Charles F. Kulpa

Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids

Ionic liquids (ILs) are novel organic salts with a wide liquid range that have enormous potential for industrial use as “green” chemicals. Varying the cationic and anionic components can alter IL properties and toxicities. Before the likely industrial release of ILs into the environment, it is necessary to determine their toxic and antimicrobial properties. As a measure of microbial toxicity of imidazolium and pyridinium ILs with varying alkyl chain lengths, we investigated Vibrio fischeri using the Microtox method. An increase in alkyl group chain length as well as an increase in the number of alkyl groups substituted on the cation ring corresponded with an increase in toxicity. Varying the anion identity did not significantly alter toxicity. We then examined the antimicrobial effects of 1000 ppm of butyl-, hexyl- and octyl- imidazolium and pyridinium bromide ILs on the growth of a group of microorganisms representing a variety of physiological and respiratory capabilities. In general, hexyl- and octyl- imidazolium and pyridinium bromides had significant antimicrobial activity to pure cultures of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas fluorescens and Saccharomyces cerevisiae. Butyl-imidazolium and pyridinium bromides were less antimicrobial than ILs with longer alkyl chain lengths to all microorganisms examined. However, the most significant antimicrobial activity was observed in tests with B. subtilis. This research provides toxicity and antimicrobial information about ILs, prior to their widespread use and release. This type of proactive approach can aid in the prevention of pollution, and avoid costs of future clean-up, and provide information about the “green” nature of practical industrial solvents.

Biodegradability of imidazolium and pyridinium ionic liquids by an activated sludge microbial community

Ionic liquids (ILs) are novel organic salts that have enormous potential for industrial use as green replacements for harmful volatile organic solvents. Varying the cationic components can alter the chemical and physical properties of ILs, including solubility, to suit a variety of industrial processes. However, to complement designer engineering, it is crucial to proactively characterize the biological impacts of new chemicals, in order to fully define them as environmentally friendly. Before introduction of ILs into the environment, we performed an analysis of the biodegradability of six ILs by activated sludge microorganisms collected from the South Bend, Indiana wastewater treatment plant. We examined biodegradability of 1-butyl, 1-hexyl and 1-octyl derivatives of 3-methyl-imidazolium and 3-methyl-pyridinium bromide compounds using the standard Organisation for Economic Cooperation and Development dissolved organic carbon Die-Away Test, changes in total dissolved nitrogen concentrations, and 1H-nuclear magnetic resonance analysis of initial and final chemical structures. Further, we examined microbial community profiles throughout the incubation period using denaturing gradient gel electrophoresis (DNA-PCR-DGGE). Our results suggest that hexyl and octyl substituted pyridinium-based ILs can be fully mineralized, but that imidazolium-based ILs are only partially mineralized. Butyl substituted ILs with either cation, were not biodegradable. Biodegradation rates also increase with longer alkyl chain length, which may be related to enhanced selection of a microbial community. Finally, DGGE analysis suggests that certain microorganisms are enriched by ILs used as a carbon source. Based on these results, we suggest that further IL design and synthesis include pyridinium cations and longer alkyl substitutions for rapid biodegradability.

Biodegradation of methyl t-butyl ether by pure bacterial cultures

Abstract Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1–2 weeks of incubation at 23–25 °C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [14C]MTBE was used to determine 14CO2 evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as 14CO2. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in 14CO2 evolution from [14C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE.

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp. (B strain) isolated from an anaerobic digester

A sulfate-reducing bacterium (SRB),Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327–332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO2. This study demonstrated the possibility of using this isolated to decontaminate the soil and water contaiminated with TNT under anaerobic conditions.

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H2 + CO2 were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system.

Biological transformation of 2,4,6-trinitrotoluene (TNT) by soil bacteria isolated from TNT-contaminated soil

Abstract Four Pseudomonas spp. were isolated from a soil consortium enriched from soil contaminated with 2,4,6-trinitrotoluene (TNT). All four species extensively transformed TNT. The rate of transformation varied among species. In isolate 4, 100% of TNT (100 ppm) was transformed in 4 days. The TNT transformation was achieved by the four isolates through a co-metabolic process with a succinate co-substrate. The four isolates produced NO 2 − from TNT. The maximum NO 2 − production, observed for isolate 1, was equal to 30% of the NO 2 − available from the nitro groups of TNT. For other isolates the NO 2 − production varied from 10 to 16%. The radiolabeling studies showed signs ring cleavage. Isolate 3 used 13% of 14 C-TNT to make cellular material, and isolate 4 converted 6% of 14 C-TNT to biomass. The production of 14 C-CO 2 was observed for all four isolates, but the amount of 14 C-CO 2 produced was quite low: isolate 4 produced 14 C-CO 2 from approximately 1% of 14 C-TNT. The rate of degradation of TNT intermediates was very slow, reflecting possible difficulties in metabolizing the intermediates of TNT to CO 2 . The main intermediates were identified as 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene.

