Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia

Biodegradation of a mixture of PAHs was assessed in forest soil microcosms performed either without or with bioaugmentation using individual fungi and bacterial and a fungal consortia. Respiratory activity, metabolic intermediates and extent of PAH degradation were determined. In all microcosms the low molecular weight PAH’s naphthalene, phenanthrene and anthracene, showed a rapid initial rate of removal. However, bioaugmentation did not significantly affect the biodegradation efficiency for these compounds. Significantly slower degradation rates were demonstrated for the high molecular weight PAH’s pyrene, benz[a]anthracene and benz[a]pyrene. Bioaugmentation did not improve the rate or extent of PAH degradation, except in the case of Aspergillus sp. Respiratory activity was determined by CO₂ evolution and correlated roughly with the rate and timing of PAH removal. This indicated that the PAHs were being used as an energy source. The native microbiota responded rapidly to the addition of the PAHs and demonstrated the ability to degrade all of the PAHs added to the soil, indicating their ability to remediate PAH-contaminated soils.     

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.     

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.     

Bioremediation of PAHs-contaminated soil through composting: Influence of bioaugmentation and biostimulation on contaminant biodegradation

The degradation of several polycyclic aromatic hydrocarbons (PAHs) in soil through composting was investigated. The selected PAHs included: fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, and chrysene, with concentrations simulating a real creosote sample. The degradation of PAHs (initial concentration 1 g of total PAHs kg⁻¹ dry soil) was assessed applying bioaugmentation with the white-rot fungi Trametes versicolor and biostimulation using compost of the source-selected organic fraction of municipal solid waste (OFMSW) and rabbit food as organic co-substrates. The process performance during 30 days of incubation was evaluated through different analyses including: dynamic respiration index (DRI), cumulative oxygen consumption during 5 days (AT₅), enzymatic activity, and fungal biomass. These analyses demonstrated that the introduced T. versicolor did not significantly enhance the degradation of PAHs. However, biostimulation was able to improve the PAHs degradation: 89% of the total PAHs were degraded by the end of the composting period (30 days) compared to the only 29.5% that was achieved by the soil indigenous microorganisms without any co-substrate (control, not amended). Indeed, the results showed that stable compost from the OFMSW has a greater potential to enhance the degradation of PAHs compared to non-stable co-substrates such as rabbit food.     

Kinetics of biodegradation of mixtures of polycyclic aromatic hydrocarbons

The kinetics of biodegradation of polycyclic aromatic hydrocarbons (PAHs) by a mixed culture were determined in a creosote-contaminated soil and in a pristine soil. A competitive-inhibition model was able to represent the kinetics of degradation of PAHs from the creosote-contaminated soil, from the lag phase through to active degradation, but not data from pristine soil with the same PAHs alone and in mixtures. The presence of phenanthrene introduced a lag phase of 4.5 days in the degradation of fluoranthene and 5 days for chrysene. Rapid degradation of pyrene followed a lag phase of circa 5 days, regardless of the presence of other PAHs. These results show that even when kinetics of PAH degradation by mixed cultures appear to follow competitive-inhibition kinetics, the underlying mechanisms may be more complex.     

Bioaugmentation and biostimulation strategies to improve the effectiveness of bioremediation processes

Bioremediation, involving bioaugmentation and/or biostimulation, being an economical and eco-friendly approach, has emerged as the most advantageous soil and water clean-up technique for contaminated sites containing heavy metals and/or organic pollutants. Addition of pre-grown microbial cultures to enhance the degradation of unwanted compounds (bioaugmentation) and/or injection of nutrients and other supplementary components to the native microbial population to induce propagation at a hastened rate (biostimulation), are the most common approaches for in situ bioremediation of accidental spills and chronically contaminated sites worldwide. However, many factors like strain selection, microbial ecology, type of contaminant, environmental constraints, as well as procedures of culture introduction, may lead to their failure. These drawbacks, along with fragmented literature, have opened a gap between laboratory trials and on-field application. The present review discusses the effectiveness as well as the limitations of bioaugmentation and biostimulation processes. A summary of experimental studies both in confined systems under controlled conditions and of real case studies in the field is presented. A comparative account between the two techniques and also the current scenario worldwide for in situ biotreatment using bioaugmentation and biostimulation, are addressed.     

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.     

Functional Establishment of Introduced Chlorobenzoate Degraders Following Bioaugmentation with Newly Activated Soil

Introduced degraders often do not survive when applied to polluted sites; however, the potential for successful bioaugmentation may be increased if newly activated soil (containing indigenous degrader populations recently exposed to the contaminant) or potentially active soil (containing indigenous degrader populations not previously exposed to the contaminant) is used as the inoculant. To investigate this concept, Madera and Oversite soils were amended with 0 or 500 micrograms of 2-, 3-, or 4-chlorobenzoate per gram soil. The Madera degraded 2-chlorobenzoate while the Oversite degraded 3- and 4-chlorobenzoate. After 22 days of incubation, non-active soils that had not degraded chlorobenzoate were bioaugmented with the appropriate activated soil that had been exposed to and degraded chlorobenzoate. Thus, Oversite soil that had not degraded 2-chlorobenzoate was bioaugmented with Madera soil that had degraded 2-chlorobenzoate. Likewise, Madera soil that had not degraded 3- or 4-chlorobenzoate was bioaugmented with the Oversite soil that had degraded 3- or 4-chlorobenzoate. Additionally, the non-active soils were bioaugmented with the corresponding potentially active soils. The Oversite soil amended with activated Madera soil degraded the 2-chlorobenzoate within 3 days of bioaugmentation. The Madera soil amended with activated Oversite soils degraded the 3- and 4-chlorobenzoate within 20 and 6 days, respectively. Large degrader populations developed in microcosms bioaugmented with activated soil, and shifts in the 3- and 4-CB degrader community structures occurred following bioaugmentation. In contrast, bioaugmentation with potentially active soil did not impact degradation. The results indicate the potential for bioaugmentation with newly activated soil to enhance contaminant degradation.     

Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 micro g/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20 degrees C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7 degrees C,53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7 degrees C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7 degrees C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Initial characterization of new bacteria degrading high-molecular weight polycyclic aromatic hydrocarbons isolated from a 2-year enrichment in a two-liquid-phase culture system

AIMS: To characterize some polycyclic aromatic hydrocarbons (PAH)-degrading microorganisms isolated from an enriched consortium degrading high molecular weight (HMW) PAHs in a two-liquid-phase (TLP) soil slurry bioreactor, and to determine the effect of low molecular weight (LMW) PAH on their growth and HMW PAH-degrading activity. METHODS AND RESULTS: Several microorganisms were isolated from a HMW-PAH (pyrene, chrysene, benzo[a]pyrene and perylene) degrading consortium enriched in TLP cultures using silicone oil as the organic phase. From 16S rRNA analysis, four isolates were identified as Mycobacterium gilvum B1 (99% identity),Bacillus pumilus B44 (99% identity), Microbacterium esteraromaticum B21 (98% identity), and to the genus Porphyrobacter B51 (96% identity). The two latter isolates have not previously been associated with PAH degradation. Isolate B51 grew strongly in the interfacial fraction in the presence of naphthalene vapours and phenanthrene compared with cultures without LMW PAHs. Benzo[a]pyrene was degraded in cultures containing a HMW PAH mixture but pyrene had no effect on its degradation. The growth of isolates B1 and B21 was improved in the aqueous phase than in the interfacial fraction for cultures with naphthalene vapours. Pyrene was required for benzo[a]pyrene degradation by isolate B1. For isolate B21, pyrene and chrysene were degraded only in cultures without naphthalene vapours. CONCLUSION: Consortium enriched in a TLP culture is composed of microorganisms with different abilities to grow at the interface or in the aqueous phase according to the culture conditions and the PAH that are present. Naphthalene vapours increased the growth of the microorganisms in TLP cultures but did not stimulate the HMW PAH degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: New HMW PAH-degrading microorganisms and a better understanding of the mechanisms involved in HMW PAH degradation in TLP cultures.     

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.     

Biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) by Trichoderma reesei FS10-C and Effect of Bioaugmentation on an Aged PAH-Contaminated Soil

ABSTRACT?The co-metabolism of benzo[a]pyrene (B[a]P) and the capacity of the fungus Trichoderma reesei FS10-C to bioremediate an aged polycyclic aromatic hydrocarbon (PAH)-contaminated soil were investigated. The fungal isolate removed about 54% of B[a]P (20 mg L^?1) after 12 days of incubation with glucose (10 g L^?1) supplementation as a co-metabolic substrate. Bioaugmented microcosms showed a 25% decrease in total PAH concentrations in soil after 28 days, and the degradation percentages of 3-, 4-, and 5(+6)-ring PAHs were 36%, 35%, and 25%, respectively. In addition, bioaugmented microcosms exhibited higher dehydrogenase (DHA) and fluorescein diacetate hydrolysis (FDAH) activities and increased average well-color development (AWCD), Shannon-Weaver index (H), and Simpson index (D) significantly. Principal component analysis (PCA) also distinguished clear differentiation between treatments, indicating that bioaugmentation restored the microbiological function of the PAH-contaminated soil. The results suggest that bioaugmentation by T. reesei FS10-C might be a promising bioremediation strategy for aged PAH-contaminated soils.     

Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848     

Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 μg/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20°C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7°C, 53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions, naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7°C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7°C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations, including members of the genera Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Biodegradation of trinitrotoluene (TNT) by indigenous microorganisms from TNT-contaminated soil,and their application in TNT bioremediation

Soil and groundwater contaminated by munitions compounds is a crucial issue in environmental protection. Trinitrotoluene (TNT) is highly toxic and carcinogenic; therefore, the control and remediation of TNT contamination is a critical environmental issue. In this study, the authors characterized the indigenous microbial isolates from a TNT-contaminated site and evaluated their activity in TNT biodegradation. The bacteria Achromobacter sp. BC09 and Citrobacter sp. YC4 isolated from TNT-contaminated soil by enrichment culture with TNT as the sole carbon and nitrogen source (strain BC09) and as the sole nitrogen but not carbon source (strain YC4) were studied for their use in TNT bioremediation. The efficacy of degradation of TNT by indigenous microorganisms in contaminated soil without any modification was insufficient in the laboratory-scale pilot experiments. The addition of strains BC09 and YC4 to the contaminated soil did not significantly accelerate the degradation rate. However, the addition of an additional carbon source (e.g., 0.25% sucrose) could significantly increase the bioremediation efficiency (ca. decrease of 200 ppm for 10 days). Overall, the results suggested that biostimulation was more efficient as compared with bioaugmentation. Nevertheless, the combination of biostimulation and bioaugmentation using these indigenous isolates is still a feasible approach for the development of bioremediation of TNT pollution.     

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

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.     

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.