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.     

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.     

Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample‐specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)‐reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.     

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.     

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.     

Bioremediation Potential of Rhodococcus pyridinivorans NT2 in Nitrotoluene-Contaminated Soils: The Effectiveness of Natural Attenuation,Biostimulation and Bioaugmentation Approaches

This work evaluated the effect of bioremediation treatments including natural attenuation, bioaugmentation, biostimulation as well as combined biostimulation and bioaugmentation on degradation of 4-nitrotoluene (4-NT), 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in soil microcosms. Bioaugmentation with a previously isolated NTs-degrading bacterium, Rhodococcus pyridinivorans NT2, showed an 86–88% decrease in 4-NT, 2,4-DNT or 2,6-DNT after 60 days. Irrespective of the substrate types, least degradation (6–6.5%) was observed in abiotic control. The addition of β-cyclodextrin or rhamnolipid significantly improved NTs degradation efficiency in soil (18.5–74%) than natural attenuation (22–25%). Exogenous addition of preselected bacterial isolate NT2 along with β-cyclodextrin/rhamnolipid resulted in the greatest number (1.8× and 2.5× high) of total heterotrophic aerobic bacteria and NT degraders, respectively, compared to natural attenuation. Irrespective of the treatment types, the population of NT degraders increased steadily in the first 5 weeks of incubation followed by a plateau within the next few weeks. The treatment BABS2 (Soil + rhamnolipid + NT2) yielded highest microbial-C and -N and dehydrogenase activity, consistent with results of NTs degradation and microbial counts in combined bioaugmentation and biostimulation. Thus the results of this study suggest that bioaugmentation by R. pyridinivorans NT2 may be a promising bioremediation strategy for nitroaromatics-contaminated soils.     

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.     

Kinetic Study of Carbon Dioxide (CO2) Respiration Rate in Bioremediation of Soil Contaminated with Spent Motor Oil

This study was carried out to investigate the kinetics of CO₂ generation from the bioremediation of soil contaminated with spent motor oil (SCSMO) in an aerobic fixed-bed bioreactor designed and fabricated using indigenous technology. Six contaminated soil samples with different compositions at the same contaminant strength were investigated under a controlled air flow rate of 10 L/h. Results obtained revealed that treatment option “6” (i.e., the biostimulation option) gave the best result, with 75% reduction in the initial oil and grease content (O&G) and CO₂ generation of 6,242 mg/kg contaminated soil (CS). Therefore, the biostimulation option was chosen as the treatment technology and kinetic investigation was based on the treatment technology. Results of kinetic investigation of the treatment technology showed that the growth rate can be represented by the popular Monod equation and the kinetic parameters are μ_max = 1.115 × 10^?16 h^?1; K _O&G = 37,490.9 mg/kg; yield of CO₂ = 42.59%; and yield of biomass = 57.41%. The rate equation and kinetic parameters can be used to design a bioreactor and monitor biodegradation reaction during bioremediation process.     

SoilCAM: soil contamination: advanced integrated characterisation and time-lapse monitoring

The SoilCAM project is aimed at improving current methods for monitoring contaminant distribution and biodegradation in the subsurface. Currently proven methods, based on invasive sampling of soil, soil water and gaseous phase, are unable to provide sufficiently accurate data with high enough resolution, resulting in inability to assess bioremediation progress and quantification of the processes involved in such bioremediation at field sites. Consequently, present assessment strategies to decide on optimal remediation approach, including design of monitoring systems, and evaluation of degradation progress, are severely flawed by uncertainty. Geophysical time-lapse measurements in combination with novel ground truthing methods give the possibility to determine contamination levels and their spatial spreading in a heterogeneous environment. Geophysical methods of data acquisition alone are presently unable to provide absolute levels of biodegradable contamination concentrations. We aim to test and develop fundamental constitutive relations between soil physical and degradation activity parameters and geophysically measurable parameters. Despite current improvements, there is a strong need to test these theories in practical field situations. The SoilCAM project is dedicated to improving both site contamination assessment and the monitoring of bioremediation processes, and changes in soil environmental conditions. We suggest combining improved conventional soil monitoring techniques with state-of-the-art geophysical approaches and modelling. Two European sites with mobile contaminants in a highly permeable subsurface are central in the project. Focus on practical field situations and strong communication with stake-holders and SMEs will ensure high relevance for society. This article is a research project funded under FP7 .     

Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation

Bioremediation of diesel oil in soil can occur by natural attenuation, or treated by biostimulation or bioaugmentation. In this study we evaluated all three technologies on the degradation of total petroleum hydrocarbons (TPH) in soil. In addition, the number of diesel-degrading microorganisms present and microbial activity as indexed by the dehydrogenase assay were monitored. Soils contaminated with diesel oil in the field were collected from Long Beach, California, USA and Hong Kong, China. After 12 weeks of incubation, all three treatments showed differing effects on the degradation of light (C12-C23) and heavy (C23-C40) fractions of TPH in the soil samples. Bioaugmentation of the Long Beach soil showed the greatest degradation in the light (72.7%) and heavy (75.2%) fractions of TPH. Natural attenuation was more effective than biostimulation (addition of nutrients), most notably in the Hong Kong soil. The greatest microbial activity (dehydrogenase activity) was observed with bioaugmentation of the Long Beach soil (3.3-fold) and upon natural attenuation of the Hong Kong sample (4.0-fold). The number of diesel-degrading microorganisms and heterotrophic population was not influenced by the bioremediation treatments. Soil properties and the indigenous soil microbial population affect the degree of biodegradation; hence detailed site specific characterization studies are needed prior to deciding on the proper bioremediation method.     

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 creosote-contaminated soil in South Africa by landfarming

AIMS: To determine the combined effects of biostimulation and bioaugmentation in the landfarming of a mispah form (lithosol; food and Agriculture Organisation (FAO)) soil contaminated with >310000 mg kg-1 creosote with a view to developing a bioremediation technology for soils heavily contaminated with creosote. METHODS AND RESULTS: The excavated soil was mixed with 2500 kg ha-1 dolomitic lime and 2000 kg ha-1 mono-ammonium phosphate (MAP) before spreading over a treatment bed of shale reinforced with clay. Sewage sludge (500 kg) was ploughed into 450 m3 of contaminated soil in the second and sixth months of treatment. A further 1000 kg ha-1 MAP was added to the soil at the end of the fifth month. Moisture was maintained at 70% field capacity. Total creosote was determined by the US Environmental Protection Agency (EPA) method 418.1 and concentrations of selected creosote components were determined by gas chromatography/flame ionisation detection (GC/FID). Total creosote was reduced by more than 90% by the 10th month of landfarming. The rate of reduction in creosote concentration was highest after the addition of sewage sludge. The three-ring PAHs were more slowly removed than naphthalene and the phenolic compounds. The four- and five-ring PAHs, although persist until the end of treatment, were reduced by 76-87% at the end of the experiment. CONCLUSIONS: A combination of biostimulation and bioaugmentation during landfarming could enhance the bioremediation of soils heavily contaminated with creosote. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides information on the management of a combination of biostimulation and bioaugmentation during landfarming, and contributes to the knowledge and database necessary for the development of a technology for bioremediating creosote-contaminated land.     

