Remediation of oil-contaminated soil using the CLEANSOIL technology

Approbation data of the innovative CLEANSOIL technology of soil purification after oil pollution are given. Drainage pipes filled with an adsorbent with microorganisms placed in the soil are used. It is revealed that the content of hydrocarbons under the technological constructions (metal columns and reservoirs) rises in comparison with the open oil-polluted areas. It is shown that the oil is destroyed quicker under the constructions versus in the open areas. The microorganisms better assimilate the n-alkanes with C₁₄ chains than the C_32–40 hydrocarbons. The application of a combined technology based on the sorption and reduction of the hydrocarbons by microorganisms makes it possible to quickly reduce the soil pollution by oil products without the soil cover’s disturbance.     

On the components of soil humic acids VII

The writers already made it clear that by active alumina the humic acids of A- and B-types contained in various soils could be separated into 3 or 4 components (1). And moreover, absorption spectra (2), nitrogen constituents (3) and colloidal nature (4) of them were studied for the purpose of examining their qualities.     

Amino sugars in fungal melanins and soil humic acids

Humic acids from six Brazilian topsoils (three Latosols and three Podzols) and five soil fungal melanins were hydrolysed and the released amino sugars qualitatively and quantitatively determined by capillary gas-liquid chromatography. Melanins were obtained from liquid culture media, synthesized with nitrate or asparagine as a source of N, of Stachybotrys atra, Aspergillus glaucus, Eurotium echinulatum and Hendersonula toruloidea. Glucosamine (48–60%), galactosamine (39–52%), and traces of mannosamine were detected in all humic acids. The total amino sugar content ranged from 1351 to 2287 mg kg^?1. In the fungal melanins analysed, mostly glucosamine (80–99%) and trace to small amounts of galactosamine and mannosamine (<10%) were found in amounts ranging from 192 to 635 mg kg^?1. The Occurrence of mannosamine in fungal melanins and fungal polysaccharides had not previously been found. The present study gives additional data to the theory that fungal melanins may play a role, as precursors, in the formation of soil organic matter.     

Analysis of polynuclear aromatic and aliphatic components in soil humic acids using ruthenium tetroxide oxidation

Although condensed aromatic components are considered to be one of the major structural units of soil humic acids (HAs) and to be responsible for the dark colour of HAs, their amount and composition remain largely unknown. In ruthenium tetroxide oxidation (RTO), condensed aromatic components are detectable as their degradation products, mainly benzenepolycarboxylic acids (BPCAs). We applied this technique to soil HAs with various degrees of humification (darkening). The yields of water‐ and dichloromethane‐soluble products from HAs upon RTO after methylation ranged from 210 to 430 mg g⁻¹ and 10–40 mg g⁻¹, respectively. Eight kinds of BPCAs with two to six carboxyl groups, and seven kinds of BPCAs with additional side chains (tentative assignment) were obtained as methylated counterparts. The yield of each BPCA and the sum of the yields of BPCAs (12–85 mg g⁻¹ HAs) increased with increasing degree of humification and aromatic C content. The compositions of BPCAs indicated that the degree of condensation was greater in the HAs with greater degrees of humification. The sum of the yields of aliphatic compounds ranged from 0.1 to 6.5 mg g⁻¹, and decreased with increasing degree of humification. The C₁₂ to C₃₀ monocarboxylic acid methyl esters accounted for > 56% of the aliphatic compounds assigned, which may be present mainly as end alkyl groups in the HA molecules. We also obtained the methylated counterparts of C₁₄ to C₂₄ dicarboxylic acids; these were possibly derived from polymethylene bridges between adjacent aromatic rings.     

