Comparison of bioremediation strategies for soil impacted with petrochemical oily sludge

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms systems were assessed during 90 days. The results showed that the highest biodegradation rates were obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms showed highest removal rate (84.1 ± 0.9%) when contaminated at 6% and bacterial consortium and nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation rate (78.7 ± 1.3%). These results demonstrate that the application of microbial consortium and nutrients favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment of the soils impacted with this important hazardous waste.     

Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture

The degradation of polycyclic aromatic hydrocarbons (PAHs) by an undefined culture obtained from a PAH-polluted soil and the same culture bioaugmented with three PAH-degrading strains was studied in carbon-limited chemostat cultures. The PAHs were degraded efficiently by the soil culture and bioaugmentation did not significantly improve the PAH degrading performance. The presence of PAHs did, however, influence the bacterial composition of the bioaugmented and non-bioaugmented soil cultures, resulting in the increase in cell concentration of sphingomonad strains. the initial enhancement of the degradation of the PAHs by biostimulation gradually disappeared and only the presence of salicylate in the additional carbon sources had a lasting slightly stimulating effect on the degradation of phenanthrene. The results suggest that bioaugmentation and biostimulation have limited potential to enhance PAH bioremediation by culture already proficient in the degradation of such contaminants.     

Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica

AIMS: To identify native Antarctic bacteria capable of oil degradation at low temperatures. METHODS AND RESULTS: Oil contaminated and pristine soils from Signy Island (South Orkney Islands, Antarctica) were examined for bacteria capable of oil degradation at low temperatures. Of the 300 isolates cultured, Pseudomonas strain ST41 grew on the widest range of hydrocarbons at 4 degrees C. ST41 was used in microcosm studies of low temperature bioremediation of oil-contaminated soils. Microcosm experiments showed that at 4 degrees C the levels of oil degradation increased, relative to the controls, with (i) the addition of ST41 to the existing soil microbial population (bioaugmentation), (ii) the addition of nutrients (biostimulation) and to the greatest extent with (iii) a combination of both treatments (bioaugmentation and biostimulation). Addition of water to oil contaminated soil (hydration) also enhanced oil degradation, although less than the other treatments. Analysis of the dominant species in the microcosms after 12 weeks, using temporal temperature gradient gel electrophoresis, showed Pseudomonas species to be the dominant soil bacteria in both bioaugmented and biostimulated microcosms. CONCLUSIONS: Addition of water and nutrients may enhance oil degradation through the biostimulation of indigenous oil-degrading microbial populations within the soil. However, bioaugmentation with Antarctic bacteria capable of efficient low temperature hydrocarbon degradation may enhance the rate of bioremediation if applied soon after the spill. SIGNIFICANCE AND IMPACT OF THE STUDY: In the future, native soil bacteria could be of use in bioremediation technologies in Antarctica.     

Influence of nutrients addition and bioaugmentation on the hydrocarbon biodegradation of a chronically contaminated Antarctic soil

Complexity involved in the transport of soils and the restrictive legislation for the area makes on-site bioremediation the strategy of choice to reduce hydrocarbons contamination in Antarctica. The effect of biostimulation (with N and P) and bioaugmentation (with two bacterial consortia and a mix of bacterial strains) was analysed by using microcosms set up on metal trays containing 2·5 kg of contaminated soil from Marambio Station. At the end of the assay (45 days), all biostimulated systems showed significant increases in total heterotrophic aerobic and hydrocarbon-degrading bacterial counts. However, no differences were detected between bioaugmented and nonbioaugmented systems, except for J13 system which seemed to exert a negative effect on the natural bacterial flora. Hydrocarbons removal efficiencies agreed with changes in bacterial counts reaching 86 and 81% in M10 (bioaugmented) and CC (biostimulated only) systems. Results confirmed the feasibility of the application of bioremediation strategies to reduce hydrocarbon contamination in Antarctic soils and showed that, when soils are chronically contaminated, biostimulation is the best option. Bioaugmentation with hydrocarbon-degrading bacteria at numbers comparable to the total heterotrophic aerobic counts showed by the natural microflora did not improve the process and showed that they would turn the procedure unnecessarily more complex.     

Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and β-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of β- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of β- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.     

