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.

Nitrogen Regulates the Distribution of Antibiotic Resistance Genes in the Soil-Vegetable System.

The increasing antibiotic resistance genes (ARGs) in fertilizer-amended soils can potentially enter food chains through their transfer in a soil-vegetable system, thus, posing threats to human health. As nitrogen is an essential nutrient in agricultural production, the effect of nitrogen (in the forms NH4 +-N and NO3 --N) on the distribution of ARGs (blaTEM-1, sul1, cmlA, str, and tetO) and a mobile genetic element (MGE; tnpA-4) in a soil-Chinese cabbage system was investigated. Not all the tested genes could transfer from soil to vegetable. For transferable ones (blaTEM-1, sul1, and tnpA-4), nitrogen application influenced their abundances in soil and vegetable but did not impact their distribution patterns (i.e., preference to either leaf or root tissues). For ARGs in soil, effects of nitrogen on their abundances varied over time, and the positive effect of NH4 +-N was more significant than that of NO3 --N. The ARG accumulation to vegetables was affected by nitrogen application, and the nitrogen form was no longer a key influencing factor. In most cases, ARGs were found to prefer being enriched in roots, and nitrogen application may slightly affect their migration from root to leaf. The calculated estimated human intake values indicated that both children and adults could intake 106-107 copies of ARGs per day from Chinese cabbage consumption, and nitrogen application affected ARG intake to varying degrees. These results provided a new understanding of ARG distribution in vegetables under the agronomic measures such as nitrogen application, which may offer knowledge for healthy vegetable cultivation in future.

Enhanced transformation capability towards benzo(a)pyrene by Fe(III)-modified manganese oxides.

Manganese oxides (Mn oxides) are ubiquitous and may coexist with Fe(III) ions in soil environments. In this study, acid birnessite, alkaline birnessite, cryptomelane, pyrolusite, manganite, and their Fe(III)-modified analogues were synthesized and used for benzo(a)pyrene transformation. Fe-modified Mn oxides show a markedly enhanced transformation capability towards benzo(a)pyrene. Specifically, the benzo(a)pyrene transformation rate constants k for Bir-H, Bir-OH, Cry, Pyr, and Man were 0.49, 0.080, 0.0071, 0.0055, and 0.0022 h-1, respectively. After Fe(III) modification, the transformation rate constants were increased to 22, 2.7, 0.25, 0.0072 and 0.0098 h-1, respectively. Fe(III)-modified layered birnessites exhibited better activity than Fe(III)-modified tunnel Mn oxides, which was attributed to their high Fe(III) contents and abundant active free radicals. Fe(III) was found to accept electrons from benzo(a)pyrene, thereby accelerating the benzo(a)pyrene transformation. Moreover, modification with Fe(III) increased the surface adsorbed water and oxygen, and promoted the generation of active free radicals. Finally, the physicochemical and biochemical properties of transformation products showed the environmental benefits of this process. Overall, the results indicate that the occurrence of Fe(III) ions could promote the removal of PAHs in Mn oxides-rich soils, and this study provides a credible understanding of PAH fates in natural soils.

Non-covalent binding interaction between phthalic acid esters and DNA.

The interaction of phthalic acid esters (PAEs) with DNA is known to be responsible for the disruption of endocrine functions and the teratogenic and carcinogenic effects. However, the binding strength and mechanism of this important process has often been neglected. Here, we confirmed the binding interaction between PAEs and DNA via fluorescence titration quenching experiment. The linear fitting curve proved that PAEs could bind to DNA, and the binding constants (KA) were 4.11 × 105, 1.04 × 105, 7.60 × 104, 1.99 × 104, and 1.42 × 103 L/mol for diethyl phthalate (DEP), bis(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), dimethyl phthalate (DMP), and benzyl butyl phthalate (BBP), respectively. DNA melting point, UV-vis spectra and Fourier transform infrared spectroscopy (FTIR) analyses revealed that PAEs interact predominantly with thymines in the DNA minor groove. Quantum chemical calculations showed that hydrogen bonding and van der Waals force formation between PAEs and DNA bases dominated the binding interaction. However, PAEs-DNA binding did not induce any DNA conformation change since the circular dichroism and FTIR spectra of B-DNA were not change. The electrostatic surface potential (ESP) might act an important role in PAEs-DNA binding interaction. This work will broaden our understanding of the interaction between PAEs and genetic materials.

Express-phytotest for choosing conditions and following process of soil remediation.