Determination of DTNB-reactive sulphhydryl groups in soil humic acids: their enrichment in humic acids from volcanic acid soils

Thirteen acid soils were collected from typical volcanic regions in Japan (S content: 0.9–4.1, mean = 2.2g kg^?1; pH (H₂O): 2.81–3.93, mean = 3.33), as well as nine reference soils (S: 0.6–1.7, mean= 1.1 gkg^?1; pH(H₂O): 4.10–4.74, mean = 4.47). Humic acids were extracted from the soils separately with 0.1 M NaOH and precipitated by acidification (pH = 2, HCl). After purification, the humic acids were subjected to colorimetric analysis using a DTNB reagent [5,5′-dithiobis(2–nitrobenzoic acid] for the active -SH group. Since humic acids have significant absorption at 4I2 nm, the coloured compound (5–mercapto-2–nitrobenzoic acid) was separated from the humic acids by ultrafiltration or solvent extraction prior to the colorimetric measurement. Humic acids also caused discoloration of the coloured compound when they coexisted in the reaction solutions. Thus, the reproducible determination of -SH was accomplished by employing a standard addition technique (-SH standard: cysteine). Although -SH contents obtained by the ultrafiltration method were considerably higher than those by the solvent-extraction method, probably due to the denaturation of humic acids by the higher buffer concentration used in the ultrafiltration method, they correlated well with each other. The humic acids from acid soils contained apparently higher concentrations of -SH (120–510, mean = 270mg S kg^?1 by the ultrafiltration method; 8–110, mean = 38mg S kg^?1 by the solvent-extraction method) compared to those from reference soils [20–260, mean = 90mg S kg^?1 by the former; not detectable (<5)-34, mean = 11 mg S kg^?1 by the latter]. This -SH enrichment in the humic acids from acid soils may result from the degradation and subsequent humification of S-rich debris of plants and micro-organisms and/or direct incorporation of volcanic acid gas (H₂S) into the humic acids.     

Hydrophobicity of electrophoretic fractions of different soil humic acids

Purpose

The fractionation of soil humic acids (HAs) according to their hydrophobicity is a common procedure in the study of this polydispersed complex natural mixture, so that reversed-phase high-performance liquid chromatography (RP-HPLC) is used resulting in humic components of differing hydrophobic/hydrophilic properties. However, a comparative study of the hydrophobicity of fractions isolated from different soil HAs have not been addressed so far.

Materials and methods

The RP-HPLC with online absorbance detection was used for analysis of International Humic Substances Society soil standard HAs, chernozem soil HAs, and their electrophoretic fractions A, B, and C?+?D, obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis. The strong relationship between hydrophobicity, electrophoretic mobility (EM), molecular size (MS), specific absorbance at 280 nm and aromaticity of HAs fractions was found.

Results and discussion

Independently of soil HAs genesis fraction A with lowest EM and highest MS is essentially more hydrophobic (60–73 % of the fraction amount remained adsorbed on the RP column) than medium EM and MS fraction B (33–47 % of the fraction amount remained adsorbed on the RP column). The lowest hydrophobicity belongs to fraction C?+?D with highest EM and lowest MS.

Conclusions

The most hydrophilic aromatic fraction C?+?D seems to have been bound with other mostly aliphatic hydrophobic fractions A and B through non covalent (possibly hydrogen) bonds. These data could be relevant to better understanding the overall makeup of soil HAs and their structural organization.     

On the components of soil humic acids (part 9)

The carbohydrates in soil organic matter seem to be derived from undecomposed or partially decomposed plant and microbial residues, In soil, these carbohydrates exist chiefly in such from as polysaccharide hemicellulose, and their polyuronide has been the chief object of investigation^1)-3). In various soil, the polyuronide is found in a large quantity in fulvic fraction of soil organic matter and has been considered as important in connection with the physical structure of soil^4),5). According to Lynch ⁵⁾, the carbohydrate content of humic acid is markedly smaller than that of fulvic acid. His work also indicates that a' considerable change is noted in the content and composition of the carbohydrate in humic acid because of the addition of some organic substances to the soils, or of the cultivation of virgin soils. Further, some investigators⁷⁾ believe that uronic acid is introduced into the aromatic structure of the humic acid by changing into pentose and furan. Accordingly, it seems that the role of carbohydrate in the formation of soil humic acid should not be overlooked.     