Aerobic bioremediation of chlorobenzene source-zone soil in flow-through columns: performance assessment using quantitative PCR

Flow-through aquifer columns were operated for 12 weeks to evaluate the benefits of aerobic biostimulation for the bioremediation of source-zone soil contaminated with chlorobenzenes (CBs). Quantitative Polymerase Chain Reaction (qPCR) was used to measure the concentration of total bacteria (16S rRNA gene) and oxygenase genes involved in the biodegradation of aromatic compounds (i.e., toluene dioxygenase, ring hydroxylating monooxygenase, naphthalene dioxygenase, phenol hydroxylase, and biphenyl dioxygenase). Monochlorobenzene, which is much more soluble than dichlorobenzenes, was primarily removed by flushing, and biostimulation showed little benefit. In contrast, dichlorobenzene removal was primarily due to biodegradation, and the removal efficiency was much higher in oxygen-amended columns compared to a control column. To our knowledge, this is the first report that oxygen addition can enhance CB source-zone soil bioremediation. Analysis by qPCR showed that whereas the biphenyl and toluene dioxygenase biomarkers were most abundant, increases in the concentration of the phenol hydroxylase gene reflected best the higher dichlorobenzene removal due to aerobic biostimulation. This suggests that quantitative molecular microbial ecology techniques could be useful to assess CB source-zone bioremediation performance.     

Bioremediation technologies for treatment of PAH-contaminated soil and strategies to enhance process efficiency

The complex and diverse structural configurations of polycyclic aromatic hydrocarbons (PAHs), combined with their low bioavailability, hydrophobic nature, strong sorption phenomena, and high persistence in soil makes the design of effective bioremediation methodologies a challenge. The multi-phasic nature of the bioremediation process, restricted mass transfer and non-availability of degrading soil microflora further compound the problem. In this direction, this communication presents a focused review of bioremediation technologies used recently for the treatment of PAH-contaminated soils. The specific roles of important factors affecting bioremediation process efficiency are discussed. Finally some of the recently used strategies to enhance bioremediation process efficiency, including bioaugmentation, biostimulation, rhizoremediation, the use of chemotaxins, the biomimetic catalytic system approach, and integrated techniques, are reviewed.     

Contributions of biosurfactants to natural or induced bioremediation

The number of studies dedicated to evaluating the influence of biosurfactants on bioremediation efficiency is constantly growing. Although significant progress regarding the explanation of mechanisms behind biosurfactant-induced effects could be observed, there are still many factors which are not sufficiently elucidated. This corresponds to the fact that although positive influence of biosurfactants is often reported, there are also numerous cases where no or negative effect was observed. This review summarizes the recent finding in the field of biosurfactant-amended bioremediation, focusing mainly on a critical approach towards potential limitations and causes of failure while investigating the effects of biosurfactants on the efficiency of biodegradation and phytoextraction processes. It also provides a summary of successive steps, which should be taken into consideration when designing biosurfactant-related treatment processes.     

Changes in the bacterial community and lin genes diversity during biostimulation of indigenous bacterial community of hexachlorocyclohexane (HCH) dumpsite soil

In this study hexachlorocyclohexane (HCH) contaminated soil (with HCH level 84 g/kg of soil) from HCH dumpsite (Ummari village, Lucknow, India) was used to demonstrate biostimulation approach for HCH bioremediation. Different nutrients (molasses and ammonium phosphate) were used in different pits having contaminated soil to stimulate the indigenous microbial community. There was a substantial reduction in the total HCH content of the soil in 12 months long experiment. Maximum reduction was seen in the pit that received a combination of molasses and ammonium phosphate. A change in the microbial community concomitant with degradation of HCH was observed. Sphingomonads, which are known degraders of HCH, were found to dominate the experimental pits. Moreover changes in linA and linB gene (primary genes involved in HCH degradation) diversity and number were also seen as revealed by T-RFLP and RT-PCR respectively. The study suggests the prospects of biostimulation in decontaminating soils heavily contaminated with HCH.     

