Super-complex mixtures of aliphatic- and aromatic acids may be common degradation products after marine oil spills: A lab-study of microbial oil degradation in a warm, pre-exposed marine environment.

When assessing oil spills in marine environments, focus has often been on describing degradation and removal of hydrocarbons. However, more and more attention is now given to the formation of mineral oil transformation products, and their potential toxicity and persistency in the environment. The aim of this study was to investigate the formation of dissolved acidic degradation products from crude oil in sea water from the Persian Gulf in a lab-experiment. A super-complex mixture of acidic degradation products was formed in the water phase and compound groups of aliphatic acids, monocyclic aromatic acids-, and polycyclic aromatic acids were identified. More specifically, alkylated PAHs were biodegraded to a high number of aromatic, carboxylic acids by hydroxylation of the alkyl side chains. These degradation products are more bioavailable than their parent compounds, and may therefore constitute a new group of contaminants that should be considered in oil spill assessments.

Full-scale bioremediation of diesel-polluted soil in an Arctic landfarm.

A full-scale, experimental landfarm was tested for the capacity to biodegrade oil-polluted soil under high-Arctic tundra conditions in northeast Greenland at the military outpost 9117 Station Mestersvig. Soil contaminated with Arctic diesel was transferred to the landfarm in August 2012 followed by yearly addition of fertilizer and plowing and irrigation to optimize microbial diesel biodegradation. Biodegradation was determined from changes in total petroleum hydrocarbons (TPH), enumeration of specific subpopulations of oil-degrading microorganisms (MPN), and changes in selected classes of alkylated isomers and isomer ratios. Sixty-four percent of the diesel was removed in the landfarm within the first year, but a recalcitrant fraction (18%) remained after five years. n-alkanes and naphthalenes were biodegraded as demonstrated by changing isomer ratios. Dibenzothiophenes and phenanthrenes showed almost constant isomer ratios indicating that their removal was mostly abiotic. Oil-degrading microorganisms were present for the major components of diesel (n-alkanes, alkylbenzenes and alkylnaphthalenes). The degraders showed very large population increases in the landfarm with a peak population of 1.2 × 109 cells g-1 of total diesel degraders. Some diesel compounds such as cycloalkanes, hydroxy-PAHs and sulfur-heterocycles had very few or no specific degraders, these compounds may consequently be degraded only by slow co-metabolic processes or not at all.

Test of vegetation-based surface exploration for detection of Arctic mineralizations: The deep buried Kangerluarsuk Zn-Pb-Ag anomaly.

The aim of our study was to test whether surficial geochemical techniques are applicable under arctic conditions where pedogenesis is slow or absent, and where the vegetation is arctic dwarf shrub tundra. To this end, we sampled vegetation and topsoil at a known Zn-Pb-Ag anomaly at Kangerluarsuk, northwest Greenland. This Zn-Pb-Ag mineralization surfaces in part of the test area and is deeply buried in other parts. The surface mineralization could readily be identified by element analysis of the omnipresent plant Salix glauca. The strongest signal came from the pathfinder element Tl. The target elements Pb and Ag gave only weak signals and Zn gave no signal, probably because the cellular concentration of these elements is actively regulated by the plant. The use of regulated plant micronutrients as reference elements gave a small reduction of analytical noise in Tl/Cu and Tl/B concentration ratios at low Tl concentrations which improved identification of the deep mineralization. Pathfinder elements in plants may thus prove useful when combined with a detailed geophysical model. Tl, Zn, Pb and Ag concentrations in topsoil identified the surface mineralization but failed to identify the deep mineralization. This difference between samples of S. glauca and topsoil is probably because target elements from the deep mineralization must be mobile to reach the surface. Mobile elements may be more accessible for ion-exchange and uptake into the plants compared to the recalcitrant and crystalline fraction in the topsoil.

Arctic and Subarctic Natural Soils Emit Chloroform and Brominated Analogues by Alkaline Hydrolysis of Trihaloacetyl Compounds.