The effects of fulvic and humic acids on soil aggregation: a micromorphological study

The influence of soil amendment with fulvic + humic acid (FH) fractions, obtained from manure or peat, upon the shape, size and numerical density of aggregates in a sandy loam and a clay soil was examined using micromorphological techniques and image analysis. The effect of the FH fraction from manure (FHM) was to produce larger aggregates from the smaller ones, while the FH from peat (FHP) gave rise to a higher numerical density of smaller aggregates. Although the type of structural change was similar in the two soil types, the extent of change per g of FH was greater in the clay soil. Aggregate shape varied with treatment and soil type, but both kinds of organic matter produced vughs and a narrowing of the planes in the two soils which can be considered as signs of structural improvement.     

Inhibition by soil humic acids of native and acetylated proteolytic enzymes

The activities of acetyltrypsin and acetylcarboxypeptidase were unaffected by neutralized solutions of soil humic acids in concentrations which markedly inhibited the non-acetylated enzymes. Further, acetyltrypsin, unlike trypsin, did not coprecipitate with humic acids. The results support the conclusion that humic acids bind the proteases by a cationexchange mechanism whereby protease amino groups are linked to humic acid carboxyl groups.     

Effects of pig slurry application on soils and soil humic acids

The effect of three annually consecutive additions of pig slurry at two rates (90 and 150 m3 x ha(-1) x year(-1) on soils and soil humic acids (HAs) was investigated in a field experiment under semiarid conditions. Soils and pig slurries were analyzed by standard methods. The HAs were isolated from soils and pig slurry by a conventional procedure based on alkaline extraction, acidic precipitation to pH 1, purification by repeated alkaline dissolutions and acidic precipitations, water washing, dialysis, and final freeze-drying. The HAs obtained were analyzed for elemental (C, H, N, S, and O) and acidic functional group (carboxylic and phenolic) composition, and by UV-vis, FT-IR, fluorescence, and ESR spectroscopies. With respect to the control soil, the pig slurry amended soils had greater pH and electrical conductivity, slightly larger total N content, and smaller values of C/N ratio. A decrease of total organic C was observed only in soils amended for 2 and 3 years at the higher slurry rate. With respect to control soil HA, pig slurry HA was characterized by larger contents of S- and N-containing groups, smaller acidic functional group and organic free radical contents, a prevalent aliphatic character, extended molecular heterogeneity, and smaller aromatic polycondensation and humification degrees. Amendment with pig slurry HA determines a number of modifications in soil HAs, including increase of C, S, and COOH contents, C/N ratios, and aliphaticity and decrease of extraction yields and N, O, phenolic OH, and organic free radical contents. These effects are generally more evident after the first year of slurry application and tend to disappear with increasing number of treatments. Most probably, over the years the slightly humified slurry HA is mineralized through extended microbial oxidation, whereas only the most recalcitrant components, such as S-containing, phenolic, and aliphatic structures, are partially accumulated by incorporation into soil HA.     

Reduction of Cr(VI) by soil humic acids

The rate of hexavalent chromium reduction by a soil humic acid (SHA) was investigated in aqueous solutions where concentrations of Cr(VI), H⁺, and SHA were independently varied. Rate experiments were done with a large excess of SHA over Cr(VI). Rates of reduction depend strongly on [H⁺], increasing with decreasing pH. Typical Cr(VI)-SHA reactions display a nonlinear reduction of Cr(VI) with time that cannot be modelled with simple first- or second-order rate equations. An empirical rate equation is developed for Cr(VI)-soil humic acid reactions over a range of experimental conditions. The model is in part based on a reactive continuum concept developed for soil fulvic acids. The rate equation describing Cr(VI) reduction by SHA is: R= -(k₀+k[H⁺]^1/2)[HCrO₄^?]^1/2X_e^?1, where k₀ is (8·3 ± 1·2) × 10^?12, s^?1k is (2·04 ± 0·05) × 10^?9 l^1/2 mol^?1/2 s^?1, and X_e is the equivalent fraction of SHA oxidized. The rate equation adequately models Cr(VI) reduction in an experiment with [Cr(VI)]₀ four times greater than the maximum concentration used in its derivation. Cr(VI) reduction at pH 3 by two other SHAs can also be modelled using the rate equation. The difference between the rate coefficients for the humic acid and the fulvic acid from the same soil was greater than the difference in the rate coefficients for humic acids from different soils.     

