Chemotactic Bacteria Facilitate the Dispersion of Nonmotile Bacteria through Micrometer-Sized Pores in Engineered Porous Media.

Recent research has demonstrated that chemotactic bacteria can disperse inside microsized pores while traveling toward favorable conditions. Microbe-microbe cotransport might enable nonmotile bacteria to be carried with motile partners to enhance their dispersion and reduce their deposition in porous systems. The aim of this study was to demonstrate the enhancement in the dispersion of nonmotile bacteria (Mycobacterium gilvum VM552, a polycyclic aromatic hydrocarbon-degrader, and Sphingobium sp. D4, a hexachlorocyclohexane-degrader, through micrometer-sized pores near the exclusion-cell-size limit, in the presence of motile Pseudomonas putida G7 cells. For this purpose, we used bioreactors equipped with two chambers that were separated with membrane filters with 3, 5, and 12 µm pore sizes and capillary polydimethylsiloxane (PDMS) microarrays (20 µm × 35 µm × 2.2 mm). The cotransport of nonmotile bacteria occurred exclusively in the presence of a chemoattractant concentration gradient, and therefore, a directed flow of motile cells. This cotransport was more intense in the presence of larger pores (12 µm) and strong chemoeffectors (γ-aminobutyric acid). The mechanism that governed cotransport at the cell scale involved mechanical pushing and hydrodynamic interactions. Chemotaxis-mediated cotransport of bacterial degraders and its implications in pore accessibility opens new avenues for the enhancement of bacterial dispersion in porous media and the biodegradation of heterogeneously contaminated scenarios.

Impact of Chemoeffectors on Bacterial Motility, Transport, and Contaminant Degradation in Sand-Filled Percolation Columns.

Chemoeffector-mediated bacterial motility and tactic swimming are major drivers for contaminant accessibility and biodegradation at submillimeter scales. In sand-filled percolated columns we tested how and to what degree chemoeffectors influenced bacterial transport and thereby promoted accessibility and degradation of distantly located 14C-naphthalene (NAH) at the centimeter scale. Sunflower root exudates and silver nanoparticles (AgNPs) were used as chemoeffectors to stimulate opposing effects of motility and tactic swimming of NAH-degrading Pseudomonas putida G7. Sunflower exudates prompted smooth bacterial movement and positive taxis, while AgNPs induced tortuous movement and repellent responses. Compared to chemoeffector-free controls exudates reduced deposition and stimulated bacterial transport during percolation experiments. AgNPs, however, provoked bacterial deposition and concomitant saturation of the collector surfaces (filter blocking) that led to progressively increased percolation of cells. Despite mechanistic differences, both motility patterns supported bacterial transport and promoted mineralization rates of NAH desorbing from a source placed at the column outlet. Observed mineralization rates in the presence of the chemoeffectors were 5-fold higher than those in their absence and similar to NAH-mineralization in well-stirred batch assays. Our results indicate that chemically mediated, small-scale bacterial motility patterns may become relevant for long-distance bacterial transport and the biodegradation of patchy contaminants at higher scales, respectively.

Mycelium-Enhanced Bacterial Degradation of Organic Pollutants under Bioavailability Restrictions.

This work examines the role of mycelia in enhancing the degradation by attached bacteria of organic pollutants that have poor bioavailability. Two oomycetes, Pythium oligandrum and Pythium aphanidermatum, were selected as producers of mycelial networks, while Mycobacterium gilvum VM552 served as a model polycyclic aromatic hydrocarbon (PAH) degrading bacterium. The experiments consisted of bacterial cultures exposed to a nondisturbed nonaqueous phase liquid (NAPL) layer containing a heavy fuel spiked with 14C-labeled phenanthrene that were incubated in the presence or absence of the mycelia of the oomycetes in both shaking and static conditions. At the end of the incubation, the changes in the total alkane and PAH contents in the NAPL residue were quantified. The results revealed that with shaking and the absence of mycelia, the strain VM552 grew by utilizing the bulk of alkanes and PAHs in the fuel; however, biofilm formation was incipient and phenanthrene was mineralized following zero-order kinetics, due to bioavailability limitations. The addition of mycelia favored biofilm formation and dramatically enhanced the mineralization of phenanthrene, up to 30 times greater than the rate without mycelia, possibly by providing a physical support to bacterial colonization and by supplying nutrients at the NAPL/water interface. The results in the static condition were very different because the bacterial strain alone degraded phenanthrene with sigmoidal kinetics but could not degrade alkanes or the bulk of PAHs. We suggest that bacteria/oomycete interactions should be considered not only in the design of new inoculants in bioremediation but also in biodegradation assessments of chemicals present in natural environments.