Phyto- and bioremediation are perspective methods for soil recultivation. In spite of resistance of plant-hyperaccumulators and degrading microorganisms to some contaminants, there are soil toxicity limits for their growth and activity. Therefore, simple and express methods are needed to estimate the soil phytotoxicity. This article is devoted to description of an express-phytotest evaluated by germination rate of white clover (Trifolium repens) (PhCG) for estimating phytotoxicity of contaminated soils. This phytotest was developed on the example of grey forest soil contaminated with diesel fuel or copper(II) and approbated during our long-year experiments on adsorptive bioremediation of petroleum-contaminated soils. The sensitivity of the phytotest values PhCG to these contaminants is much higher compared to those phytotests evaluated by germination of larger seeds: cress (Lepidium sativum), and wheat (Triticum vulgare). A significant increase of PhCG in those soils by 10% was already recorded at 50-100 mg of available Cu2+ kg-1 and 1-5 g total petroleum hydrocarbons kg-1, depending on the hydrocarbon composition. The sensitivity of the standard phytotests evaluated by root length of wheat seedlings or by plant (T. vulgare or T. repens) biomass is higher than that of PhCG determination. However, bio- and phytoremediation are mostly applied for heavily contaminated soils. Therefore, use of the simple and cheap express phytotest for choosing optimal conditions of the soil remediation and following the process is quite justified. Besides, measuring an additional parameter-root length of the white clover seedlings may significantly increase the sensitivity of the express phytotest for lower contaminated soils.

Sorption of benzo[a]pyrene by Chernozem and carbonaceous sorbents: comparison of kinetics and interaction mechanisms.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon, highly persistent and toxic and a widespread environmental pollutant. Although various technologies have been developed to remove BaP from the environment, its sorption through solid matrixes has received increasing attention due to cost-effectiveness. The present research compares the adsorption capacity of Haplic Chernozem, granular activated carbon and biochar in relation to BaP from water solution. Laboratory experiments with different initial BaP concentrations in the liquid phase and different ratios of the solid and liquid phases show that Freundlich model describes well the adsorption isotherms of BaP by the soil and both sorbents. Moreover, the BaP isotherm sorption by the Haplic Chernozem is better illustrated by the Freundlich model than the Langmuir equation. The results reveal that the sorption capacity of the carbonaceous adsorbents at a ratio 1:20 (solid to liquid phases) is orders of magnitude higher (13 368 ng mL-1 of activated carbon and 3 578 ng mL-1 of biochar) compared to the soil (57.8 ng mL-1). At the ratio of 0.5:20, the adsorption capacity of the carbonaceous sorbents was 17-45 times higher than that of the soil. This is due to the higher pore volume and specific surface area of the carbonaceous sorbents than soil particles, assessed through scanning electron microscopy. The sorption kinetic of BaP by Chernozem was compared with the adsorption kinetics by the carbonaceous sorbents. Results indicate that the adsorption dynamic involves two steps. The first one is associated with a fast BaP adsorption on the large available surface and inside macro- and meso-pores of the sorbent particles of the granular activated carbon and biochar. Then, the adsorption is followed by a slower process of BaP penetration into the microporous space and/or redistribution into a hydrophobic fraction. The effectiveness of the sorption process depends on both the sorbent properties and the solvent competition. Overall, the granular activated carbon and biochar are highly effective adsorbents for BaP, whereas the Haplic Chernozem has a rather limited capacity to remove BaP from contaminated solutions.

The Effectiveness of Biostimulation, Bioaugmentation and Sorption-Biological Treatment of Soil Contaminated with Petroleum Products in the Russian Subarctic.

The effectiveness of different bioremediation methods (biostimulation, bioaugmentation, the sorption-biological method) for the restoration of soil contaminated with petroleum products in the Russian Subarctic has been studied. The object of the study includes soil contaminated for 20 years with petroleum products. By laboratory experiment, we established five types of microfungi that most intensively decompose petroleum hydrocarbons: Penicillium canescens st. 1, Penicillium simplicissimum st. 1, Penicillum commune, Penicillium ochrochloron, and Penicillium restrictum. One day after the start of the experiment, 6 to 18% of the hydrocarbons decomposed: at 3 days, this was 16 to 49%; at 7 days, 40 to 73%; and at 10 days, 71 to 87%. Penicillium commune exhibited the greatest degrading activity throughout the experiment. For soils of light granulometric composition with a low content of organic matter, a more effective method of bioremediation is sorption-biological treatment using peat or granulated activated carbon: the content of hydrocarbons decreased by an average of 65%, which is 2.5 times more effective than without treatment. The sorbent not only binds hydrocarbons and their toxic metabolites but is also a carrier for hydrocarbon-oxidizing microorganisms and prevents nutrient leaching from the soil. High efficiency was noted due to the biostimulation of the native hydrocarbon-oxidizing microfungi and bacteria by mineral fertilizers and liming. An increase in the number of microfungi, bacteria and dehydrogenase activity indicate the presence of a certain microbial potential of the soil and the ability of the hydrocarbons to produce biochemical oxidation. The use of the considered methods of bioremediation will improve the ecological state of the contaminated area and further the gradual restoration of biodiversity.

