Bioremediation of creosote-contaminated soil: a pilot-scale landfarming evaluation

A pilot-scale landfarming investigation of the effects of biostimulation and bioaugmentation on a creosote-contaminated (258.3 g kg^–1) mispah form (FAO: lithosol) soil, with a view to developing a cost-effective bioremediation methodology for creosote-contaminated soils was conducted in nine duplicate reactors, including two controls (Treatments 1 and 2). Treatments 3–9 were watered and aerated daily and Treatment 4–9 were monthly amended with mono-ammonium phosphate. Treatment 5–9 received further amendments as follows: Treatment 5, hydrogen peroxide; Treatment 6, indigenous microbial biosupplement; Treatment 7, sewage sludge; Treatment 8, cow manure; Treatment 9, poultry manure. Residual concentrations of creosote ranged between 29 and 215 g kg^–1 after sixteen weeks. The phenolics and the 2- and 3-ringed polyaromatic hydrocarbons (PAHs) were removed below detectable levels or to very low levels. The 4- and 5-ringed PAHs were removed by between 68 and 83%. Indigenous microbial biosupplement and sewage sludge were the most effective in creosote removal. Hydrogen peroxide did not significantly enhance microbial population and creosote removal. There was no significant difference between the results obtained from the treatments amended with organic manures. However, there was a significant difference between the effects of the organic manures and the indigenous microbial biosupplement. Results from this study suggests that a combination of the two treatment techniques (biostimulation and bioaugmentation) would be a better approach to treating soil contaminated with very high concentrations of creosote.     

Natural Attenuation, Biostimulation, and Bioaugmentation in 4-Chloroaniline-Contaminated Soil

Bioremediation treatments including natural attenuation (NA), biostimulation (BS), and bioaugmentation (BA) were performed and compared regarding the degradation of 4-chloroaniline (4CA) contaminating two types of agricultural soil collected from Nakornnayok (NN) and Chiangmai (CM) provinces, Thailand. Despite the different soil properties, both soil types exhibited intrinsic potential for biodegradation. 4CA degradation by NA in loam soil-NN was fairly effective (ca. 40%), while in sandy-clay loam soil-CM it occurred poorly (<10%). Compared to NA, BS with aniline and BA with 4CA-degrading Klebseilla sp. CA17 were comparatively more effective techniques, although the degradation occurred differently in each soil type. In soil-NN, the biodegradation of 4CA took place at a higher rate, achieving biodegradation of 70–75% within 4 weeks, than in soil-CM, i.e., up to 40–46% within 8 weeks. During each treatment, changes in soil microbial activity, numbers of 4CA-degrading micro-organisms, and dynamic modification of soil microbial community structure were also monitored. The results suggest that both BS and BA are feasible techniques for bioremediation of 4CA accumulated in soil, although the biodegrading efficiency in soil environment depends not only on site characteristics but also on the characteristics of either indigenous microbial population or the survival and stability of bioaugmented cultures.     

Potential of Bioaugmentation and Biostimulation for Enhancing Intrinsic Biodegradation in Oil Hydrocarbon-Contaminated Soil

Studies were conducted using a 10-chamber Micro-Oxymax (Columbus, OH, USA) respirometer to determine the effect of bioaugmentation and biostimulation (by diverse ways of O₂ supply) on enhancing biodegradation of oil hydrocarbons to reduce risk at a former military airport in Kluczewo, Poland. Indigenous or exogenous bacteria bioaugmentation was used to degrade hydrocarbons. Aerated water and/or aqueous solutions of H₂O₂ or KMnO₄ were used to supply O₂. The intrinsic and enhanced biodegradation was evaluated by the O₂ uptake and CO₂ production rates obtained using a linear regression of the cumulative O₂ uptake and CO₂ production curves. Generally, in all cases biodegradation rates enhanced by bioaugmentation were two to four times higher than the rates of intrinsic biodegradation. Moreover, application of indigenous bacteria was more efficient in comparison to the exogenous consortia. The highest CO₂ production rates were achieved when aqueous solution of KMnO₄ was applied, as the increase of CO₂ production rates were about 71% to 97% higher compared to a control. The aqueous solution of H₂O₂ did not cause any significant improvement of the biodegradation rates. Compared to a control, the addition of aerated water resulted in a decrease of CO₂ production rates. Most probably the excessive soil moisture could reduce the air-filled porosity and, consequently, the oxygen contents in soil.     