Synergistic Effect of Yeast-Bacterial Co-Culture on Bioremediation of Oil-Contaminated Soil

The effects of various treatments, including biostimulation, bioaugmentation with bacterial consortium, yeast, and yeast-bacterial co-culture, on oil biodegradation were systematically compared. Synergistic effects were observed on the removal of total petroleum hydrocarbon and polycyclic aromatic hydrocarbons via the amendment of co-culture, with a 48-day degradation of 56% for total petroleum hydrocarbon and 32% for polycyclic aromatic hydrocarbons, respectively. Yeast played an important role in the removal of polycyclic aromatic hydrocarbons with 4–6 rings. The synergistic effect of yeast-bacteria was further evidenced by the increase of biomass and enzyme activities in soil. In comparison with the bacterial community, the yeast community was more sensitive to the inoculated cultures, which was indexed by the changes of diversity, abundance, and evenness in polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis.     

Bioremediation strategies for diesel and biodiesel in oxisol from southern Brazil

Leaks and spillages during the extraction, transport and storage of petroleum and its derivatives may result in environmental contamination. Biodiesel is an alternative energy source that can contribute to a reduction in environmental pollution. The aim of the present work was to evaluate biodegradation of diesel, biodiesel, and a 20% biodiesel-diesel mixture in oxisols from southern Brazil, using two bioremediation strategies: natural attenuation and bioaugmentation/biostimulation. Fuel biodegradation was monitored over 60 days by dehydrogenase activity, CO₂ evolution and gas chromatography. The bacterial inoculum employed for bioaugmentation/biostimulation consisted of Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia and PCR-DGGE using 16S RNAr primers showed that some members of this consortium survived in the soil after 60 days. The biodegradation of pure biodiesel was higher for bioaugmentation/biostimulation than for natural attenuation, suggesting that the addition of the microbial consortium, together with adjustment of the macronutrient ratio, increased biodiesel degradation. The results of dehydrogenase and respiratory activity, together with GC analysis, suggested that the presence of biodiesel may, by stimulating general microbial degradative metabolism, increase the biodegradation of petroleum diesel. The microbial community was altered by both treatments, with natural attenuation producing a lower diversity index than the amended soil. The bioaugmentation/biostimulation strategy was showed to have a high potential for cleaning up soils contaminated with diesel and biodiesel blends.     

Studies on Biodegradation of Kerosene in Soil under Different Bioremediation Strategies

The effectiveness of bioremediation is often a function of the microbial population and how they can be enriched and maintained in an environment. Strategies for inexpensive in situ bioremediation of soil contaminated with petroleum hydrocarbons include stimulation of the indigenous microorganisms by introduction of nutrients (biostimulation) and/or through inoculation of an enriched mixed microbial culture into soil (bioaugmentation). To demonstrate the potential use of bioremediation in soil contaminated with kerosene, a laboratory study with the objective of evaluating and comparing the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation was performed. The present study dealt with the biodegradation of kerosene in soil under different bioremediation treatment strategies: bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation, respectively. Each treatment strategy contained 10% (w/w) kerosene in soil as a sole source of carbon and energy. After 5 weeks of remediation, the results revealed that bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation exhibited 44.1%, 67.8%, 83.1%, and 87.3% kerosene degradation, respectively. Also, the total hydrocarbon-degrading bacteria (THDB) count in all the treatments increased with time up till the second week after which it decreased. The highest bacterial growth was observed for combined biostimulation and bioaugmentation treatment strategy. A first-order kinetic model equation was fitted to the biodegradation data to further evaluate the rate of biodegradation and the results showed that the specific degradation rate constant (k) value was comparatively higher for combined biostimulation and bioaugmentation treatment strategy than the values for other treatments. Therefore, value of the kinetic parameter showed that the degree of effectiveness of these bioremediation strategies in the clean up of soil contaminated with kerosene is in the following order: bioattenuation < bioaugmentation < biostimulation < combined biostimulation and bioaugmentation. Conclusively, the present work has defined combined biostimulation and bioaugmentation treatment strategy requirements for kerosene oil degradation and thus opened an avenue for its remediation from contaminated soil.     

Pyrosequence analyses of bacterial communities during simulated in situ bioremediation of polycyclic aromatic hydrocarbon-contaminated soil

