Temporal change in culturable phenanthrene degraders in response to long-term exposure to phenanthrene in a soil column system

Widespread environmental contamination by polycyclic aromatic hydrocarbons (PAH) has led to increased interest in the use of natural attenuation as a clean-up strategy. However, few bioremediation studies have investigated the behaviour of the indigenous PAH-degrading community after long-term exposure to a PAH. In this study, a column packed with sandy loam soil was exposed to a solution saturated with phenanthrene ( approximately 1.2 mg l-1) for a 6-month period to examine the temporal response of the indigenous phenanthrene-degrading community. Initial soil, effluent, and final soil samples were collected and analysed for phenanthrene concentration and culturable phenanthrene degraders. Phenanthrene-degrading isolates were grouped by colony morphology. For each unique group, 16S rDNA polymerase chain reaction was performed, and then sequencing analysis was used to identify the isolate at the genus level. Twenty-five phenanthrene-degrading isolates, potentially representing 19 genera, were obtained from this analysis. Of these, eight genera have not been reported previously to degrade phenanthrene, including Afipia, Janthinobacterium, Leptothrix, Massilia, Methylobacterium, Rhizobium, Sinorhizobium and Thiobacillus. Results indicate that the dominant phenanthrene-degrading population changed over the course of this 6-month experiment. Specifically, the isolates obtained initially from the soil were not subsequently found in either effluent samples or the soil at the end of the experiment. Furthermore, several isolates that were found in the soil at the end of the experiment were not observed in the soil initially or in the effluent samples. This study confirms earlier findings indicating that a diverse community participates in phenanthrene degradation in the environment, and also suggests that the composition of this community is temporally variable.     

Factors affecting the microbial degradation of phenanthrene in soil

Summary Because phenanthrene was mineralized more slowly in soils than in liquid media, a study was conducted to determine the environmental factors that may account for the slow biodegradation in soil. Mineralization was enhanced by additions of phosphate but not potassium, and it was reduced by additions of nitrate. Aeration or amending the soil with glucose affected the rate of mineralization, although not markedly. Phenanthrene was sorbed to soil constituents, the extent of sorption being directly related to the percentage of organic matter in the soil. Soluble phenanthrene was not detected after addition of the compound to a muck soil. The rate of mineralization was slow in the organic soil and higher in mineral soils with lower percentages of organic matter. We suggest that sorption by soil organic matter slows the biodegradation of polycyclic aromatic hydrocarbons that are otherwise readily metabolized. Offprint requests to: M. Alexander     

Relationship between bacterial diversity and function under biotic control: the soil pesticide degraders as a case study

In soil, the way biotic parameters impact the relationship between bacterial diversity and function is still unknown. To understand these interactions better, we used RNA-based stable-isotope probing to study the diversity of active atrazine-degrading bacteria in relation to atrazine degradation and to explore the impact of earthworm-soil engineering with respect to this relationship. Bulk soil, burrow linings and earthworm casts were incubated with ¹³C-atrazine. The pollutant degradation was quantified by liquid chromatography–mass spectrometry for 8 days, whereas active atrazine degraders were identified at 2 and 8 days by sequencing the 16S ribosomal RNA in the ¹³C-RNA fractions from the three soil microsites. An original diversity of atrazine degraders was found. Earthworm soil engineering greatly modified the taxonomic composition of atrazine degraders with dominance of α-, β- and γ-proteobacteria in burrow linings and of Actinobacteria in casts. Earthworm soil bioturbation increased the γ-diversity of atrazine degraders over the soil microsites generated. Atrazine degradation was enhanced in burrow linings in which primary atrazine degraders, closely related to Pelomonas aquatica, were detected only 2 days after atrazine addition. Atrazine degradation efficiency was not linearly related to the species richness of degraders but likely relied on keystone species. By enhancing soil heterogeneity, earthworms sustained high phylogenetic bacterial diversity and exerted a biotic control on the bacterial diversity–function relationships. Our findings call for future investigations to assess the ecological significance of biotic controls on the relationships between diversity and function on ecosystem properties and services (for example, soil detoxification) at larger scales.     