Mobilization of Pollutant-Degrading Bacteria by Eukaryotic Zoospores.

The controlled mobilization of pollutant-degrading bacteria has been identified as a promising strategy for improving bioremediation performance. We tested the hypothesis whether the mobilization of bacterial degraders may be achieved by the action of eukaryotic zoospores. We evaluated zoospores that are produced by the soil oomycete Pythium aphanidermatum as a biological vector, and, respectively, the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria Mycobacterium gilvum VM552 and Pseudomonas putida G7, acting as representative nonflagellated and flagellated species. The mobilization assay was performed with a chemical-in-capillary method, in which zoospores mobilized bacterial cells only when they were exposed to a zoospore homing inducer (5% (v/v) ethanol), which caused the tactic response and settlement of zoospores. The mobilization was strongly linked to a lack of bacterial motility, because the nonflagellated cells from strain M. gilvum VM552 and slightly motile, stationary-phase cells from P. putida G7 were mobilized effectively, but the actively motile, exponentially grown cells of P. putida G7 were not mobilized. The computer-assisted analysis of cell motility in mixed suspensions showed that the swimming rate was enhanced by zoospores in stationary, but not in exponentially grown, cells of P. putida G7. It is hypothesized that the directional swimming of zoospores caused bacterial mobilization through the thrust force of their flagellar propulsion. Our results suggest that, by mobilizing pollutant-degrading bacteria, zoospores can act as ecological amplifiers for fungal and oomycete mycelial networks in soils, extending their potential in bioremediation scenarios.

Impact of dissolved organic matter on bacterial tactic motility, attachment, and transport.

Bacterial dispersal is a key driver of the ecology of microbial contaminant degradation in soils. This work investigated the role of dissolved organic matter (DOM) in the motility, attachment, and transport of the soil bacterium Pseudomonas putida G7 in saturated porous media. The study is based on the hypothesis that DOM quality is critical to triggering tactic motility and, consequently, affects bacterial transport and dispersal. Sunflower root exudates, humic acids (HA), and the synthetic oleophilic fertilizer S-200 were used as representatives of fresh, weathered, and artificially processed DOM with high nitrogen and phosphorus contents, respectively. We studied DOM levels of 16-130 mg L(-1), which are representative of DOM concentrations typically found in agricultural soil pore water. In contrast to its responses to HA and S-200, strain G7 exhibited a tactic behavior toward root exudates, as quantified by chemotaxis assays and single-cell motility observations. All DOM types promoted bacterial transport through sand at high concentrations (∼ 130 mg L(-1)). At low DOM concentrations (∼ 16 mg L(-1)), the enhancement occurred only in the presence of sunflower root exudates, and this enhancement did not occur with G7 bacteria devoid of flagella. Our results suggest that tactic DOM effectors strongly influence bacterial transport and the interception probability of motile bacteria by collector surfaces.

Role of desorption kinetics in the rhamnolipid-enhanced biodegradation of polycyclic aromatic hydrocarbons.