Barcoded amplicon pyrosequencing was used to generate libraries of partial 16S rRNA genes from two columns designed to simulate in situ bioremediation of polycyclic aromatic hydrocarbons (PAHs) in weathered, contaminated soil. Both columns received a continuous flow of artificial groundwater but one of the columns additionally tested the impact of biostimulation with oxygen and inorganic nutrients on indigenous soil bacterial communities. The penetration of oxygen to previously anoxic regions of the columns resulted in the most significant community changes. PAH-degrading bacteria previously determined by stable-isotope probing (SIP) of the untreated soil generally responded negatively to the treatment conditions, with only members of the Acidovorax and a group of uncharacterized PAH-degrading Gammaproteobacteria maintaining a significant presence in the columns. Additional groups of sequences associated with the Betaproteobacterial family Rhodocyclaceae (including those associated with PAH degradation in other soils), and the Thiobacillus, Thermomonas, and Bradyrhizobium genera were also present in high abundance in the biostimulated column. Similar community responses were previously observed during biostimulated ex situ treatment of the same soil in aerobic, slurry-phase bioreactors. While the low relative abundance of many SIP-determined groups in the column libraries may be a reflection of the slow removal of PAHs in that system, the similar response of known PAH degraders in a higher-rate bioreactor system suggests that alternative PAH-degrading bacteria, unidentified by SIP of the untreated soil, may also be enriched in engineered systems.     

Bioremediation of petroleum hydrocarbon contaminated soil: Effects of strategies and microbial community shift

Biodegradation of petroleum hydrocarbon oil (14,000 mg kg⁻¹) were investigated in six biopiles batches, differing in the remediation strategy: bioaugmentation (selected consortium and kitchen waste were introduced), biostimulation (added with rhamnolipid, high-level, or low-level nutrient), and bioaugmentation plus biostimulation (added both with rhamnolipid and bacterial consortia). After the 140-day operation, the kitchen waste (KW) and the low-level nutrient (NEL) batches achieved the highest total petroleum hydrocarbon degradation efficiency (>80%). The result of the hydrocarbon analysis revealed that the bioaugmentation approaches were the most effective ones in removing aromatic component (64% and 68%), and KW and NEL were the only two approaches that can remove the polar component with positive efficiency, 11% and 21%, respectively. The terminal-restriction fragment length polymorphism percentage (T-RFLP) abundance applied with nonmetric multidimensional scaling indicated a similarity of the bacterial communities during the early fastest remediation stage. The results of the oligonucleotide array targeting the ribosomal internal transcribed spacer (ITS) region, along with the hydrocarbon fractional analysis, indicated a successive degradation completed by the bacterial-fungi consortia. Before Day 70, the bacterial community was dominant in decomposing the saturated and partially aromatic hydrocarbons. After Day 70, the fungal community found to be dynamic and responsible for degradation of the polar hydrocarbons composing of recalcitrant metabolites.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

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.     

Bacterial Diversity of a Consortium Degrading High-Molecular-Weight Polycyclic Aromatic Hydrocarbons in a Two-Liquid Phase Biosystem

High-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs) are pollutants that persist in the environment due to their low solubility in water and their sequestration by soil and sediments. Although several PAH-degrading bacterial species have been isolated, it is not expected that a single isolate would exhibit the ability to degrade completely all PAHs. A consortium composed of different microorganisms can better achieve this. Two-liquid phase (TLP) culture systems have been developed to increase the bioavailability of poorly soluble substrates for uptake and biodegradation by microorganisms. By combining a silicone oil–water TLP system with a microbial consortium capable of degrading HMW PAHs, we previously developed a highly efficient PAH-degrading system. In this report, we characterized the bacterial diversity of the consortium with a combination of culture-dependent and culture-independent methods. Polymerase chain reaction (PCR) of part of the 16S ribosomal RNA gene (rDNA) sequences combined with denaturing gradient gel electrophoresis was used to monitor the bacterial population changes during PAH degradation of the consortium when pyrene, chrysene, and benzo[a]pyrene were provided together or separately in the TLP cultures. No substantial changes in bacterial profiles occurred during biodegradation of pyrene and chrysene in these cultures. However, the addition of the low-molecular-weight PAHs phenanthrene or naphthalene in the system favored one bacterial species related to Sphingobium yanoikuyae. Eleven bacterial strains were isolated from the consortium but, interestingly, only one—IAFILS9 affiliated to Novosphingobium pentaromativorans—was capable of growing on pyrene and chrysene as sole source of carbon. A 16S rDNA library was derived from the consortium to identify noncultured bacteria. Among 86 clones screened, 20 were affiliated to different bacterial species–genera. Only three strains were represented in the screened clones. Eighty-five percent of clones and strains were affiliated to Alphaproteobacteria and Betaproteobacteria; among them, several were affiliated to bacterial species known for their PAH degradation activities such as those belonging to the Sphingomonadaceae. Finally, three genes involved in the degradation of aromatic molecules were detected in the consortium and two in IAFILS9. This study provides information on the bacterial composition of a HWM PAH-degrading consortium and its dynamics in a TLP biosystem during PAH degradation.     