Anaerobic biodegradation of explosives and related compounds by sulfate-reducing and methanogenic bacteria : a review.

In recent years, research on microbial degradation of explosives and nitroaromatic compounds has increased. Most studies of the microbial metabolism of nitroaromatic compounds have used aerobic microorganisms. Ecological observations suggest that sulfate-reducing and methanogenic bacteria might metabolize nitroaromatic compounds under anaerobic conditions if appropriate electron donors and electron acceptors are present in the environment, but this ability had not been demonstrated until recently. Few review papers exist, and those deal mainly with aerobic bacterial degradation of explosives; none deals with anaerobic bacteria. In this paper, we review the anaerobic metabolic processes in the degradation of explosives and nitroaromatic compounds under sulfate-reducing and methanogenic conditions.

A laboratory study of the bioremediation of 2,4,6-trinitrotoluene-contaminated soil using aerobic/anoxic soil slurry reactor

The successful operation of an aerobic/anoxic laboratory-scale soil slurry reactor showed that soil contaminated with 2,4,6-trinitrotoluene (TNT) and hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) could be treated in batches or semicontinuously. Batch treatment resulted in the transformation of TNT. Semicontinuous treatment resulted in complete degradation of TNT. In addition to removing TNT, the slurry reactor also removed contaminants such as trinitrobenzene, 2,4-dinitrotoluene, RDX, and octahydro-l,3,5,7-tetranitro-l,3,5,7-tetraazocine (HMX). Radiolabeled TNT incubated with reactor biomass showed that 23% of [{sup 14}C]TNT was mineralized, 27% was converted to biomass, and 8% was adsorbed onto the soil. The rest of the [{sup 14}C]TNT was accounted for as metabolites, including a ring cleavage product identified as 2,3-butanediol. Increasing the frequency of soil addition from once to two or three times weekly did not affect the TNT removal rates. The soil slurry reactor also maintained the bacterial population fairly well, needing only 0.3% molasses as a cosubstrate.

Biotransformation of 2,4,6-trinitrotoluene (TNT) by co-metabolism with various co-substrates: A laboratory-scale study

Abstract Previous studies on the biotransformation of 2,4,6-trinitrotoluene (TNT) have shown that many aerobic bacterial consortia can transform TNT by co-metabolism. In this study various co-substrates have been used with the main objective of finding an inexpensive carbon source for large-scale biotreatment of TNT. Succinate, citrate, malic acid, acetate, glucose, sucrose, and molasses were used as carbon sources for an aerobic bacterial consortium transforming TNT. The results indicated that, among the various carbon sources studied, the cultures that received molasses at a concentration of 0·3% transformed 100 ppm of TNT within 12 h of incubation at ambient temperature, whereas the cultures with other carbon sources took more than 100 h to transform 100 ppm of TNT. The major intermediates identified were 4-amino-2,6-dinitrotoluene and its isomer, 2-amino-4,6-dinitrotoluene. Studies with [ 14 C]TNT provided no significant evidence that TNT was mineralized to CO 2 . The bacterial consortium was composed of various microorganisms, primarily Gram-negative rods. Molasses is an inexpensive carbon source that can be used in large-scale application for the biotreatment of TNT-contaminated soil and water.

Application of the polymerase chain reaction (PCR) and reverse transcriptase/PCR for determining the fate of phenol-degrading Pseudomonas putida ATCC 11172 in a bioaugmented sequencing batch reactor

Abstract The impact of bioaugmentation on the efficacy of an existing microbial population to detoxify phenol in a laboratory-scale sequencing batch reactor was evaluated. Phenol degradation and the persistence and expression of the catabolic dmpN gene were studied for 44 days using a combination of conventional monitoring approaches and molecular techniques. Following addition of the phenol-degrading bacterium, Pseudomonas putida ATCC 11172, which converts phenol to catechol by the aerobic meta-cleavage pathway, phenol removal␣in the bioaugmented reactor increased and was maintained at 95 %–100 %. In the unaugmented control reactor, decreased phenol removal was observed. Correspondingly, dmpN DNA, characteristic of P. putida ATCC 11172, and its expression were detected in activated sludge biomass from the bioaugmented reactor for over 41 days. The results of this study show that (i) bioaugmentation provides stability in phenol degradation, and (ii) monitoring with molecular techniques such as the polymerase chain reaction (PCR) and reverse transcriptase/PCR can successfully assess the state of a bacterium used in bioaugmentation.