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.

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Elemental and redox analysis of single bacterial cells by x-ray microbeam analysis.

High-energy x-ray fluorescence measurements were used to make elemental maps and qualitative chemical analyses of individual Pseudomonas fluorescens strain NCIMB 11764 cells. Marked differences between planktonic and adhered cells were seen in the morphology, elemental composition, and sensitivity to Cr(VI) of hydrated cells at spatial scales of 150 nm. This technology can be applied to natural geomicrobiological systems.

Direct extraction of DNA from soils for studies in microbial ecology.

Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.

Hydrogen isotopic composition of individual n-alkanes as an intrinsic tracer for bioremediation and source identification of petroleum contamination.

The isotopic signatures of crude oil hydrocarbons are potentially powerful intrinsic tracers to their origins and the processes by which the oils are modified in the environment. Stable carbon isotopic data are of limited use for studying petroleum contaminants because of the relatively small amount of isotopic fractionation that occurs during natural processes. Hydrogen isotopes, in contrast, are commonly fractionated to a much greater extent and as a result display larger variations in delta values. We studied the effect of in vitro aerobic biodegradation on the hydrogen isotopic composition of individual n-alkanes from crude oil. The isotopic analysis was conducted using gas chromatography-thermal conversion-isotope ratio mass spectrometry. In general, biodegradation rates decreased with increasing hydrocarbon chain length, consistent with previous studies. More importantly the n-alkanes that were degraded at the fastest rates (n-C15 to n-C18) also showed the largest overall isotopic fractionation (approximately 12-25 per thousand deuterium enrichment), suggesting that the lower molecular weight n-alkanes can be used to monitor in-situ bioremediation of crude oil contamination. The hydrogen isotopic compositions of the longer chain alkanes (n-C19 to n-C27) were relatively stable during biodegradation (<5%o overall deuterium enrichment), indicating that these compounds are effective tracers for oil-source identification studies.

Elucidation of the metabolic pathway for dibenzothiophene desulphurization by Rhodococcus sp. strain IGTS8 (ATCC 53968).

Rhodococcus sp. strain IGTS8 (ATCC 53968) is able to utilize dibenzothiophene (DBT) as a sole source of sulphur. The carbon skeleton of DBT is not metabolized and is conserved as 2-hydroxybiphenyl (HBP), which accumulates in the medium. This phenotype is due to the expression of the plasmid-encoded DBT-desulphurization (dsz) operon, which encodes three proteins, DszA, B and C. In this paper it is shown, using [35S]DBT radiolabelling studies, that sulphur is released in the form of inorganic sulphite. The pathway of DBT desulphurization is described in detail. In summary, DszC catalyses the stepwise S-oxidation of DBT, first to dibenzothiophene 5-oxide (DBTO) and then to dibenzothiophene 5,5-dioxide (DBTO2); DszA catalyses the conversion of DBTO2 to 2-(2'-hydroxyphenyl)benzene sulphinate (HBPSi-) and DszB catalyses the desulphination of HBPSi- to give HBP and sulphite. Studies with cell-free extracts show that DszA and DszC, but not DszB, require NADH for activity. 18O2-labelling studies show that each incorporated oxygen atom is derived directly from molecular oxygen. These results are consistent with the role of DszC as a mono-oxygenase, of DszA as an apparently unique enzyme which catalyses the reductive hydroxylation of DBTO2 leading to cleavage of the thiophene ring, and of DszB as an aromatic sulphinic acid hydrolase.