Kinetic Modelling and Half-Life Study on Bioremediation of Soil Co-Contaminated with Lubricating Motor Oil and Lead Using Different Bioremediation Strategies

The focus of this study was to investigate the effect of nutrient supplement (urea fertilizer) and microbial species augmentation (mixed culture of Aeromonas, Micrococcus, and Serratia sp.) on biodegradation of lubricating motor oil (LMO) and lead uptake by the autochthonous microorganism in LMO and lead-impacted soil were investigated. The potential inhibitory effects of lead on hydrocarbon utilization were investigated over a wide range of lead concentrations (25–200 mg/kg) owing to the complex co-contamination problem frequently encountered in most sites. Under aerobic conditions, total petroleum hydrocarbons (TPH) removal was 45.3% in the natural attenuation microcosm while a maximum of 72% and 68.2% TPH removal was obtained in biostimulation and bioaugmentation microcosms, respectively. Lead addition, as lead nitrate, to soil samples reduced the number of hydrocarbon degraders in all samples by a wide range (11–52%) depending on concentration and similarly, the metabolic activities were affected as observed in mineralization of LMO (3–60%) in soils amended with various lead concentrations. Moreover, the uptake of lead by the autochthonous microorganisms in the soil reduced with increase in the initial lead concentration. First-order kinetics described the biodegradation of LMO very well. The biodegradation rate constants were 0.015, 0.033, and 0.030 day^?1 for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The presence of varying initial lead concentration reduced the biodegradation rate constant of LMO degradation in the biostimulation treatment microcosm. Half-life times were 46.2, 21, and 23 days for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The half-life time in the biostimulation treatment microcosm was increased with a range between 10.7 and 39.2 days by the presence of different initial lead concentration. The results have promising potential for effective remediation of soils co-contaminated with hydrocarbons and heavy metals.     

Bacterial Community Dynamics during Bioremediation of Diesel Oil-Contaminated Antarctic Soil

The effect of nutrient and inocula amendment in a bioremediation field trial using a nutrient-poor Antarctic soil chronically contaminated with hydrocarbons was tested. The analysis of the effects that the treatments caused in bacterial numbers and hydrocarbon removal was combined with the elucidation of the changes occurring on the bacterial community, by 16S rDNA-based terminal restriction fragment length polymorphism (T-RFLP) typing, and the detection of some of the genes involved in the catabolism of hydrocarbons. All treatments caused a significant increase in the number of bacteria able to grow on hydrocarbons and a significant decrease in the soil hydrocarbon content, as compared to the control. However, there were no significant differences between treatments. Comparison of the soil T-RFLP profiles indicated that there were changes in the structure and composition of bacterial communities during the bioremediation trial, although the communities in treated plots were highly similar irrespective of the treatment applied, and they had a similar temporal dynamics. These results showed that nutrient addition was the main factor contributing to the outcome of the bioremediation experiment. This was supported by the lack of evidence of the establishment of inoculated consortia in soils, since their characteristic electrophoretic peaks were only detectable in soil profiles at the beginning of the experiment. Genetic potential for naphthalene degradation, evidenced by detection of nahAc gene, was observed in all soil plots including the control. In treated plots, an increase in the detection of catechol degradation genes (nahH and catA) and in a key gene of denitrification (nosZ) was observed as well. These results indicate that treatments favored the degradation of aromatic hydrocarbons and probably stimulated denitrification, at least transiently. This mesocosm study shows that recovery of chronically contaminated Antarctic soils can be successfully accelerated using biostimulation with nutrients, and that this causes a change in the indigenous bacterial communities and in the genetic potential for hydrocarbon degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Optimization of high-molecular-weight polycyclic aromatic hydrocarbons' degradation in a two-liquid-phase bioreactor

A microbial consortium degrading the high-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) pyrene, chrysene, benzo[a]pyrene and perylene in a two-liquid-phase reactor was studied. The highest PAH-degrading activity was observed with silicone oil as the water-immiscible phase; 2,2,4,4,6,8, 8-heptamethylnonane, paraffin oil, hexadecane and corn oil were much less, or not efficient in improving PAH degradation by the consortium. Addition of surfactants (Triton X-100, Witconol SN70, Brij 35 and rhamnolipids) or Inipol EAP22 did not promote PAH biodegradation. Rhamnolipids had an inhibitory effect. Addition of salicylate, benzoate, 1-hydroxy-2-naphtoic acid or catechol did not increase the PAH-degrading activity of the consortium, but the addition of low-molecular-weight (LMW) PAHs such as naphthalene and phenanthrene did. In these conditions, the degradation rates were 27 mg l-1 d-1 for pyrene, 8.9 mg l-1 d-1 for chrysene, 1.8 mg l-1 d-1 for benzo[a]pyrene and 0.37 mg l-1 d-1 for perylene. Micro-organisms from the interface were slightly more effective in degrading PAHs than those from the aqueous phase.     