中国黑土上腐殖酸和腐殖物质的提取及其描述

Twenty-three progressive extractions were performed to study individual humic acids (HAs) and humin fractions from a typical black soil (Mollisol) in Heilongjiang Province, China using elemental analysis and spectroscopic techniques. After 23 HA extractions the residue was separated into high and low organic carbon humin fractions. HA yield was the highest for the first extraction and then gradually decreased with further extractions. Organic carbon (OC) of the humin fractions accounted for 58% of total OC even after 23 successive HA extractions. In addition, the atomic C/H ratio decreased during the course of extraction while C/O increased; the E4/E6 ratio from the UV analysis decreased with further extraction while E~/E3 increased; the band assigned to aliphatic carbon (2 930 cm-1) in the diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) spectra gradually increased with progressive extraction; the calculated ratio of the sum of aromatic carbon peak heights to that of aliphatic carbon peak heights from DRIFTS spectra declined with extractions; and nuclear magnetic resonance (NMR) data suggested that HA aliphatic carbons increased with extractions while aromatic carbons decreased. Thus, hydrophobicity and aliphaticity of HAs increased with extractions while polarity and aromaticity decreased. These data showed substantial chemical, structural, and molecular differences among the 23 HAs and two humin fractions. Therefore, these results may help explain why soil and sediment humin fractions have high sorption capacity for organic contaminants.     

Long term effects of a brown coal-based amendment on the properties of soil humic acids

In this study a typical grey-brown podzolic soil was amended with different doses of a brown coal-based preparation called Rekulter (R) largely used in Poland. After seven years, soils were analyzed and humic acids (HAs) were extracted both from the control soil and from the amended soils. All HAs were characterized by Fourier transform infrared spectroscopy and fluorescence spectroscopy both in emission, excitation and synchronous-scan mode and as Excitation-Emission-Matrix (EEM) contour maps. A higher carbon content was observed in the amended soils whereas significant differences were highlighted between the unamended and the amended soil HAs. HAs from amended soils showed a higher content of carboxyl groups and a more aromatic character, particularly HA extracted from the soil amended with the highest dose of R.     

Gas chromatographic analysis of amino acids from the action of proteolytic enzymes on soil humic acids

Six proteases released more amino compounds from albumin than from a humic acid. Pronase and thermolysin released about one-sixth of the acid-hydrolysable amino acids from a humic acid, papain had slight activity while chymotrypsin, trypsin and subtilopeptidase did not attack humic acid. Thermolysin hydrolysates of four humic acids contained peptides which on acid hydrolysis broke down to increase three fold the yield of α-amino N. Net amounts of amino compounds released on incubation of thermolysin and humic acids were directly related to incubation time, substrate concentration and enzyme concentration.     

Physico-chemical investigations of interaction mechanisms between s-triazine herbicides and soil humic acids

Multiple binding mechanisms that may occur upon interactions between three soil humic acids of different origins and four s-triazine herbicides, differing in chemical structure and properties, have been investigated.Results of elemental, thermal, infrared and electron spin resonance analyses of the products of interactions demonstrate that different processes are involved and that they are controlled mainly by the content of the acidic functional groups and hydrogen binding capacity of humic acid and by the basicity of s-triazines.Experimental and theoretical evidence is given on the occurrence of electron donor—acceptor processes involving free rardical intermediates and leading to stable charge-transfer complexes between the adsorbant and the adsorbate. Parallels are suggested between the biological and chemical behaviour of s-triazines towards quinone-like structures in electron donor—acceptor systems.The feasible formation of covalent bonds, which leads to interaction products of enhanced molecular complexity, capable of stabilizing free radical intermediates, is discussed.Analysis of our data suggests that the higher the capacity of humic acids to form ionic and hydrogen bonds with s-triazines, the lower their effectiveness in forming electron-transfer complexes, as related to the lower ability to generate free radicals.Finally, our results show that the basicity of s-triazines, and hence their tendency to form ionic bonds, is not the main factor governing adsorption. Indeed the most basic prometone is not the most adsorbed; nevertheless, it appears to be the most efficient among s-triazines in giving rise to electron donor—acceptor processes with humic acids.     