Characterization of Different Molecular Size Fractions of Glomalin-Related Soil Protein From Forest Soil and Their Interaction With Phenanthrene.

As a natural organic compound secreted by arbuscular mycorrhizal fungi (AMF), glomalin-related soil protein (GRSP) is an important part in soil, affecting the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in it. Previous research have demonstrated that GRSP could enhance the availability of PAHs in the soil and favor their accumulation in plant roots. However, a scarcity of research exists on the different molecular weights of GRSP interacting with PAHs due to their complexation and heterogeneity. In this research, the extracted GRSP in soil was divided into three molecular weight (Mw) fractions of GRSP (<3,000, 3,000-10,000, and >10,000 Da), whose characteristics and binding capacity of PAHs were conducted by using UV-visible absorption, quenching fluorometry and, Fourier transform infrared spectroscopy. The results showed that the GRSP was composed of abundant compounds, it has a wide distribution of molecular weight, and the >10,000 Da Mw fraction was dominant. For three Mw fractions of GRSP, they have some difference in spectral features, for example, the >10,000 Da fraction showed higher dissolved organic carbon (DOC) contents, more phenolic hydroxyl groups, and stronger UV adsorption capacity than the low and middle Mw fractions. In addition, the interaction between GRSP and phenanthrene is related to the characteristics of the Mw fractions, especially the phenolic hydroxyl group, which has a significantly positive correlation with a binding coefficient of K A (k = 0.992, p < 0.01). Simultaneously, hydrophobic, NH-π, and H-bound also played roles in the complexation of phenanthrene with GRSP. These findings suggested that different GRSP Mw fractions could influence the fate, availability, and toxicity of PAHs in soil by their interaction.

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.

Benzo[a]pyrene degradation and bioaccumulation in soil-plant system under artificial contamination.

The involvement of benzo[a]pyrene (BaP) one of the most toxic polycyclic aromatic hydrocarbons (PAHs) in the soil-plant system causes its potential carcinogenicity and mutagenicity for human health. The aim of this article is benzo[a]pyrene (BaP) degradation and bioaccumulation in soil-plant system under artificial contamination in model experiment with Haplic Chernozem and that spiked with various doses of BaP (20, 200, 400 and 800µgkg-1) equivalent to 1, 10, 20 and 40 levels of maximal permissible concentrations (MPC) planted with spring barley (Hordeum sativum distichum). The experimental soil samples were planted every spring and incubated outdoor during 4years. The express-method of subcritical water extraction was used for BaP extraction from samples. It was established the values of BaP period of semi-degradation in soil (T50, y) contaminated with 10, 20 and 40MPC deviated from 1.4 to 1.8years, while these values in low contaminated soils deviated from 2.9 to 5.4years. It was found the BaP concentrations in plants depended on initial BaP contamination and reduced simultaneously with diminish of BaP concentration in the related spiked samples. Growing of spring barley in the BaP spiked soils lead to BaP accumulation in plants. The bioaccumulation factors for BaP in roots and vegetative part of barley plants (BAFr and BAFv respectively) fluctuated within 0.035-0.065 and 0.015-0.025 respectively at the 1st season and then reduced about twice to the 4th season. Meantime those values in control soils vice-versa increased twice from 0.03 and 0.01 respectively.

Effects of polyaromatic hydrocarbons on photosystem II activity in pea leaves.