Pilot Scale Bioremediation of Creosote-Contaminated Soil—Efficacy of Enhanced Natural Attenuation and Bioaugmentation Strategies

In this study, the efficacy of bioremediation strategies (enhanced natural attenuation with nitrate and phosphate addition [ENA] and bioaugmentation) for the remediation of creosote-contaminated soil (7767 ± 1286 mg kg^?1 of the 16 EPA priority PAHs) was investigated at pilot scale. Bioaugmentation of creosote-contaminated soil with freshly grown or freeze dried Mycobacterium sp. strain 1B (a PAH degrading microorganism) was applied following bench scale studies that indicated that the indigenous soil microflora had a limited PAH metabolic activity. After 182 days, the total PAH concentration in creosote-contaminated soil was reduced from 7767 ± 1286 mg kg^?1 to 5579 ± 321 mg kg^?1, 2250 ± 71 mg kg^?1, 2050 ± 354 mg kg^?1 and 1950 ± 70 mg kg^?1 in natural attenuation (no additions) and ENA biopiles and biopiles augmented with freshly grown or freeze dried Mycobacterium sp. strain 1B respectively. In ENA and bioaugmentation biopiles, between 82% and 99% of three-ring compounds (acenaphthene, anthracene, fluorene, phenanthrene) were removed while four-ring PAH removal ranged from 33 to 81%. However, the extent of PAH degradation did not vary significantly between the ENA treatment and biopiles augmented with Mycobacterium sp. strain 1B. Four-ring PAH removal followed the order fluoranthene > pyrene > benz[a]anthracene > chrysene. The high residual concentration of some four-ring PAHs may be attributable to bioavailability issues rather than a lack of microbial catabolic activity. Comparable results between ENA and bioaugmentation at pilot scale were surprising given the limited degradative capacity of the microbial consortia enriched from the creosote-contaminated soil.     

Bioremediation (Natural Attenuation and Biostimulation) of Diesel-Oil-Contaminated Soil in an Alpine Glacier Skiing Area

We investigated the feasibility of bioremediation as a treatment option for a chronically diesel-oil-polluted soil in an alpine glacier area at an altitude of 2,875 m above sea level. To examine the efficiencies of natural attenuation and biostimulation, we used field-incubated lysimeters (mesocosms) with unfertilized and fertilized (N-P-K) soil. For three summer seasons (July 1997 to September 1999), we monitored changes in hydrocarbon concentrations in soil and soil leachate and the accompanying changes in soil microbial counts and activity. A significant reduction in the diesel oil level could be achieved. At the end of the third summer season (after 780 days), the initial level of contamination (2,612 ± 70 μg of hydrocarbons g [dry weight] of soil⁻¹) was reduced by (50 ± 4)% and (70 ± 2)% in the unfertilized and fertilized soil, respectively. Nonetheless, the residual levels of contamination (1,296 ± 110 and 774 ± 52 μg of hydrocarbons g [dry weight] of soil⁻¹ in the unfertilized and fertilized soil, respectively) were still high. Most of the hydrocarbon loss occurred during the first summer season ([42 ± 6]% loss) in the fertilized soil and during the second summer season ([41 ± 4]% loss) in the unfertilized soil. In the fertilized soil, all biological parameters (microbial numbers, soil respiration, catalase and lipase activities) were significantly enhanced and correlated significantly with each other, as well as with the residual hydrocarbon concentration, pointing to the importance of biodegradation. The effect of biostimulation of the indigenous soil microorganisms declined with time. The microbial activities in the unfertilized soil fluctuated around background levels during the whole study.     