There has been increasing recognition of the occurrence of natural, halogenated organic compounds in marine and terrestrial environments. Chloroform is an example of a halogenated organic compound with natural formation as its primary source. Chloroform emission from soil has been reported from diverse Arctic, temperate, and (sub)tropical ecosystems. The terrestrial environment is a significant source to the atmosphere, but little is known about the formation pathway of chloroform in soil. Here, we present evidence that chloroform is formed through the hydrolysis of trichloroacetyl compounds in natural, organic-rich soils. In situ emissions of chloroform from soil in nine Arctic and subarctic ecosystems were linked to soil trichloroacetyl turnover. The residence time from formation of the trichloroacetyl compounds in soil to the release of chloroform to the atmosphere varied between 1 and 116 active months in unfrozen topsoil, depending on soil pH. Nonspecific halogenation that leads to trihaloacetyl formation does not discriminate between chloride and bromide, and brominated analogues were formed alongside chloroform. Soil may therefore be a previously unrecognized, natural source of brominated haloforms. The formation pathway of haloforms through trihaloacetyl compounds can most likely be extended to other ecosystems with organic topsoils.

Biodegradation of crude oil in Arctic subsurface water from the Disko Bay (Greenland) is limited.

Biological degradation is the main process for oil degradation in a subsurface oil plume. There is, however, little information on the biodegradation potential of Arctic, marine subsurface environments. We therefore investigated oil biodegradation in microcosms at 2 °C containing Arctic subsurface seawater from the Disko Bay (Greenland) and crude oil at three concentrations of 2.5-10 mg/L. Within 71 days, the total petroleum hydrocarbon concentration decreased only by 18 ± 18% for an initial concentration of 5 mg/L. The saturated alkanes nC13-nC30 and the isoprenoids iC18-iC21 were biodegraded at all concentrations indicating a substantial potential for biodegradation of these compound classes. Polycyclic aromatic compounds (PACs) disappeared from the oil phase, but dissolution was the main process of removal. Analysis of diagnostic ratios indicated almost no PAC biodegradation except for the C1-naphthalenes. To conclude, the marine subsurface microorganisms from the Disko Bay had the potential for biodegradation of n-alkanes and isoprenoids while the metabolically complex and toxic PACs and their alkylated homologs remained almost unchanged.

Marine biodegradation of crude oil in temperate and Arctic water samples.

Despite increased interest in marine oil exploration in the Arctic, little is known about the fate of Arctic offshore oil pollution. Therefore, in the present study, we examine the oil degradation potential for an Arctic site (Disko Bay, Greenland) and discuss this in relation to a temperate site (North Sea, Denmark). Biodegradation was assessed following exposure to Oseberg Blend crude oil (100 mg L(-1)) in microcosms. Changes in oil hydrocarbon fingerprints of polycyclic aromatic hydrocarbons (PAHs), alkyl-substituted PAHs, dibenzothiophenes, n-alkanes and alkyltoluenes were measured by gas chromatography-mass spectrometry (GC-MS). In the Disko Bay sample, the degradation order was n-alkanes>alkyltoluenes (para->meta->ortho-isomers)>PAHs and dibenzothiophenes, whereas, the degradation order in the North Sea samples was PAHs and dibenzothiophenes>alkyltoluenes>n-alkanes. These differences in degradation patterns significantly affect the environmental risk of oil spills and emphasise the need to consider the specific environmental conditions when conducting risk assessments of Arctic oil pollution.

The potential for bioaugmentation of sand filter materials from waterworks using bacterial cultures degrading 4-chloro-2-methylphenoxyacetic acid.

BACKGROUND: The herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) is found frequently in Danish groundwater in concentrations exceeding the EU threshold limit of 0.1 µg L(-1) . Groundwater is used for drinking water, and one potential remediation strategy is bioaugmentation using inoculation of sand filters at affected waterworks with degrader bacteria. Numerous bacteria degrading phenoxyacetic acid herbicide have previously been isolated, and they may be candidates for bioaugmentation processes. Designing the optimum inoculum, however, requires knowledge of the capacity for degrading realistically low herbicide concentrations and the robustness of the bacteria when inoculated into sand filter materials. RESULTS: Testing a range of different MCPA-mineralising bacterial combinations, using a high-throughput microplate radiorespirometric mineralisation assay, highlighted three efficient cocultures for mineralising low MCPA concentrations. Cocultures demonstrating a shorter time delay before initiation of (14) C-ring-labelled MCPA mineralisation to (14) CO2 , and a more extensive mineralisation of MCPA, compared with those of single strains, were found. When inoculated into different sand filter materials, the coculture effect was diminished, but several single strains enhanced MCPA mineralisation significantly at low MCPA concentrations. CONCLUSION: This study shows that an increase in the potential for mineralisation of low herbicide concentrations in sand filter materials can be achieved by inoculating with bacterial degrader cultures. © 2014 Society of Chemical Industry.

Polar metabolites of polycyclic aromatic compounds from fungi are potential soil and groundwater contaminants.

