcoides sp. DNA in 20 of the 33 evaluated samples. In parallel, the analysis of changes in the bacterial community composition in the aquifers using canonical correspondence analysis (CCA) indicated the predominant influence of the chlorinated ethene concentrations (56.3% of the variance, P=0.005). The integrated approach may open new prospects for the assessment of spatial and temporal functioning of bioattenuation in contaminated groundwater systems.     

Study of tropaeolin degradation by iron--proposition of a reaction mechanism

The degradation of tropaeolin by iron was studied under oxidizing and inert atmospheres. The products were identified by various chromatographic and spectroscopic methods. Under inert atmosphere, the proposed model of mechanism is based on the adsorption of tropaeolin by the sulfonate function on the solid iron, followed by a reduction of the –N=N– bond with formation of 1-amino-2-naphthol and sulfanilic acid. These two intermediaries were reduced to 1,2-dihydroxynaphthalene and benzene with NH₄⁺ and HSO₃⁻ liberation. Under oxidizing atmosphere, besides the reductions of tropaeolin, water and ferric ions, the existence of a homogeneous degradation was found. This process was explained (on the basis of surface iron potential monitoring) by a stepwise reduction of oxygen giving hydrogen peroxide. The initial stages of the mechanism were similar to those under inert atmosphere, but the degradation of the 1,2-dihydroxynaphthalene continued giving 1,2-naphthalenedione, then various polar monocyclic compounds leading to three unidentified volatile final products.     

Influence of electron donor, oxygen, and redox potential on bacterial perchlorate degradation

Experiments were conducted to assess the influence of electron donor, redox potential, and dissolved oxygen on bacterial perchlorate degradation. Microcosms containing a diverse, perchlorate-acclimated, bacterial culture fed lactate at a 1:1 electron donor-to-perchlorate ratio (electron-equivalent basis) degraded perchlorate more slowly (k = 0.038 mg ClO4-/mg VSS h) and to a lesser extent than microcosms fed lactate at 2:1 and 4:1 ratios (k = 0.045 mg ClO4-/mg VSS h). The optimal COD/ClO4- ratio to consume all perchlorate and all electron donor was approximately 1.2 mg COD/mg ClO4-. In experiments where the redox potential was held constant, the extent of perchlorate degradation increased with decreasing redox potential, and 100% removal was only achieved at the lowest redox potential examined (-220 mV); however, perchlorate degradation (32% of added perchlorate) was observed as high as +180 mV. Additions of oxygen to actively degrading treatments did not adversely effect perchlorate degradation. It appears, therefore, that addition of excess electron donor is sufficient to negate potential inhibitory effects of molecular oxygen. If the redox conditions are more oxidized, however, the rate and extent of perchlorate degradation will be significantly decreased. This is the first report of perchlorate degradation under oxidized conditions using an environmentally relevant, diverse, bacterial enrichment culture, and this is also the first report of perchlorate reduction occurring at appreciable dissolved oxygen concentrations in a batch system.     

Atrazine degradation in anaerobic environment by a mixed microbial consortium

Atrazine degradation by anaerobic mixed culture microorganism in co-metabolic process and in absence of external carbon and nitrogen source was studied at influent atrazine concentration range of 0.5-15 mg/l. Wastewater of desired characteristic was prepared by the addition of various constituents in distilled water spiked with atrazine. In co-metabolic process, dextrose of various concentrations (150-2000 mg/l) was supplied as external carbon source. The reactors were operated in sequential batch mode in which 20% of treated effluent was replaced by the same amount of fresh wastewater everyday, thus maintaining a hydraulic retention time (HRT) equal to 5 days. In co-metabolic process, 40-50% of influent atrazine degradation was observed. First-order atrazine degradation rate (expressed in day(-1)) was better in co-metabolic process (5.5 x 10(-4)) than in absence of external carbon source (2.5 x 10(-5)) or carbon and nitrogen source (1.67 x 10(-5)). In presence of 2000 mg/l of dextrose, atrazine degradation was between 8% and 15% only. Maximum atrazine degradation was observed from wastewater containing 300 mg/l of dextrose and 5mg/l of atrazine. Influent atrazine concentration did not have much effect on the methanogenic bacteria which was clear from methane gas production and specific methanogenic activity (SMA).     

Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium

A bacterium termed 7CY, capable of decomposing cyanobacterial toxins, was isolated from surface water sample of Lake Suwa and degradation of microcystin-RR and nodularin-Har was investigated. The isolated 7CY was a gram-negative, aerobic bacillus, and a member of a genus Sphingomonas. The strain degraded microcystin-LY, -LW, and -LF completely as well as microcystin-LR within 4 days after their addition (6 microg/ml) whereas degradation of nodularin-Har did not occur at all during experiment. On the contrary, the strain was capable of degrading nodularin-Har in the presence of microcystin-RR and both toxins were completely decomposed within 6 days. The strain scarcely degraded nodularin-Har in the presence of microcystin-RR when glucose and ammonium chloride were added to the medium. The degradation of nodularin-Har did not occur in the medium from which bacterial cells had been removed after degradation of microcystin-RR. Furthermore, when microcystin-RR and nodularin-Har were added to the cytoplasm fraction of 7CY cells, microcystin-RR was rapidly degraded within 18 h, but nodularin-Har was not. The strain 7CY may require an enzyme(s) induced during the degradation of microcystin-RR in order to utilize nodularin-Har as nutrition.     