The main aim of this study was to investigate the effect of a rhamnolipid biosurfactant on biodegradation of (14)C-labeled phenanthrene and pyrene under desorption-limiting conditions. The rhamnolipid caused a significant solubilization and enhanced biodegradation of PAHs sorbed to soils. The enhancement was, however, negatively influenced by experimental conditions that caused an enrichment of slow desorption fractions. These conditions included aging, a higher organic matter content in soil, and previous extraction with Tenax to remove the labile-desorbing chemical. The decline in bioavailability caused by aging on sorbed (14)C-pyrene was partially reversed by rhamnolipids, which enhanced mineralization of the aged compound, although not so efficiently like with the unaged chemical. This loss in biosurfactant efficiency in promoting biodegradation can be explained by intra-aggregate diffusion of the pollutant during aging. We suggest that rhamnolipid can enhance biodegradation of soil-sorbed PAHs by micellar solubilization, which increase the cell exposure to the chemicals in the aqueous phase, and partitioning into soil organic matter, thus enhancing the kinetics of slow desorption. Our study show that rhamnolipid can constitute a valid alternative to chemical surfactants in promoting the biodegradation of slow desorption PAHs, which constitutes a major bottleneck in bioremediation.

Risk reductions during pyrene biotransformation and mobilization in a model plant-bacteria-biochar system.

The productive application of motile microorganisms for degrading hydrophobic contaminants in soil is one of the most promising processes in modern remediation due to its sustainability and low cost. However, the incomplete biodegradation of the contaminants and the formation of the intermediary metabolites in the process may increase the toxicity in soil during bioremediation, and motile inoculants may mobilize the pollutants through biosorption. Therefore, controlling these factors should be a fundamental part of soil remediation approaches. The aim of this study was to evaluate the sources of risk associated with the cometabolism-based transformation of 14C-labeled pyrene by inoculated Pseudomonas putida G7 and identify ways to minimize risk. Our model scenario examined the increase in bioaccessibility to a distant source of contamination facilitated by sunflower (Helianthus annuus L.) roots. A biochar trap for mobilized pollutant metabolites and bacteria has also been employed. The experimental design consisted of pots filled with a layer of sand with 14C-labeled pyrene (88 mg kg-1) as a contamination focus located several centimeters from the inoculation point. Half of the pots included a biochar layer at the bottom. The pots were incubated in a greenhouse with sunflower plants and P. putida G7 bacteria. Pots with sunflower plants showed a higher biodegradation of pyrene, its mobilization as metabolites through the percolate and the roots, and bacterial mobilization toward the source of contamination, also resulting in increased pyrene transformation. In addition, the biochar layer efficiently reduced the concentrations of pyrene metabolites collected in the leachates. Therefore, the combination of plants, motile bacteria and biochar safely reduced the risk caused by the biological transformation of pyrene.

Chemical effectors cause different motile behavior and deposition of bacteria in porous media.

We tested the hypothesis whether chemically induced motility patterns of bacteria may affect their transport in porous media. Naphthalene-degrading Pseudomonas putida G7 cells were exposed to glucose, salicylate, and silver nanoparticles (AgNPs) and their motility was assessed by computer-assisted, quantitative swimming and capillary-based taxis determinations. Exposure to salicylate induced smooth movement with few acceleration events and positive taxis, whereas cells exposed to AgNPs exhibited tortuous movement and a repellent response. Although metabolized by strain G7, glucose did not cause attraction and induced a hyper-motile mode of swimming, characterized by a high frequency of acceleration events, high swimming speed (>60 µm s(-1)), and a high tortuosity in the trajectories. Chemically induced motility behavior correlated with distinct modes of attachment to sand in batch assays and breakthrough curves in percolation column experiments. Salicylate significantly reduced deposition of G7 cells in column experiments whereas glucose and AgNPs enhanced attachment and caused filter blocking that resulted in a progressive decrease in deposition. These findings are relevant for bioremediation scenarios that require an optimized outreach of introduced inoculants and in other environmental technologies, such as water disinfection and microbially enhanced oil recovery.

Nature-based approaches to reducing the environmental risk of organic contaminants resulting from military activities.