Metabolic diversity in bacterial degradation of aromatic compounds

Aromatic compounds pose a major threat to the environment, being mutagenic, carcinogenic, and recalcitrant. Microbes, however, have evolved the ability to utilize these highly reduced and recalcitrant compounds as a potential source of carbon and energy. Aerobic degradation of aromatics is initiated by oxidizing the aromatic ring, making them more susceptible to cleavage by ring-cleaving dioxygenases. A preponderance of aromatic degradation genes on plasmids, transposons, and integrative genetic elements (and their shuffling through horizontal gene transfer) have lead to the evolution of novel aromatic degradative pathways. This enables the microorganisms to utilize a multitude of aromatics via common routes of degradation leading to metabolic diversity. In this review, we emphasize the exquisiteness and relevance of bacterial degradation of aromatics, interlinked degradative pathways, genetic and metabolic regulation, carbon source preference, and biosurfactant production. We have also explored the avenue of metagenomics, which opens doors to a plethora of uncultured and uncharted microbial genetics and metabolism that can be used effectively for bioremediation.     

Coevolution with phages does not influence the evolution of bacterial mutation rates in soil

Coevolution with phages drive the evolution of high bacterial mutation rates in vitro, but the relevance of this finding to natural populations is unclear. Here, we investigated how coevolution affects mutation rate evolution in soil, in the presence and absence of the rest of the natural microbial community. Although mutation rate on average increased threefold, neither coevolving phages nor the rest of natural community significantly affected mutation rates. Our results suggest that features of the soil over and above directly interacting organisms constrain the evolution of strong mutators, helping to explain their relatively low frequency compared with some laboratory and clinical settings.     

Effect of copper on the degradation of phenanthrene by soil micro-organisms

AIMS: The effect of copper on the degradation by soil micro-organisms of phenanthrene, a polycyclic aromatic hydrocarbon, was investigated. METHODS AND RESULTS: Inert nylon filters were incubated in the soil for 28 days at 25 degrees C. Each filter was inoculated with a soil suspension, phenanthrene (400 ppm), copper (0, 70, 700 or 7000 ppm) and nitrogen/phosphorus sources. The filters were assessed for phenanthrene degradation, microbial respiration and colonization. Phenanthrene degradation proceeded even at toxic copper levels (700/7000 ppm), indicating the presence of phenanthrene-degrading, copper-resistant and/or -tolerant microbes. However, copper at these high levels reduced microbial activity (CO2 evolution). CONCLUSION: High levels of copper caused an incomplete mineralization of phenanthrene and possible accumulation of its metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of heavy metals in soils could seriously affect the bioremediation of PAH-polluted environments.     

Strong impact on the polycyclic aromatic hydrocarbon (PAH)-degrading community of a PAH-polluted soil but marginal effect on PAH degradation when priming with bioremediated soil dominated by mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.     

Light availability affects stream biofilm bacterial community composition and function,but not diversity

Changes in riparian vegetation or water turbidity and browning in streams alter the local light regime with potential implications for stream biofilms and ecosystem functioning. We experimented with biofilms in microcosms grown under a gradient of light intensities (range: 5–152 μmole photons s^?1 m^?2) and combined 454‐pyrosequencing and enzymatic activity assays to evaluate the effects of light on biofilm structure and function. We observed a shift in bacterial community composition along the light gradient, whereas there was no apparent change in alpha diversity. Multifunctionality, based on extracellular enzymes, was highest under high light conditions and decoupled from bacterial diversity. Phenol oxidase activity, involved in the degradation of polyphenolic compounds, was twice as high on average under the lowest compared with the highest light condition. This suggests a shift in reliance of microbial heterotrophs on biofilm phototroph‐derived organic matter under high light availability to more complex organic matter under low light. Furthermore, extracellular enzyme activities correlated with nutrient cycling and community respiration, supporting the link between biofilm structure–function and biogeochemical fluxes in streams. Our findings demonstrate that changes in light availability are likely to have significant impacts on biofilm structure and function, potentially affecting stream ecosystem processes.     

Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment

Forty six bacterial isolates able to grow on crude oil were isolated from various hydrocarbon-contaminated sites in Kuwait. The extent of crude oil degradation varied over a wide range (1–87%) among the isolates. Isolates were predominantly Gram-positive bacteria (79% of total isolates) belonging to the genera Bacillus (93%) and Paenibacillus (7%). Among the few Gram-negative isolates were from the genera Acinetobacter, Alcaligenes, Klebsiella, Burkholderia, Pseudomonas, and Williamsia. Analyses of their cell-surface hydrophobicity (CSH) by various methods equally showed a wide variation among the isolates. About 74% of isolates that degraded significant amounts of crude oil (>40% degradation) possessed high level of CSH, while 58% of all the isolates exhibited high levels of CSH. Statistical analyses showed significantly high correlation between the ability to degrade crude oil and CSH. The ability of the isolates to bind to polystyrene and salt-aggregation test as measures of CSH were more strongly correlated with hydrocarbon-degrading ability than adherence to hydrocarbons.     

The role of salicylate and biosurfactant in inducing phenanthrene degradation in batch soil slurries

The majority of polycyclic aromatic hydrocarbons (PAHs) sorb strongly to soil organic matter posing a complex barrier to biodegradation. Biosurfactants can increase soil-sorbed PAHs desorption, solubilisation, and dissolution into the aqueous phase, which increases the bioavailability of PAHs for microbial metabolism. In this study, biosurfactants, carbon sources, and metabolic pathway inducers were tested as stimulators of microorganism degradation. Phenanthrene served as a model PAH and Pseudomonas putida ATCC 17484 was used as the phenanthrene degrading microorganism for the liquid solutions and soil used in this investigation. Bench-scale trials demonstrated that the addition of rhamnolipid biosurfactant increases the apparent aqueous solubility of phenanthrene, and overall degradation by at least 20% when combined with salicylate or glucose in liquid solution, when compared to solutions that contained salicylate or glucose with no biosurfactant. However, salicylate addition, with no biosurfactant addition, increased the total degradation of phenanthrene 30% more than liquid systems with only biosurfactant addition. In soil slurries, small amounts of biosurfactant (0.25 g/L) showed a significant increase in total removal when only biosurfactant was added. In soil slurries containing salicylate, the effects of biosurfactant additions were negligible as there was greater than 90% removal, regardless of the biosurfactant concentration. The results of experiments performed in this study provide further evidence that an in situ enhancement strategy for phenanthrene degradation could focus on providing additional carbon substrates to induce metabolic pathway catabolic enzyme production, if degradation pathway intermediates are known.     

Spatial distribution of bacterial communities and phenanthrene degradation in the rhizosphere of Lolium perenne L

Rhizodegradation of organic pollutants, such as polycyclic aromatic hydrocarbons, is based on the effect of root-produced compounds, known as exudates. These exudates constitute an important and constant carbon source that selects microbial populations in the plant rhizosphere, modifying global as well as specific microbial activities. We conducted an experiment in two-compartment devices to show the selection of bacterial communities by root exudates and phenanthrene as a function of distance to roots. Using direct DNA extraction, PCR amplification, and thermal gradient gel electrophoresis screening, bacterial population profiles were analyzed in parallel to bacterial counts and quantification of phenanthrene biodegradation in three layers (0 to 3, 3 to 6, and 6 to 9 mm from root mat) of unplanted-polluted (phenanthrene), planted-polluted, and planted-unpolluted treatments. Bacterial community differed as a function of the distance to roots, in both the presence and the absence of phenanthrene. In the planted and polluted treatment, biodegradation rates showed a strong gradient with higher values near the roots. In the nonplanted treatment, bacterial communities were comparable in the three layers and phenanthrene biodegradation was high. Surprisingly, no biodegradation was detected in the section of planted polluted treatment farthest from the roots, where the bacterial community structure was similar to those of the nonplanted treatment. We conclude that root exudates and phenanthrene induce modifications of bacterial communities in polluted environments and spatially modify the activity of degrading bacteria.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