This study investigated the sorption to soil of water-soluble metabolites from polycyclic aromatic compounds (PACs). The soil fungus Cunninghamella elegans was used to produce PAC metabolites from two un-substituted PACs (phenanthrene, pyrene), three alkyl-substituted PACs (2-methylnaphthalene, 1-methylphenanthrene, 1-methylpyrene), and one sulfur-containing heterocyclic PAC (dibenzothiophene). Fifty-eight metabolites were tentatively identified; metabolites from the un-substituted PACs were hydroxylated and sulfate conjugated, whereas metabolites from alkyl-substituted PACs were sulfate conjugated and either hydroxylated or oxidized to carboxylic acids at the methyl group. The metabolism of the sulfur-containing heterocyclic PAC resulted in sulfate conjugates. The sorption of the PAC metabolites to three soils was determined using a batch equilibrium method, and partition coefficients (Kd's) were calculated for fourteen representative metabolites. Sulfate conjugated metabolites displayed Kd's below 70 whereas the metabolites with both a sulfate and a carboxylic acid group had Kd's below 2.8. The low Kd's of water-soluble PAC metabolites indicate high mobility in soil and a potential for leaching to surface- and groundwaters.

Does microbial centimeter-scale heterogeneity impact MCPA degradation in and leaching from a loamy agricultural soil?

The potential for pesticide degradation varies greatly at the centimeter-scale in agricultural soil. Three dimensional numerical simulations were conducted to evaluate how such small-scale spatial heterogeneity may affect the leaching of the biodegradable pesticide 2-methyl-4-chlorophenoxyacetic acid (MCPA) in the upper meter of a variably-saturated, loamy soil profile. To incorporate realistic spatial variation in degradation potential, we used data from a site where 420 mineralization curves over 5 depths have been measured. Monod kinetics was fitted to the individual curves to derive initial degrader biomass values, which were incorporated in a reactive transport model to simulate heterogeneous biodegradation. Six scenarios were set up using COMSOL Multiphysics to evaluate the difference between models having different degrader biomass distributions (homogeneous, heterogeneous, or no biomass) and either matrix flow or preferential flow through a soil matrix with a wormhole. MCPA leached, within 250 days, below 1m only when degrader biomass was absent and preferential flow occurred. Both biodegradation in the plow layer and the microbially active lining of the wormhole contributed to reducing MCPA-leaching below 1m. The spatial distribution of initial degrader biomass within each soil matrix layer, however, had little effect on the overall MCPA-leaching.

Quantification of small-scale variation in the size and composition of phenanthrene-degrader populations and PAH contaminants in traffic-impacted topsoil.

Small-scale colocalisation of microbial polycyclic aromatic hydrocarbon (PAH) degraders and PAHs in contaminated soil is a prerequisite for efficient biodegradation of the PAHs. We therefore tested the hypothesis that phenanthrene-degrading bacteria are colocalised with PAHs at the millimetre-to-centimetre-scale. Microbial populations and PAH concentrations were determined for 40-mg samples from a 112-mm transect of a traffic-impacted topsoil. The spatial distribution of cultivable phenanthrene degraders (0.3 × 10(5) -7.2 × 10(5) cells g(-1) ) mirrored neither the distribution of PAHs, nor the distribution of the total cultivable heterotrophic populations. Quantitative real-time PCR (qPCR) analysis of PAH dioxygenase genes (2 × 10(6) -4 × 10(6) cells g(-1) ) from a second transect showed distributions similar to the cultivable phenanthrene degraders, but at a 20-fold higher level. The omnipresence of high densities of PAH degraders at the millimetre scale indicate that PAH persistence may not be caused by local lack of degrader cells. To the best of our knowledge, this is the first time that either MPN of pollutant degraders, qPCR of functional genes, CFU of heterotrophic micro-organisms, or the content of PAHs have been determined with such high spatial resolution.

The gompertz function can coherently describe microbial mineralization of growth-sustaining pesticides.

Mineralization of (14)C-labeled tracers is a common way of studying the environmental fate of xenobiotics, but it can be difficult to extract relevant kinetic parameters from such experiments since complex kinetic functions or several kinetic functions may be needed to adequately describe large data sets. In this study, we suggest using a two-parameter, sigmoid Gompertz function for parametrizing mineralization curves. The function was applied to a data set of 252 normalized mineralization curves that represented the potential for degradation of the herbicide MCPA in three horizons of an agricultural soil. The Gompertz function fitted most of the normalized curves, and trends in the data set could be visualized by a scatter plot of the two Gompertz parameters (rate constant and time delay). For agricultural topsoil, we also tested the effect of the MCPA concentration on the mineralization kinetics. Reduced initial concentrations lead to shortened lag-phases, probably due to reduced need for bacterial growth. The effect of substrate concentration could be predicted by simply changing the time delay of the Gompertz curves. This delay could to some extent also simulate concentration effects for 2,4-D mineralization in agricultural soil and aquifer sediment and 2,6-dichlorobenzamide mineralization in single-species, mineral medium.