As is the case with many other industrial activities, the organic contaminants at military-impacted sites may pose significant hazards to the environment and human health. Given the expected increase in defense investments globally, there is a need to make society aware of the risks of emissions of organic contaminants generated by military activities and to advance risk minimization approaches. The most recent advances in environmental analytical chemistry, persistence, bioavailability and risk assessment of organic contaminants indicate that efficient risk reductions through biological means are possible. This review debates the organic contaminants of interest associated with military activities, the methodology used to extract and analyze these contaminants, and the nature-based remediation technologies available to recover these sites. In addition, we revise the military environmental regulatory frameworks designed to sustain such actions. Military activities that potentially release organic contaminants on land could be classified as infrastructure and base operations, training exercises and armed conflicts; additionally, chemicals may include potentially toxic compounds, energetic compounds, chemical warfare agents and military chemical compounds. Fuel components, PFASs, TNT, RDX and dyphenylcyanoarsine are examples of organic contaminants of environmental concern. Particularly in the case of potentially toxic and energetic compounds, bioremediation and phytoremediation are considered eco-friendly and low-cost technologies that can be used to remediate these contaminated sites. In addition, this article identifies implementing the bioavailability of organic contaminants as a justifiable approach to facilitate the application of these nature-based approaches and to reduce remediation costs. More realistic risk assessment in combination with new and economically feasible remediation methods that reduce risk by reducing bioavailability (instead of lowering the total contaminant concentration) will serve as an incentive for the military and regulators to accept nature-based approaches.

Role of tactic response on the mobilization of motile bacteria through micrometer-sized pores.

A major cause of high bioremediation endpoints is the limited bioaccessibility to residual contaminants resting in soil pores with diameters close to the size exclusion limit of bacterial cells. Under nongrowing conditions and in the absence of hydraulic flow, we examined how the tactic behavior of motile, contaminant-degrading Pseudomonas putida G7 cells (2 × 1 µm) influenced passage through membranes with pores ranging in size from 1 µm to 12 µm. The bacteria were spontaneously retained by the membranes - even those with the largest pore size. However, the cells were mobilized through 5 µm and 12 µm pores after the application of an attractant (salicylate). Mobilization also occurred by attraction to the common root exudate constituents γ-aminobutyric acid and citrate and repellence (or negative taxis) to zero-valent iron nanoparticles. The observed pore size threshold for tactic mobilization (5 µm) and unaltered cell fluxes and effective cell diffusion against different chemoeffector strengths and concentrations suggest that there is a physical constraint on the gradient sensing mechanism at the pores that drives the tactic response. Our results indicate that chemically mediated, small-scale tactic reactions of motile bacteria may become relevant to enhance the bioaccessibility of the residual contaminants present in micrometer-sized soil pores.

Determining the bioavailability of benzo(a)pyrene through standardized desorption extraction in a certified reference contaminated soil.

There is a strong need for certified reference materials in the quality assurance of nonionic soil contaminant bioavailability estimations through physicochemical methods. We applied desorption extraction, a method recently standardized as ISO16751, to determine the bioavailable concentration of the most commonly regulated polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (BaP), in the reference industrial soil BCR-524 with a certified BaP total concentration of 8.60 mg kg-1. This concentration represented BaP levels found in many PAH-polluted soils. The method, based on single-point extraction of the analyte desorbed into the aqueous phase by a receiving phase (Tenax or cyclodextrin), was applied ten times. The data fulfilled highly demanding quality criteria based on recovery and repeatability. The bioavailable BaP concentration detected through Tenax extraction, 1.82 mg kg-1, was comparable to bioavailable concentrations determined in field-contaminated soils and to environmental quality standards based on previously observed total BaP concentrations. There was good agreement (Student's t-test, P ≤ 0.05) with the bioavailable BaP concentration determined by cyclodextrin extraction (1.53 mg kg-1). The methods were extended to four other certified 4- and 5-ringed PAHs for comparative purposes. We suggest ways of improving of the ISO16751 standard related to further systematic assessment of the Tenax-to-soil ratio and incorporation of mass balances. Furthermore, BCR-524 is suitable for quality-assurance protocols with these methods when used in site-specific risk assessments of PAH-polluted environments.

Use of natural sorbents for accelerated bioremediation of grey forest soil contaminated with crude oil.