Influence of arbuscular mycorrhizal mycelial exudates on soil bacterial growth and community structure

Plant root systems colonized by arbuscular mycorrhizal (AM) fungi have previously been shown to influence soil bacterial populations; however, the direct influence of the AM extraradical mycelium itself on bacterial growth and community composition is not well understood. In this study, we investigated the effects of exudates produced by AM extraradical mycelia on the growth and development of an extracted soil bacterial community in vitro. The chemical composition of the mycelial exudates was analysed using proton nuclear magnetic resonance spectrometry. Following the addition of exudates to a bacterial community extracted from soil, bacterial growth and vitality were determined using a bacterial vitality stain and fluorescence microscopy. Changes in community composition were also analysed at various times over the course of 3 days by terminal restriction fragment length polymorphism analysis, in combination with cloning and sequencing of 16S rRNA genes. Mycelial exudates increased bacterial growth and vitality and changed bacterial community composition. Several Gammaproteobacteria, including a taxon within the Enterobacteriaceae, increased in frequency of occurrence in response to AM mycelial exudates. This study is the first attempt to identify carbohydrates from the extraradical mycelium of an AM fungus, and demonstrates the direct effects of mycelial exudates on a soil bacterial community.     

Anticonvulsants modify inactivation but not activation processes of sodium channels in Myxicola axons

The effects of pronase and the anticonvulsant drugs diphenylhydantoin, bepridil, and sodium valproate on fast and slow Na+ inactivation were examined in cut-open Myxicola giant axons with loose patch-clamp electrodes applied to the internal surface. Pronase completely eliminated fast Na+ inactivation without affecting the kinetics of Na+ activation or the maximum Na+ conductance. The time and voltage dependences of slow inactivation following pronase treatment were identical to those measured before enzyme application in the same axons. All three anticonvulsants slowed the time course of recovery from fast Na+ inactivation in untreated axons, and shifted the steady-state fast inactivation curve in the hyperpolarizing direction along the voltage axis. Anticonvulsants enhanced steady-state slow inactivation and retarded recovery from slow inactivation in both untreated and pronase-treated axons. Although some quantitative differences were seen, the order of potency of the anticonvulsants on slow Na+ inactivation was the same as that for recovery from fast inactivation.     

Decomposition of root exudates in soil

The conversion of synthetic root exudates, i.e. of a mixture of amino acids, organic acids and sugars, added to soil in a single dose or continuously, was studied. After the addition of a single dose, the root exudates were gradually mineralized and after 76 hours, 85% of carbon had been released in the form of carbon dioxide. The extent and rate of mineralization was not influenced by the simultaneous addition of ammonium phosphate. The continuous addition of substrate formed a model artificial rhizosphere. In the steady state, 93% of the carbon in the added substrate was mineralized to carbon dioxide. The conversion of organic acids, sugars and amino acids and the mineralization of nitrogen was studied simultaneously by chromatography. In soil continuously enriched with root exudates, phenomena similar to the rhizosphere effect in nature were observed both in the numbers of microorganisms and in the relative incidence of the nutritional groups of bacteria.     

Reclamation of abandoned saline-alkali soil increased soil microbial diversity and degradation potential

Plant and Soil - Reclamation of saline-alkali soils to grow cotton (Gossypium spp.) is very common in the arid Manas River Basin in Northwest China. However, little is known about the degradation...