Natural Attenuation of Trichloroethylene in Rhizosphere Soils at the Savannah River Site

Extensive trichloroethylene (TCE) groundwater contamination has resulted from discharges to a former seepage basin in the A/M Area at the Department of Energy's Savannah River Site. The direction of groundwater flow has been determined and a seep line where the contaminated groundwater is estimated to emerge as surface water has been identified in a region of the Southern Sector of the A/M Area. This study was undertaken to estimate the potential of four rhizosphere soils along the seep line to naturally attenuate TCE. Microcosms were setup to evaluate both biotic and abiotic attenuation of TCE. Results demonstrated that sorption to soil was the dominant mechanism during the first week of incubation, with as much as 90% of the TCE removed from the aqueous phase. Linear partitioning coefficients (K_d) ranged from 0.83 to 7.4?mL/g, while organic carbon partition coefficients (K_oc) ranged from 72 to 180?mL/gC. Diffu-sional losses from the microcosms appeared to be a dominant fate mechanism during the remainder of the experiment, as indicated by results from the water controls. A limited amount of TCE biodegradation was observed, and attempts to stimulate TCE biodegradation by either methanotrophic or methanogenic activity through amendments with methane, oxygen, and methanol were unsuccessful. The appearance of cis-1,2-dichloroethylene (c-DCE), and trans-1,2-dichloroethylene (t-DCE) confirmed the potential for anaerobic reductive dechlorination. However, these daughter products represented less than 5% of the initial TCE added. The sorption results indicate that natural attenuation may represent a viable remediation option for the TCE plume as it passes through the rhizosphere.     

Biodegradation of 4-nitrotoluene with biosurfactant production by Rhodococcus pyridinivorans NT2: metabolic pathway, cell surface properties and toxicological characterization

A novel 4-nitrotoluene-degrading bacterial strain was isolated from pesticides contaminated effluent-sediment and identified as Rhodococcus pyridinivorans NT2 based on morphological and biochemical properties and 16S rDNA sequencing. The strain NT2 degraded 4-NT (400 mg l^?1) with rapid growth at the end of 120 h, reduced surface tension of the media from 71 to 29 mN m^?1 and produced glycolipidic biosurfactants (45 mg l^?1). The biosurfactant was purified and characterized as trehalose lipids. The biosurfactant was stable in high salinity (10 % w/v NaCl), elevated temperatures (120 °C for 15 min) and a wide pH range (2.0–10.0). The noticeable changes during biodegradation were decreased hydrophobicity; an increase in degree of fatty acid saturation, saturated/unsaturated ratio and cyclopropane fatty acid. Biodegradation of 4-NT was accompanied by the accumulation of ammonium (NH₄ ⁺) and negligible amount of nitrite ion (NO₂ ^?). Product stoichiometry showed a carbon (C) and nitrogen (N) mass balance of 37 and 35 %, respectively. Biodegradation of 4-NT proceeded by oxidation at the methyl group to form 4-nitrobenzoate, followed by reduction and hydrolytic deamination yielding protocatechuate, which was metabolized through β-ketoadipate pathway. In vitro and in vivo acute toxicity assays in adult rat (Rattus norvegicus) showed sequential detoxification and the order of toxicity was 4-NT >4-nitrobenzyl alcohol >4-nitrobenzaldehyde >4-nitrobenzoate >> protocatechuate. Taken together, the strain NT2 could be used as a potential bioaugmentation candidate for the bioremediation of contaminated sites.     