Solar power enhancement of electrokinetic bioremediation of phenanthrene by Mycobacterium pallens

Enhanced bioremediation of phenanthrene-contaminated soil with Mycobacterium pallens was conducted. Kaolinite was used in the tests as a soil matrix and was artificially contaminated with phenanthrene at a concentration of 2 mg phenanthrene per gram dry soil. Mycobacterim pallens at concentration of 10⁸ colony-forming units (CFU) per milliliter was used as a potential microorganism to degrade phenanthrene. Aspects of the study included evaluating efficacy of using Mycobacterium pallens for degrading phenanthrene, electrokinetics for delivering nutrients and microorganisms to contaminated soil, and solar panels for generating power for electrokinetic bioremediation. A novel anode-cathode configuration, in which the anode and cathode are placed in the same compartment, was implemented to control/minimize changes in pH during electrokinetic bioremediation. The nutrients (NO₃^?), electrical current, temperature, Mycobacterium pallens (CFU), and phenatherene concentration were evaluated. The results showed that solar panels generated sufficient power for electrokinetic bioremediation. The highest current obtained was generated when bacteria and nutrients were added to the soil. This was associated with the highest phenanthrene removal from the soil (50% of the initial concentration). Additionally, we determined that the novel anode-cathode configuration in the electrokinetic bioremediation cell was successful in delivering the bacteria and nutrients to the contaminated soil and in maintaining a relatively neutral pH around the electrode compartments, which improved the remediation. Overall, this study showed that the use of solar power with electrokinetic bioremediation can provide a cost-effective approach to reduce and remove hydrocarbon contaminations in soil.     

Comparative Mesocosm Study of Biostimulation Efficiency in Two Different Oil-Amended Sub-Antarctic Soils

Biological treatment has become increasingly popular as a remediation method for soils and groundwater contaminated with petroleum hydrocarbon, chlorinated solvents, and pesticides. Bioremediation has been considered for application in cold regions such as Arctic and sub-Arctic climates and Antarctica. Studies to date suggest that indigenous microbes suitable for bioremediation exist in soils in these regions. This paper reports on two case studies at the sub-Antarctic Kerguelen Island in which indigenous bacteria were found that were capable of mineralizing petroleum hydrocarbons in soil contaminated with crude oil and diesel fuel. All results demonstrate a serious influence of the soil properties on the biostimulation efficiency. Both temperature elevation and fertilizer addition have a more significant impact on the microbial assemblages in the mineral soil than in the organic one. Analysis of the hydrocarbons remaining at the end of the experiments confirmed the bacterial observations. Optimum temperature seems to be around 10 degrees C in organic soil, whereas it was higher in mineral soil. The benefit of adding nutrients was much stronger in mineral than in the organic soil. Overall, this study suggests that biostimulation treatments were driven by soil properties and that ex situ bioremediation for treatment of cold contaminated soils will allow greater control over soil temperature, a limiting factor in cold climates.     

Chemical and microbial community analysis during aerobic biostimulation assays of non-sulfonated alkyl-benzene-contaminated groundwater

A chemical and microbial characterization of lab-scale biostimulation assays with groundwater samples taken from an industrial site in which the aquifer had been contaminated by linear non-sulfonate alkyl benzenes (LABs) was carried out for further field-scale bioremediation purposes. Two lab-scale biodegradability assays were performed, one with a previously obtained gas-oil-degrading consortium and another with the native groundwater flora. Results for the characterization of the groundwater microbial population of the site revealed the presence of an important LAB-degrading microbial population with a strong degrading capacity. Among the microorganisms identified at the site, the detection of Parvibaculum lavamentivorans, which have been described in other studies as alkyl benzene sulfonates degraders, is worth mentioning. Incubation of P. lavamentivorans DSMZ13023 with LABs as reported in this study shows for the first time the metabolic capacity of this strain to degrade such compounds. Results from the biodegradation assays in this study showed that the indigenous microbial population had a higher degrading capacity than the gas-oil-degrading consortium, indicating the strong ability of the native community to adapt to the presence of LABs. The addition of inorganic nutrients significantly improved the aerobic biodegradation rate, achieving levels of biodegradation close to 90%. The results of this study show the potential effectiveness of oxygen and nutrients as in situ biostimulation agents as well as the existence of a complex microbial community that encompasses well-known hydrocarbon- and LAS-degrading microbial populations in the aquifer studied.