Due to the extensive oil extraction and transportation that occurs in oil-producing countries, many lands remain contaminated because of accidental leakages. Despite its low cost and environmentally safe nature, bioremediation technology is not always successful, mainly because of the soil toxicity to the degrading microbial populations and plants. Here we report a three-year microfield experiment on the influence of natural sorbents of mineral (zeolite, kaolinite, vermiculite, diatomite), organic (peat), carbonaceous (biochar) origin, and a mixed sorbent ACD (composed of granular activated carbon and diatomite) on the bioremediation of grey forest soil contaminated with weathered crude oil (40.1 g total petroleum hydrocarbons (TPH) kg-1). Optimal doses of the sorbents significantly accelerated bioremediation of petroleum-contaminated soil through bioaugmentation followed by phytoremediation. The main reason for the influence of the sorbent amendments relied upon the creation of optimal conditions for the activation of hydrocarbon-utilizing bacteria and plant growth due to the reduction of soil toxicity, as well as maintaining an optimal pH and water-air regime in the soil. That happened because of reducing the soil hydrophobicity, increasing porosity and water holding capacity. The content of the TPH in the best samples (2% biochar or ACD) reduced to their local permissible concentration accepted for remediated soils in the Russian Federation (≤5 g kg-1) after two warm seasons compared to that after three warm seasons in the other samples. Although some sorbents decelerated biodegradation of highly condensed polycyclic aromatic hydrocarbons (PAHs, including benzo(a)pyrene) in the soil, the overall risk from the residual contaminants present in the remediated soil and plants was minimized. The final total content of the main PAHs in the sorbent-amended soils did not exceed the maximal permissible levels that are accepted in most EU countries (1000-40,000 µg kg-1), and they did not accumulate in the aboveground phytomass of grasses in dangerous concentrations.

Scientific concepts and methods for moving persistence assessments into the 21st century.

The evaluation of a chemical substance's persistence is key to understanding its environmental fate, exposure concentration, and, ultimately, environmental risk. Traditional biodegradation test methods were developed many years ago for soluble, nonvolatile, single-constituent test substances, which do not represent the wide range of manufactured chemical substances. In addition, the Organisation for Economic Co-operation and Development (OECD) screening and simulation test methods do not fully reflect the environmental conditions into which substances are released and, therefore, estimates of chemical degradation half-lives can be very uncertain and may misrepresent real environmental processes. In this paper, we address the challenges and limitations facing current test methods and the scientific advances that are helping to both understand and provide solutions to them. Some of these advancements include the following: (1) robust methods that provide a deeper understanding of microbial composition, diversity, and abundance to ensure consistency and/or interpret variability between tests; (2) benchmarking tools and reference substances that aid in persistence evaluations through comparison against substances with well-quantified degradation profiles; (3) analytical methods that allow quantification for parent and metabolites at environmentally relevant concentrations, and inform on test substance bioavailability, biochemical pathways, rates of primary versus overall degradation, and rates of metabolite formation and decay; (4) modeling tools that predict the likelihood of microbial biotransformation, as well as biochemical pathways; and (5) modeling approaches that allow for derivation of more generally applicable biotransformation rate constants, by accounting for physical and/or chemical processes and test system design when evaluating test data. We also identify that, while such advancements could improve the certainty and accuracy of persistence assessments, the mechanisms and processes by which they are translated into regulatory practice and development of new OECD test guidelines need improving and accelerating. Where uncertainty remains, holistic weight of evidence approaches may be required to accurately assess the persistence of chemicals. Integr Environ Assess Manag 2022;18:1454-1487. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Effect of a nonionic surfactant on biodegradation of slowly desorbing PAHs in contaminated soils.