Using population dynamics analysis by DGGE to design the bacterial consortium isolated from mangrove sediments for biodegradation of PAHs

There are many PAH-degrading bacteria in mangrove sediments and in order to explore their degradation potential, surface sediment samples were collected from a mangrove area in Fugong, Longhai, Fujian Province of China. A total of 53 strains of PAH-degrading bacteria were isolated from the mangrove sediments, consisting of 14 strains of phenanthrene (Phe), 13 strains of pyrene (Pyr), 13 strains of benzo[a]pyrene (Bap) and 13 strains of mixed PAH (Phe + Pyr + Bap)-degrading bacteria. All of the individual colonies were identified by 16S rDNA sequencing. Based on the information of bacterial PCR-DGGE profiles obtained during enrichment batch culture, Phe, Pyr, Bap and mixed PAH-degrading consortia consisted of F1, F2, F3, F4 and F15 strains, B1, B3, B6, B7 and B13 strains, P1, P2, P3, P5 and P7 strains, M1, M2, M4, M12 and M13 strains, respectively. In addition, the degradation ability of these consortia was also determined. The results showed that both Phe and mixed PAH-degrading consortia had the highest ability to degrade the Phe in a liquid medium, with more than 91% being degraded in 3 days. But the biodegradation percentages of Pyr by Pyr-degrading consortium and Bap by Bap-degrading consortium were relatively lower than that of the Phe-degrading consortium. These results suggested that a higher degradation of PAHs depended on both the bacterial consortium present and the type of PAH compound. Moreover, using the bacterial community structure analysis method, where the consortia consist of different PAH-degrading bacteria, the information from the PCR-DGGE profiles could be used in the bioremediation of PAHs in the future.     

Effects of the Inoculant Strain Sphingomonas paucimobilis 20006FA on Soil Bacterial Community and Biodegradation in Phenanthrene-contaminated Soil

The effects of the inoculant strain Sphingomonas paucimobilis 20006FA (isolated from a phenanthrene-contaminated soil) on the dynamics and structure of microbial communities and phenanthrene elimination rate were studied in soil microcosms artificially contaminated with phenanthrene. The inoculant managed to be established from the first inoculation as it was evidenced by denaturing gradient gel electrophoresis analysis, increasing the number of cultivable heterotrophic and PAH-degrading cells and enhancing phenanthrene degradation. These effects were observed only during the inoculation period. Nevertheless, the soil biological activity (dehydrogenase activity and CO₂ production) showed a late increase. Whereas gradual and successive changes in bacterial community structures were caused by phenanthrene contamination, the inoculation provoked immediate, significant, and stable changes on soil bacterial community. In spite of the long-term establishment of the inoculated strain, at the end of the experiment, the bioaugmentation did not produce significant changes in the residual soil phenanthrene concentration and did not improve the residual effects on the microbial soil community.     

Long-term simulation of in situ biostimulation of polycyclic aromatic hydrocarbon-contaminated soil

A continuous-flow column study was conducted to evaluate the long-term effects of in situ biostimulation on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil from a manufactured gas plant site. Simulated groundwater amended with oxygen and inorganic nutrients was introduced into one column, while a second column receiving unamended groundwater served as a control. PAH and dissolved oxygen (DO) concentrations, as well as microbial community profiles, were monitored along the column length immediately before and at selected intervals up to 534?days after biostimulation commenced. Biostimulation resulted in significantly greater PAH removal than in the control condition (73% of total measured PAHs vs. 34%, respectively), with dissolution accounting for a minor amount of the total mass loss (~6%) in both columns. Dissolution was most significant for naphthalene, acenaphthene, and fluorene, accounting for >20% of the total mass removed for each. A known group of PAH-degrading bacteria, 'Pyrene Group 2' (PG2), was identified as a dominant member of the microbial community and responded favorably to biostimulation. Spatial and temporal variations in soil PAH concentration and PG2 abundance were strongly correlated to DO advancement, although there appeared to be transport of PG2 organisms ahead of the oxygen front. At an estimated oxygen demand of 6.2?mg O(2)/g dry soil and a porewater velocity of 0.8?m/day, it took between 374 and 466?days for oxygen breakthrough from the 1-m soil bed in the biostimulated column. This study demonstrated that the presence of oxygen was the limiting factor in PAH removal, as opposed to the abundance and/or activity of PAH-degrading bacteria once oxygen reached a previously anoxic zone.