Bioremediation of Soil Artificially Contaminated with Petroleum Hydrocarbon Oil Mixtures: Evaluation of the Use of Animal Manure and Chemical Fertilizer

The search for cheaper and environmentally friendly options of enhancing petroleum hydrocarbon degradation has continued to elicit research interest. One of such options is the use of animal manure as biostimulating agents. A combination of treatments consisting of the application of poultry manure, piggery manure, goat manure, and chemical fertilizer was evaluated in situ during a period of 4 weeks of remediation. Each treatment contained petroleum hydrocarbon mixture (kerosene, diesel oil, and gasoline mixtures) (10% w/w) in soil as a sole source of carbon and energy. After 4 weeks of remediation, the results showed that poultry manure, piggery manure, goat manure, and NPK (nitrogen, phosphorous, and potash [potassium]) fertilizer exhibited 73%, 63%, 50%, and 39% total petroleum hydrocarbon degradation, respectively. Thus, all the biostimulating treatment strategies showed the ability to enhance petroleum hydrocarbon microbial degradation. However, poultry manure, piggery manure, and goat manure treatments showed greater petroleum hydrocarbon reductions than NPK fertilizer treatment. A first-order kinetic equation was fitted to the biodegradation data and the specific degradation rate constant (k) values obtained showed that the order of effectiveness of these biostimulating strategies in the cleanup of soil contaminated with petroleum hydrocarbon mixtures (mixture of kerosene, diesel oil, and gasoline) is NPK fertilizer < goat manure < piggery manure < poultry manure. Therefore, this present work has indicated that the application of poultry manure, piggery manure, goat manure, and chemical fertilizer could enhance petroleum hydrocarbon degradation with poultry manure, showing a greater effectiveness and thus could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil.     

Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: Field-scale processes in Poland

Research was conducted to estimate impact of the multiple bioaugmentation on the treatment of soil contaminated by fuels - diesel oil and aircraft fuel. The bacteria used to inoculate the remediation plots were isolated from the polluted soil and proliferated in field conditions. The amount of biomass applied to the polluted soil was set to ensure the total number of bacteria in soil 10⁷-10⁸ cfu/g d.w. The multiple inoculation of soil with indigenous bacteria active in diesel oil and engine oil (plot A) degradation increased bioremediation effectiveness by 50% in comparison to the non-inoculated control soil and by 30% in comparison to the soil that was inoculated only once. The multiple inoculation of soil with indigenous microorganisms was then applied in bioremediation of the soil polluted with double high concentration of diesel oil (soil B) and in bioremediation of the soil polluted with aircraft fuel (soil C). The process efficiency was 80% and 98% removal of TPH for soil B and C, respectively.     

Fundamentals and Application Potential of Arsenic-Resistant Bacteria for Bioremediation in Rhizosphere: A Review

As a hazardous environmental metalloid toxicant, arsenic (As)—at elevated levels in water and soil—has created a major public health concern through its entry into the food chain by accumulation in crops. Among the various methods reported thus far for reclamation of As-contaminated crop fields, bioremediation using bacteria with plant-growth-promoting traits has been found to be a most promising solution. There is every possibility that bacterial isolates with the ability to remove or immobilize As could be used for successful bioremediation. However, bioremediation needs to define its boundaries between promise and field application, as most studies have been restricted to laboratory results only. Rhizosphere interactions play a critical role in monitoring As bioavailability to crop plants, thus a better understanding of it might improve rhizoremediation technologies. The challenges rely on the application of these novel approaches under field conditions. Despite some limitations, the prospect for successful stimulation and exploitation of microbial metabolism for As rhizoremediation appears to be very promising.     

Treatability Testing for Weathered Hydrocarbons in Soils: Bioremediation,Soil Washing,Chemical Oxidation,and Thermal Desorption

Soils previously treated with landfarming to reduce petroleum hydrocarbon concentrations are often left with a less biodegradable residual fraction that can present challenges for additional treatment. Four possible polishing technologies were tested on the bench scale for weathered hydrocarbons present in fine-grain soils obtained from a previously landfarmed area at an active oil refinery. The technologies included additional bioremediation (both biostimulation and bioaugmentation tested), soil washing, chemical oxidation, and low-temperature thermal desorption. Multiple parameters were tested separately for each technology to identify possible factors that were relevant across technologies. Extractable hydrocarbons comprised only approximately 35% of the organic carbon in the soils, and this component was considerably less affected by biological, surfactant, and oxidant treatment than organic materials that are not quantified by the TEH analysis. Treatment testing of thermal desorption indicated removal of large quantities of extractable hydrocarbons despite the presence of high organic matter. The additional demand to the system would likely result in considerably large timeframes (biological treatment), reagent quantities (soil washing and oxidation), or energy input (thermal desorption) for treatment of target hydrocarbons on a full scale.     