The influence of the nonionic surfactant Brij 35 on biodegradation of slowly desorbing polycyclic aromatic hydrocarbons (PAHs) was determined in contaminated soils. We employed a soil originated from a creosote-polluted site, and a manufactured gas plant soil that had been treated by bioremediation. The two soils differed in their total content in five indicator 3-, 4-, and 5-ring PAHs (2923 mg kg(-1) and 183 mg kg(-1) in the creosote-polluted and bioremediated soils, respectively) but had a similar content (140 mg kg(-1) vs 156 mg kg(-1)) of slowly desorbing PAHs. The PAHs present in the bioremediated soil were highly recalcitrant. The surfactant at a concentration above its critical micelle concentration enhanced the biodegradation of slowly desorbing PAHs in suspensions of both soils, but it was especially efficient with bioremediated soil, causing a 62% loss of the total PAH content. An inhibition of biodegradation was observed with the high-molecular-weight PAHs pyrene and benzo[a]pyrene in the untreated soil, possibly due to competition effects with other solubilized PAHs present at relatively high concentrations. We suggest that nonionic surfactants may improve bioremediation performance with soils that have previously undergone extensive bioremediation to enrich for a slowly desorbing profile.

Root-mediated bacterial accessibility and cometabolism of pyrene in soil.

Partial transformation of pollutants and mobilization of the produced metabolites may contribute significantly to the risks resulting from biological treatment of soils polluted by hydrophobic chemicals such as polycyclic aromatic hydrocarbons (PAHs). Pyrene, a four-ringed PAH, was selected here as a model pollutant to study the effects of sunflower plants on the bacterial accessibility and cometabolism of this pollutant when located at a spatially distant source within soil. We compared the transformation of passively dosed 14C-labeled pyrene in soil slurries and planted pots that were inoculated with the bacterium Pseudomonas putida G7. This bacterium combines flagellar cell motility with the ability to cometabolically transform pyrene. Cometabolism of this PAH occurred immediately in the inoculated and shaken soil slurries, where the bacteria had full access to the passive dosing devices (silicone O-rings). Root exudates did not enhance the survival of P. putida G7 cells in soil slurries, but doubled their transport in column tests. In greenhouse-incubated soil pots with the same pyrene sources instead located centimeters from the soil surface, the inoculated bacteria transformed 14C-labeled pyrene only when the pots were planted with sunflowers. Bacterial inoculation caused mobilization of 14C-labeled pyrene metabolites into the leachates of the planted pots at concentrations of approximately 1 mg L-1, ten times greater than the water solubility of the parent compound. This mobilization resulted in a doubled specific root uptake rate of 14C-labeled pyrene equivalents and a significantly decreased root-to-fruit transfer rate. Our results show that the plants facilitated bacterial access to the distant pollutant source, possibly by increasing bacterial dispersal in the soil; this increased bacterial access was associated with cometabolism, which contributed to the risks of biodegradation.

Influence of low oxygen tensions and sorption to sediment black carbon on biodegradation of pyrene.

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of (14)C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by Mycobacterium gilvum VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (K(om)) of 14.1 microM and a dissolved pyrene half-saturation constant (K(pm)) of 6 nM. The fluorescence of (14)C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of (14)CO(2) in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.

Effect of electrokinetics on the bioaccessibility of polycyclic aromatic hydrocarbons in polluted soils.

Bioaccessibility is one of the most relevant aspects to be considered in the restoration of soils using biological technologies. Polycyclic aromatic hydrocarbons (PAH) usually have residual fractions that are resistant to biodegradation at the end of the biological treatment. In some situations, these residual concentrations could still be above legal standards. Here, we propose that the available knowledge about electroremediation technologies could be applied to enhance bioremediation of soils polluted with PAH. The main objective of this study was to show that a previous electrokinetic treatment could reduce the PAH residual fractions when the soil is subsequently treated by means of a bioremediation process. The approach involved the electrokinetic treatment of PAH-polluted soils at a potential drop of 0.9 to 1.1 V/cm and the subsequent estimations of bioaccessibility of residual PAHs after slurry-phase biodegradation. Bioaccessibility of PAH in two creosote-polluted soils (clay and loamy sand, total PAH content averaging 300 mg/kg) previously treated with an electric field in the presence of nonionic surfactant Brij 35 was often higher than in untreated controls. For example, total PAH content remaining in clay soil after bioremediation was only 62.65 +/- 4.26 mg/kg, whereas a 7-d electrokinetic pretreatment had, under the same conditions, a residual concentration of 29.24 +/- 1.88 mg/kg after bioremediation. Control treatments without surfactant indicated that the electrokinetic treatment increased bioaccessibility of PAHs. A different manner of electric field implementation (continuous current vs. current reversals) did not induce changes in PAH bioaccessibility. We suggest that this hybrid technology may be useful in certain bioremediation scenarios, such as soils rich in clay and black carbon, which show limited success due to bioavailability restrictions, as well as in highly heterogeneous soils.