Centimeter-scale spatial variability in 2-methyl-4-chlorophenoxyacetic Acid mineralization increases with depth in agricultural soil.

Mineralization of organic chemicals in soil is typically studied using large homogenized samples, but little is known about the small-scale spatial distribution of mineralization potential. We studied centimeter-scale spatial distribution of 2-methyl-4-chlorophenoxyacetic acid (MCPA) mineralization activity at different depths (8-115 cm) in a Danish agricultural soil profile using a 96-well microplate C-radiorespirometric method for small-volume samples. The heterotrophic microbial population and specific MCPA degraders decreased 10- to 100-fold from the plow layer to a depth of 115 cm. MCPA was mineralized in all samples in the plow layer, but only about 60% in the transition zone immediately below the plow layer showed mineralization; at greater depth even fewer samples showed mineralization. A patchy spatial distribution of mineralization activity was observed from right below the plow layer and in the subsoil, with a few clearly defined active zones surrounded by areas devoid of mineralization activity. Due to the patchy distribution of mineralization activity at the centimeter scale just beneath the plow layer, MCPA and presumably other weakly sorbing pesticides might be at risk of leaching to the groundwater if transported from the plow layer into the subsoil.

Protozoan predation in soil slurries compromises determination of contaminant mineralization potential.

Soil suspensions (slurries) are commonly used to estimate the potential of soil microbial communities to mineralize organic contaminants. The preparation of soil slurries disrupts soil structure, however, potentially affecting both the bacterial populations and their protozoan predators. We studied the importance of this "slurry effect" on mineralization of the herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA, (14)C-labelled), focussing on the effects of protozoan predation. Mineralization of MCPA was studied in "intact" soil and soil slurries differing in soil:water ratio, both in the presence and absence of the protozoan activity inhibitor cycloheximide. Protozoan predation inhibited mineralization in dense slurry of subsoil (soil:water ratio 1:3), but only in the most dilute slurry of topsoil (soil:water ratio 1:100). Our results demonstrate that protozoan predation in soil slurries may compromise quantification of contaminant mineralization potential, especially when the initial density of degrader bacteria is low and their growth is controlled by predation during the incubation period.

Centimetre-scale vertical variability of phenoxy acid herbicide mineralization potential in aquifer sediment relates to the abundance of tfdA genes.

Centimetre-scale vertical distribution of mineralization potential was determined for 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) by 96-well microplate radiorespirometric analysis in aquifer sediment sampled just below the groundwater table. Mineralization of 2,4-D and MCPA was fastest in sediment samples taken close to the groundwater table, whereas only minor mineralization of MCPP was seen. Considerable variability was exhibited at increasing aquifer depth, more so with 2,4-D than with MCPA. This suggests that the abundance of MCPA degraders was greater than that of 2,4-D degraders, possibly due to the fact that the overlying agricultural soil had long been treated with MCPA. Mineralization of 2,4-D and MCPA was followed by increased abundance of tfdA class I and class III catabolic genes, which are known to be involved in the metabolism of phenoxy acid herbicides. tfdA class III gene copy number was approximately 100-fold greater in samples able to mineralize MCPA than in samples able to mineralize 2,4-D, suggesting that tfdA class III gene plays a greater role in the metabolism of MCPA than of 2,4-D. Degradation rate was found to correlate positively with tfdA gene copy number, as well as with the total organic carbon content of the sediment.

Triclosan affects the microbial community in simulated sewage-drain-field soil and slows down xenobiotic degradation.