On-Site Treatment of Contaminated Soils: An Approach to Bioremediation of Weathered Petroleum Compounds

A bench-scale investigation was conducted prior to on-site bioremediation of 52,000 cubic yards of contaminated soil containing weathered, structurally complex petroleum compounds from an inactive oil refinery. Addition of bulking agents was required to improve soil physical properties. A supplemental study was also conducted to evaluate the effectiveness of bio-enhancement products. Loss of n-alkanes was rapid in soil mixtures containing a high nitrogen sludge compost, but very slow in mixtures containing wood products as bulking agents. By completion of the study at day 110, the isoprenoids pristane and phytane had nearly disappeared from mixtures containing sludge compost. Clearly, pristane and phytane are inadequate biomarkers when conditions favor an advanced stage of biodegradation. Nearly half the complex branched and cyclic alkanes in the unresolved complex mixture also degraded. After 70 days, depletion of dibenzo-thiophenes and phenan-threnes was 75 and 90%, respectively. The most stable PAHs within each group were the highly methylated homologues. Because of their complex structures, both steranes and hopanes were stable in all soil mixtures. Data were normalized to hopanes as a conserved internal standard or biomarker. Use of hopane-normalized data successfully eliminated much of the data variability and permitted a more accurate assessment of biodegradation. A relatively slow decline in total hydrocarbons occurred later in the study. This slowing tendency of microbial utilization is caused not only by substrate depletion, but also because remaining hydrocarbons are structurally more complex and persistent. Because of this, it is important to avoid using kinetic data from early stages of bioremediation to predict later hydrocarbon losses, such as the time required to attain a cleanup standard. In the supplemental study, an oleophilic fertilizer product accelerated hydrocarbon degradation when compared with a conventional fertilizer. This product will be tested in combination with organic bulking agents under field conditions to determine its cost effectiveness.     

Developments in Bioremediation of Soils and Sediments Polluted with Metals and Radionuclides: 2. Field Research on Bioremediation of Metals and Radionuclides

Bioremediation of metals and radionuclides has had many field tests, demonstrations, and full-scale implementations in recent years. Field research in this area has occurred for many different metals and radionuclides using a wide array of strategies. These strategies can be generally characterized in six major categories: biotransformation, bioaccumulation/bisorption, biodegradation of chelators, volatilization, treatment trains, and natural attenuation. For all field applications there are a number of critical biogeochemical issues that most be addressed for the successful field application. Monitoring and characterization parameters that are enabling to bioremediation of metals and radionuclides are presented here. For each of the strategies a case study is presented to demonstrate a field application that uses this strategy.     

Haloalkane hydrolysis by Rhodococcus erythropolis cells: comparison of conventional aqueous phase dehalogenation and nonconventional gas phase dehalogenation