Adsorptive bioremediation of soil highly contaminated with crude oil.

Due to the extended oil extraction and transportation in Russia and other oil-producing countries, many lands remain contaminated because of accidental spills. This situation requires the cost-effective and efficient remediation of petroleum-contaminated soils. Bioremediation of soils contaminated with high concentrations of crude oil is usually hampered by high toxicity thresholds for microbial degraders. We have performed a two-year microfield experiment on the influence of a mixed adsorbent (ACD) composed of granular activated carbon and diatomite on bioremediation of a grey forest soil contaminated with crude oil at concentrations (5-15 % w/w) that would theoretically not result in a successful pollutant removal due to toxicity. Remediation of these soils was evaluated after treating with the ACD adsorbent (from 4 to 12% w/w) and a biopreparation (BP) containing hydrocarbon-degrading bacteria, separately or in combination. Reduction of total petroleum hydrocarbons content was significantly greater in highly contaminated soils with the combined amendments than in the respective controls (through the activation of indigenous degrading microorganisms by fertilizing and mixing) by 9-10% and 5-8% at the end of the first and second years, respectively, depending on the contamination level. Significantly higher counts of petroleum-degrading microorganisms (as indigenous and introduced by the BP), as well as much less phytotoxicity was detected in the ACD-amended soils, as compared with the samples without adsorbent. In addition, the ACD mixture drastically reduced the wash-out of polar petroleum metabolites (evidently oxidized hydrocarbons) and the phytotoxicity of the lysimetric waters, especially in highly contaminated soils. The results indicate that the mixture of activated carbon and diatomite is a prospective adsorbent for the in situ bioremediation of soils highly contaminated with crude oil.

Rhizosphere-enhanced biosurfactant action on slowly desorbing PAHs in contaminated soil.

We studied how sunflower plants affect rhamnolipid biosurfactant mobilization of slowly desorbing fractions of polycyclic aromatic hydrocarbons (PAHs) in soil from a creosote-contaminated site. Desorption kinetics of 13 individual PAHs revealed that the soil contained initially up to 50% slowly desorbing fractions. A rhamnolipid biosurfactant was applied to the soil at the completion of the sunflower cycle (75 days in greenhouse conditions). After this period, the PAHs that remained in the soil were mainly present in a slowly desorbing form as a result of the efficient biodegradation of fast-desorbing PAHs by native microbial populations. The rhamnolipid enhanced the bioavailable fraction of the remaining PAHs by up to 30%, as evidenced by a standardized desorption extraction with Tenax, but the enhancement occurred with only planted soils. The enhanced bioavailability did not decrease residual PAH concentrations under greenhouse conditions, possibly due to ecophysiological limitations in the biodegradation process that were independent of the bioavailability. However, biodegradation was enhanced during slurry treatment of greenhouse planted soils that received the biosurfactant. The addition of rhamnolipids caused a dramatic shift in the soil bacterial community structure, which was magnified in the presence of sunflower plants. The stimulated groups were identified as fast-growing and catabolically versatile bacteria. This new rhizosphere microbial biomass possibly interacted with the biosurfactant to facilitate intra-aggregate diffusion of PAHs, thus enhancing the kinetics of slow desorption. Our results show that the usually limited biosurfactant efficiency with contaminated field soils can be significantly enhanced by integrating the sunflower ontogenetic cycle into the bioremediation design.