Separation and characterization of the constituents of compost and soil humic acids by two-dimensional electrophoresis

To investigate the chemical heterogeneity of humic acids (HAs), we applied two-dimensional (2-D) electrophoresis to HAs from a compost and two types of soils. In this method, HAs are first separated by isoelectric focusing (IEF) and then separated by polyacrylamide gel electrophoresis (PAGE). IEF and PAGE were carried out in the presence of 7?M urea. Upon 2-D electrophoresis of HAs, dark-colored substances were spread out across the gel mainly in the isoelectric point (pI) range of 3.0–4.5. Green fluorescence was observed in the smaller molecular size region of the gel, especially in the pI range of 3.0–4.5, and the most intense fluorescence was found at the moving front. The gels were divided into 36 sections, and then HA constituents were extracted from the individual sections and recovered by precipitation with acid. The distribution of organic carbon (C) among the gel sections coincided with that of the dark-colored substances on the gel. The total C recoveries were only 43–50%, suggesting that a considerable amount of HA constituents was lost during the extraction from the gels and purification. High-performance size-exclusion chromatography confirmed that the constituents of HAs were separated based on their molecular sizes by PAGE. The measurement of diffuse reflectance infrared Fourier transform (DRIFT) spectra indicated that the chemical properties of the HA constituents differed depending on the position on the gels and were affected by the molecular size rather than the pI. The fractions of the compost HA were characterized by higher proportions of aliphatic, proteinous and polysaccharide moieties and by the presence of lignin-derived structures. For the soil HAs, the fractions were characterized by a high proportion of the carboxyl group and a low proportion of aliphatic moieties. The proportion of proteinous and polysaccharide moieties in the fractions of soil HAs decreased with decreasing molecular size. The chemical properties of the green fluorescent substances remained unclear, since there was not enough of the substances to measure the DRIFT spectra. The present study showed that 2-D electrophoresis in the presence of concentrated urea offers an effective method for fractionating and isolating the constituents of HAs.     

Effects of sewage sludge amendment on humic acids and microbiological properties of a semiarid Mediterranean soil

The residual effects of adding 40 t ha^–1 sewage sludge (SL) to a degraded soil cropped with barley were investigated after 9 and 36 months in a field experiment under semiarid conditions. The principal soil properties were apparently still affected by SL amendment 9 months after application but the effects disappeared after 36 months. With respect to control soil humic acids (HAs), the SL-HA was characterized by higher contents of S- and N-containing groups, smaller contents of acidic groups, a prevalent aliphaticity, extended molecular heterogeneity, and smaller degrees of aromatic polycondensation and humification. Amendment with SL caused an increase in N, H, S and aliphaticity contents and a decrease in C/N ratios and O and acidic functional group contents in soil HAs isolated 9 months after SL application. These effects tended to decrease after 36 months, most probably because the slightly humified SL-HA was mineralised over time through extended microbial oxidation, while only the most recalcitrant components such as S-containing and aromatic structures were partially accumulated by incorporation into soil HA. Microbial biomass, basal respiration, metabolic quotient and enzymatic activities increased in soil 9 months after SL application, possibly because of increased soil microbial metabolism and enhanced mineralisation processes. After 36 months these properties returned to values similar to those of the unamended soil, presumably due to the loss of energy sources.     

Bioremediation of oil-contaminated soil by combination of soil conditioner and microorganism

Purpose

Oil hydrocarbons are widespread pollutants in soil which pose serious threats to ecological environment. Thus, this study carried out the bioremediation of oil-contaminated soil by using the efficient petroleum-degrading bacteria and soil conditioner, to investigate the changes of physicochemical properties of contaminated soil during bioremediation, reveal the relationship among the exogenous degradation strains and indigenous microbe, and finally illuminate the effects of soil conditioner and microbe on the bioremediation of oil-contaminated soil.