Biodegradation of nitrilotriacetate (NTA) by bacteria—II. Cultivation of an NTA-degrading bacterium in anaerobic medium

Bacterial strains with the ability to utilize nitrilotriacetate (NTA) under aerobic and anaerobic conditions have been isolated from natural waters exposed to NTA. One of these strains (NTA-A2) is a facultative anaerobe which grows under anaerobic conditions on NTA if nitrate is available in the medium. (Under aerobic conditions this strain can utilize acetate, glucose and some other sugars as well as NTA but not EDTA (ethylenediaminotetraacetate) or lactose as the sole carbon source.) The properties and characteristics of the strain NTA-A2 are described.     

Retention of inorganic arsenic by coryneform mutant strains

The natural resistance mechanisms of corynebacteria to respond to the environments containing high levels of arsenic were successfully adopted to develop inexpensive and selective extractants for submicrogram amounts of arsenic. Kinetic and equilibrium characteristics were evaluated, and a preliminary exploration of the capability of these strains to be used for arsenic speciation was also made in this work. Three kinetics models were used to fit the experimental data. It was found that the pseudo-first-order kinetics model was not quite adequate to describe the retention process, while the intraparticle diffusion and the pseudo-second-order kinetics models provide the best fits. The equilibrium isotherm showed that the retention of arsenic was consistent with the Langmuir equation and that the Freundlich and Dubinin-Radushkevich models provided poorer fits to the experimental data. The maximum effective retention capacity for arsenic was about 15.4 ng As/mg biomass. The amount of arsenic retained was directly measured in the biomass by forward planning a slurry electrothermal atomic absorption spectrometric procedure.     

The determination of nitrilotriacetic acid (NTA) in sewage and sewage effluent

Published methods for the determination of NTA have been examined and all have been found to be subject to interference in analysis of sewage and sewage effluents. The inadequacies of the various methods are described.

Passage of the sample through a chelating resin column as a pre-treatment to eliminate metal interference is proposed as a modification of the zinc-zincon method of determination. The procedure thus modified has given quantitative recovery of NTA from the samples analysed.     

The degradation of polychlorinated biphenyls by micro-organisms

An investigation is described concerning the effects of two micro-organisms cultures on single compounds and multi-component mixtures of polychlorinated biphenyls (PCBs).Several low chlorine PCB compounds can be degraded easily with these cultures and, in favourable circumstances, some compounds containing up to six chlorine atoms per molecule can be degraded.In certain multi-component commercial mixtures some PCB compounds are degraded more readily than if present singly. The significance of this is discussed, particularly the fact that work on single compounds does not enable predictions to be made about the behaviour of commercial mixtures in the environment. Some tentative explanations are suggested for the behaviour of the mixtures exposed to these cultures.     

2-fluorophenol degradation by aerobic granular sludge in a sequencing batch reactor

Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by aerobic granules, as shown through the recovering of the strain from the granular sludge at the end of the experiment. Overall, the granular SBR has shown to be robust, exhibiting a high performance after bioaugmentation with the 2-FP degrading strain. This study corroborates the fact that bioaugmentation is often needed in cases where biodegradation of highly recalcitrant compounds is targeted.     

Atrazine degradation by anodic Fenton treatment

Anodic Fenton treatment (AFT), an hydroxyl radical oxidation process recently developed for the degradation of aqueous pesticide waste, was applied to the degradation of atrazine, seven degradation products, and a formulated atrazine product. Using AFT, degradation of the parent compound occurred in 3 min. The concentration profiles of seven degradation products formed during treatment were measured, and degradation pathways are proposed for the treatment. The primary termination product after 10 min was dechlorinated ammeline. Three different ¹⁴C labeled atrazine compounds (ethyl, isopropyl and U-triazine ring labeled atrazine) were also treated in an air-tight AFT apparatus and the mass balance was calculated. The triazine ring was not cleaved during this treatment process. Formulated atrazine was 70% degraded in 3 min. AFT holds promise as an effective pesticide-laden water treatment technology.     

Cyclophosphamide degradation by advanced oxidation processes

Cyclophosphamide is a hazardous cytostatic drug, which should be removed from wastewaters before disposals. Ozonation slowly inactivates this substance, requiring long reaction times to obtain degradation. In this paper, different advanced oxidation processes (AOPs) are studied for cyclophosphamide oxidative degradation in aqueous medium: ozone at several basic pH values, ozone/hydrogen peroxide at neutral and basic pHs with the addition of the peroxide at the beginning of ozonation. The influence of ozone concentration in the gas phase and gas flow is also discussed. Results show that, under the experimental conditions studied, the radical mechanism acting in the AOPs rapidly degrades cyclophosphamide. Additionally, second-order rate constants k_d and k_OH were determined for direct (molecular) and indirect (radical) reactions. Finally, reactions were carried out with the aim of isolating and identifying some of the degradation products. From ozonation at pH 9, 4-ketocyclophosphamide was identified as the main reaction product.     

High performance degradation of azo dye Acid Orange 7 and sulfanilic acid in a laboratory scale reactor after seeding with cultured bacterial strains

Bacterial strains 1CX and SAD4i--previously isolated from the mixed liquor of a municipal sewage treatment plant--are capable of degrading the azo dye Acid Orange 7 (AO7) and sulfanilic acid, respectively. A rotating drum bioreactor (RDBR), operating under continuous flow and nutrient conditions designed to simulate the effluent from a dye manufacturing plant, was seeded with strains 1CX and SAD4i, forming a biofilm capable of degrading AO7 and sulfanilic acid. In addition, an RDBR containing a pre-existing biofilm capable of degrading AO7, but not sulfanilic acid, was seeded with strain SAD4i alone. Strain SAD4i was incorporated into the existing biofilm and degraded the sulfanilic acid resulting from the degradation of AO7 by indigenous members of the biofilm. The ability to seed a bioreactor with bacterial strains capable of degrading azo dyes, and resulting by-products, in a mixed microbial community suggests that this process could have commercial applications.