Effects of the common antibacterial agent triclosan on microbial communities and degradation of domestic xenobiotics were studied in simulated sewage-drain-field soil. Cultivable microbial populations decreased 22-fold in the presence of 4 mg kg⁻¹ of triclosan, and triclosan-resistant Pseudomonas strains were strongly enriched. Exposure to triclosan also changed the general metabolic profile (Ecoplate substrate profiling) and the general profile (T-RFLP) of the microbial community. Triclosan degradation was slow at all concentrations tested (0.33-81 mg kg⁻¹) during 50-days of incubation. Mineralization experiments (¹4C-tracers) and chemical analyses (LC-MS/MS) showed that the persistence of a linear alkylbenzene sulfonate (LAS) and a common analgesic (ibuprofen) increased with increasing triclosan concentrations (0.16-100 mg kg⁻¹). The largest effect was seen for LAS mineralization which was severely reduced by 0.16 mg kg⁻¹ of triclosan. Our findings indicate that environmentally realistic concentrations of triclosan may affect the efficiency of biodegradation in percolation systems.

Quantification of centimeter-scale spatial variation in PAH, glucose and benzoic acid mineralization and soil organic matter in road-side soil.

The aim of the study was to determine centimeter-scale spatial variation in mineralization potential in diffusely polluted soil. To this end we employed a 96-well microplate method to measure the mineralization of 14C-labeled organic compounds in deep-well microplates and thereby compile mineralization curves for 348 soil samples of 0.2-cm3. Centimeter-scale spatial variation in organic matter and the mineralization of glucose, benzoic acid, and PAHs (phenanthrene and pyrene) was determined for urban road-side soil sampled as arrays (7×11cm) of 96 subsamples. The spatial variation in mineralization was visualized by means of 2-D contour maps and quantified by means of semivariograms. The geostatistical analysis showed that the easily degradable compounds (glucose and benzoic acid) exhibited little spatial variation in mineralization potential, whereas the mineralization was highly heterogeneous for the PAH compounds that require specialized degraders. The spatial heterogeneity should be taken into account when estimating natural attenuation rates.

Presence of psychrotolerant phenanthrene-mineralizing bacterial populations in contaminated soils from the Greenland High Arctic.

Fuel-contaminated soils from Station Nord (St. Nord) in Greenland were sampled with the aim of assessing the intrinsic attenuation potential by quantifying the presence and activity of degradative microbial populations using phenanthrene as a model compound. Mineralization of [(14)C]phenanthrene to (14)CO(2) was measured in contaminated soils at temperatures down to 0 degrees C and sizable naphthalene-, undecane-, biphenyl- and phenanthrene-degrading populations were measured by microplate-based most-probable-number analysis. Cloning and 16S rRNA gene sequencing, focused on the dominant phenanthrene-degrading bacteria, revealed strains related to bacteria previously found in cold and contaminated environments. Overall, we provide evidence for the presence and potential activity of phenanthrene-degrading bacteria in polluted St. Nord soils and this study is the first to indicate an intrinsic bioremediation potential in hydrocarbon-contaminated soils from the Greenland High Arctic.

Fungal PAH-metabolites resist mineralization by soil microorganisms.

This study investigated the mineralization of water-soluble polycyclic aromatic hydrocarbon (PAH) metabolites produced by the soil fungus Cunninghamella elegans. Eleven soil fungi were screened for their ability to metabolize (14)C-phenanthrene, (14)C-fluoranthene, and (14)C-pyrene into water-soluble compounds. Eight fungi produced water-soluble metabolites from all or some of the PAHs. The composition of the water-soluble PAH-metabolites from the most effective solubilizer C. elegans was analyzed by an ultraperformance liquid chromatograph interfaced to a quadrupole time-of-flight mass spectrometer. Thirty-eight metabolites were detected. All of 34 identified metabolites were sulfate-conjugated. The mineralization of (14)C-metabolites, produced by C. elegans, was compared to mineralization of the parent (14)C-PAHs in soil slurries. It was hypothesized that the increased bioavailability and metabolic activation of the metabolites would increase mineralization in soil slurries compared to mineralization of the parent PAHs. Unexpectedly, the mineralization of the (14)C-metabolites was in all cases extremely slow compared to the mineralization of the parent (14)C-PAHs. Slow (14)C-metabolite mineralization was not caused by metabolite toxicity, neither was cometabolic mineralization of (14)C-metabolites stimulated by the presence of active PAH-degraders. High water solubility, low lipophilicity, and extremely slow mineralization of the metabolites indicate a potential problem of leaching of fungal PAH-metabolites to the groundwater.

A radiorespirometric method for measuring mineralization of [14C]-compounds in a 96-well microplate format.

A radiorespirometric method was developed for quantifying mineralization of [14C]-labeled substrates in deep-well microplates. Ninety-six 14CO2-traps were placed on an adhesive microplate sealing tape which made it possible to trap 14CO2 simultaneously from all wells. Trapped radioactivity was quantified by digital autoradiography.