Materials and methods

A PAH-degrading strain named Stenotrophomonas maltophilia was used in this study, which was isolated from an e-waste dismantling area. The soil conditioner in this present study was developed previously by using agricultural wastes, which was in a powdered form and rich in N, P, and K. The simulated experiments were conducted under the control environmental conditions of greenhouse, to study the effects of inoculation and soil conditioner on bioremediation of oil-contaminated soil. Then, the physicochemical properties of soil and the degradation rates of oil were measured at different set times to evaluate the bioremediation effect.

Results and discussion

Adding 1% soil conditioner could significantly improve the soil conditions and offer microorganism enough N, P, and K, which would promote microbial growth and played a key role on bioremediation of oil-contaminated soil. Although in polluted soil, the microorganism could maintain metabolic activity and use the petroleum as carbon source. The soil indigenous microbe was more easily to adapt to the contaminated surrounding. However, when both of them co-existed in soil, they would restrain each other, and go against the oil decomposition. Thus, making two types of microorganisms work to achieve synergy was the key to gain much better remediation effect. Because the indigenous microbe was good at decomposing low molecular compounds and saturated hydrocarbons, while the oil-degrading strains can effectively decompose high molecular weight aromatics.

Conclusions

The soil nutrient and microorganism, including the exogenous oil-degrading strains and the soil indigenous microbe, had an important effect on degradation of petroleum. The addition of soil conditioner, presence of indigenous microbe, and inoculation of oil-degrading strains all were conducive to bioremediation of oil-contaminated site, but the key was to control the proportion and relationship of the three.

Graphical abstract

     

Comparison of carbon skeletal structures in black humic acids from different soil origins

Andosols are characterized by an abundance of black humic acids (HAs) belonging to Type A with a high content of aromatic carbon (C) in particular condensed aromatic C. Black HAs are also observed in other soils, such as Chernozems and the subsoil of paddy field, and extracted after washing with an acid or using chelating agent such as sodium pyrophosphate (Na₄P₂O₇). However, contribution of condensed aromatic structures to those soil HAs are unknown. To obtain the information about C skeletal structures of black HAs in soils other than Andosols, HAs were obtained from 2 Chinese Chernozem samples, 2 subsoil samples from Japanese paddy fields (Fulvisols), and a Rendzina-like soil (Cambisols) as well as an Andosol sample (reference) by successive extraction with 0.1 M NaOH (HAs₁) and 0.1 M Na₄P₂O₇ (HAs₂), and ¹³C nuclear magnetic resonance and X-ray diffraction 11-band profile analyses were applied. In the black HAs₂ from the non-Andosol samples, the proportion of C present as aromatic C, size of C layer planes, and relative C layer plane content ranged from 52 to 59%, 0.48 to 1.92 nm (mean size, 0.76–0.91 nm), and 58 to 100 AU (arbitrary unit) mg^?1, respectively, with a positive correlation between total C layer plane content and the degree of humification. Those ranges were similar to the distribution ranges of Andosols HAs₁ reported by our previous study.     

Competitive sorption between imidacloprid and imidacloprid-urea on soil clay minerals and humic acids

Soil organic matter and clay minerals are responsible for the adsorption of many pesticides. Adsorption and competitive sorption of imidacloprid on clay minerals and humic acids (HA) were determined using the batch equilibration method. The sorption coefficient of imidacloprid on humic acids was significantly higher than that on Ca-clay minerals, indicating that soil organic matter content was a more important property in influencing the adsorption of imidacloprid. Competitive sorption was investigated between imidacloprid and its main metabolite imidacloprid-urea on HA and Ca-clay minerals. The results showed that the sorption capacity of imidacloprid on clay minerals and HA was reduced in the presence of the metabolite, implying that imidacloprid-urea could occupy or block adsorption sites of imidacloprid on soil, potentially affecting the fate, transport, and bioavailability of imidacloprid in the environment. The interactions between a Ca-clay or HA-clay mixture and adsorption of imidacloprid and imidacloprid-urea were studied using IR differential spectra on thin films made of the adsorbent. The possible mechanisms were discussed from the shift of characteristic IR absorption bands of imidacloprid and imidacloprid-urea after sorption.