Biofiltration of air polluted by volatile organic compounds is now recognized by the industrial and research communities as an effective and viable alternative to standard environmental technologies. Whereas many studies have focused on solid/liquid/gas biofilters, there have been fewer reports on waste air treatment using other biological processes, especially in a solid/gas biofilter. In this study, a comparison was made of the hydrolysis of halogenated compounds (such as 1-chlorobutane) by lyophilized Rhodococcus erythropolis cells in a novel solid/gas biofilter and in the aqueous phase. We first determined the culture conditions for the production of R. erythropolis cells with a strong dehalogenase activity. Four different media were studied and the amount of 1-chlorobutane was optimized. Next, we report the possibility to use R. erythropolis cells in a solid/gas biofilter in order to transform halogenated compounds in corresponding alcohols. The effect of experimental parameters (total flow into the biofilter, thermodynamic activity of the substrates, temperature, carbon chain length of halogenated substrates) on the activity and stability of lyophilized cells in the gas phase was determined. A critical water thermodynamic activity (a(w)) of 0.4 is necessary for the enzyme to become active and optimal dehalogenase activity for the lyophilized cells is obtained for an a(w) of 0.9. A temperature of reaction of 40 degrees C represents the best compromise between stability and activity. Activation energy of the reaction was determined and found equal to 59.5 KJ/mol. The pH effect on the dehalogenase activity of R. erythropolis cells was also studied in the gas phase and in the aqueous phase. It was observed that pH 9.0 provided the best activity in both systems. We observed that in the aqueous phase R. erythropolis cells were less sensitive to the variation in pH than R. erythropolis cells in the gas phase. Finally, the addition of volatile Lewis base (triethylamine) in the gaseous phase and the action of the lysozyme in order to permeabilize the cells was found to be highly beneficial to the effectiveness of the biofilter.     

Bioremediation of 6 % [w/w] Diesel‐Contaminated Mainland Soil in Singapore: Comparison of Different Biostimulation and Bioaugmentation Treatments

The efficacy of indigenous microorganisms to degrade diesel oil in contaminated mainland sites in Singapore was investigated. A semi‐scale trial was made by spiking topsoil with 6 % [w/w] of diesel oil. The results indicated that in the presence of NPK commercial (Rosasol®) fertilizer a 53 % reduction in contaminant concentration was recorded after 60 days compared to untreated controls while the addition of a mixture of urea and K₂HPO₄ effected a 48 % reduction in the Total Recoverable Petroleum Hydrocarbons. A commercial culture and an enriched/isolated microbial association proved to be the least effective with 25 and 9 % reductions, respectively. The results confirmed the bioremediation potential of indigenous microorganisms for diesel‐oil contaminated mainland soil. Identification of the persistent compounds was done and perceived as a tool in decision‐making on strategies for speeding up of the degradation process to achieve clean‐up standards in shorter remediation periods.     

Aerobic Biodegradation of DDT by Advenella Kashmirensis and Its Potential Use in Soil Bioremediation

The aim of this study was to select a bacterial strain able to degrade 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT), and to use it for bioaugmentation in order to decontamination soil. Advenella Kashmirensis MB-PR (A. Kashmirensis MB-PR) was isolated from DDT contaminated soil, and the degradation ability of DDT by this strain in the mineral salt medium was screened by gas chromatography. The efficiency of degradation was 81% after 30 days of bacterial growth. The study of intermediary products during the degradation of DDT showed the appearance and accumulation of DDD and DDE, which emerged from the first days of the experiment. Other metabolites were detected at a lower number of chlorine atoms, such as DBH. DNA samples were isolated and screened for the linA gene, encoding dehydrochlorinase. The bioaugmentation by A. Kashmirensis MB-PR of polluted sterile soil showed that 98% of DDT disappeared after 20 days of experience. This study demonstrates the significant potential use of A. Kashmirensis MB-PR for the bioremediation of DDT in the environment.     

Developments in Bioremediation of Soils and Sediments Polluted with Metals and Radionuclides. 3. Influence of Chemical Speciation and Bioavailability on Contaminants Immobilization/Mobilization Bio-processes

The biotransformation of metals is an exciting, developing strategy to treat metal contamination, especially in environments that are not accessible to other remediation technologies. However, our ability to benefit from these strategies hinges on our ability to monitor these transformations in the environment. That’s why remediation of contaminated sediments and soil requires detailed in situ characterization of the speciation of the toxic substances and their transformations with respect to time and spatial distribution. The present paper gives an overview of the literature regarding research performed in the laboratory as well as in the field.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄ ^?) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.