The acute effects of three typical polyaromatic hydrocarbons (PAHs): naphthalene (Naph), phenanthrene (Phen) and fluoranthene (Flu) on photochemical activity of photosystem II (PSII) in detached leaves of 3-week-old pea plants were studied. The leaves were exposed in water with PAHs under white light for 0.5-72 h. The activity of PSII was examined by prompt and delayed chlorophyll a (Chl a) fluorescence. The effects of PAHs depended on their concentration and exposure time. This dependency was more significant in the presence of chemical stressors (Triton X-100 or acetone) or under high intensity irradiance. Increased content of PAHs and long-term exposure (24-72 h) led to significant reduction of the maximum photochemical quantum efficiency (Fv/Fm) of PS II, changes in the polyphasic fluorescence induction (OJIP), and to decreasing amplitudes of fast and slow components of delayed Chl a fluorescence. The damage of PSII depended on water solubility of a given type of PAHs, their concentration and exposure time. During short-time exposure the compound with highest water-solubility - naphthalene - revealed the strongest effect. During long-time exposure the compounds with low water-solubility -Phen, Flu-revealed the strongest effect as the corresponding PAH accumulates in the thylakoids especially when the solution is oversaturated containing a solid phase. The reduction of PSII activity at the presence of naphthalene (30 mg L(-1)) was accompanied by transient generation of H2O2 as well as swelling of thylakoids and distortion of cell plasma membranes, which was indicated by electron microscopy images. Distortion of thylakoid membranes due to accumulation of PAHs as well as the development of oxidative stress seems to be the main pathways of PAHs influencing the photochemical activity of PS II.

Non-invasive panel tests for gastrointestinal motility monitoring within the MARS-500 Project.

AIM: To develop an integrated approach for monitoring gastrointestinal motility and inflammation state suitable for application in long-term spaceflights. METHODS: Breath tests based on the oral administration of ¹³C-labeled or hydrogen-producing substrates followed by the detection of their metabolites (¹³CO2 or H2) in breath were used to measure gastrointestinal motility parameters during the 520-d spaceflight ground simulation within the MARS-500 Project. In particular, the gastric emptying rates of solid and liquid contents were evaluated by ¹³C-octanoic acid and ¹³C-acetate breath tests, respectively, whereas the orocecal transit time was assessed by an inulin H2-breath test, which was performed simultaneously with the ¹³C-octanoic acid breath test. A ready-to-eat, standardized pre-packaged muffin containing 100 mg of ¹³C-octanoic acid was used in the ¹³C-octanoic acid breath test to avoid the extemporaneous preparation of solid meals. In addition, a cassette-type lateral flow immunoassay was employed to detect fecal calprotectin, a biomarker of intestinal inflammation. Because no items could be introduced into the simulator during the experiment, all materials and instrumentation required for test performance during the entire mission simulation had to be provided at the beginning of the experiment. RESULTS: The experiments planned during the simulation of a manned flight to Mars could be successfully performed by the crewmembers without any external assistance. No evident alterations (i.e., increasing or decreasing trends) in the gastric emptying rates were detected using the ¹³C-breath tests during the mission simulation, as the gastric emptying half-times were in the range of those reported for healthy subjects. In contrast to the ¹³C-breath tests, the results of the inulin H2-breath test were difficult to interpret because of the high variability of the H2 concentration in the breath samples, even within the same subject. This variability suggested that the H2-breath test was strongly affected by external factors, which may have been related to the diet of the crewmembers or to environmental conditions (e.g., the accumulation of hydrogen in the simulator microenvironment). At least in closed microenvironments such as the MARS-500 simulator, ¹³C-breath tests should therefore be preferred to H2-breath tests. Finally, the fecal calprotectin test showed significant alterations during the mission simulation: all of the crewmembers were negative for the test at the beginning of the simulation but showed various degrees of positivity in at least one of the subsequent tests, thus indicating the onset of an intestinal inflammation. CONCLUSION: Breath tests, especially those ¹³C-based, proved suitable for monitoring gastrointestinal motility in the 520-d isolation experiment within MARS-500 project and can be applied in long-term spaceflights.

Dynamics of PCB removal and detoxification in historically contaminated soils amended with activated carbon.

Activated carbon (AC) can help overcome toxicity of pollutants to microbes and facilitate soil bioremediation. We used this approach to treat a Histosol and an Alluvial soil historically contaminated with PCB (4190 and 1585 mg kg(-1), respectively; primarily tri-, tetra- and pentachlorinated congeners). Results confirmed PCB persistence; reductions in PCB extractable from control and AC-amended soils were mostly due to a decrease in tri- and to some extent tetrachlorinated congeners as well as formation of a bound fraction. Mechanisms of PCB binding by soil and AC were different. In addition to microbial degradation of less chlorinated congeners, we postulate AC catalyzed dechlorination of higher chlorinated congeners. A large decrease in bioavailable PCB in AC-amended soils was demonstrated by greater clover germination and biomass. Phytotoxicity was low in treated soils but remained high in untreated soils for the duration of a 39-month experiment. These observations indicate the utility of AC for remediation of soils historically contaminated with PCB.

TNT biotransformation and detoxification by a Pseudomonas aeruginosa strain.

Successful microbial-mediated remediation requires transformation pathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L(-1) in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate 2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and 2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.