Microbial community structure changes during bioremediation of PAHs in an aged coal-tar contaminated soil by in-vessel composting

The microbial community structure changes of an aged-coal-tar soil contaminated with polycyclic aromatic hydrocarbons (PAHs) were investigated during simulated bioremediation at the laboratory-scale using an in-vessel composting approach. The composting reactors were operated using a logistic three-factor factorial design with three temperatures (T=38, 55 or 70 °C), four soil to green-waste amendment ratios (S:GW=0.6:1, 0.7:1, 0.8:1 or 0.9:1 on a dry weight basis) and three moisture contents (MC=40%, 60% or 80%). Relative changes in microbial populations were investigated by following the dynamics of phospholipid fatty acid (PLFA) signatures using a ¹³C-labeled palmitic acid internal standard and sensitive GC/MS analysis during in-vessel composting over 98 days. The results of this investigation indicated that fungal to bacterial PLFA ratios were significantly influenced by temperature (p<0.05), and Gram-positive to Gram-negative bacterial ratios were significantly influenced by temperature (p<0.001) and S:GW ratio (p<0.01) during in-vessel composting. Additionally, the Gram-positive to Gram-negative bacterial ratios were correlated to the extent of PAH losses (p<0.005) at 70 °C.     

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.     

Co-composting of PAH-contaminated soil with poultry manure

AIMS: To investigate the effects of co-composting poultry manure with soil contaminated with different concentrations of polycyclic aromatic hydrocarbons (PAHs), on the degradation of selected PAHs in a static-pile compost system. METHODS: Mispah form (Food & Agricultural Organisation, FAO : lithosol) soil contaminated with PAHs was co-composted with poultry manure for 19 months. The soil was mixed with wood chips in a ratio of 1:1 to improve aeration and then mixed with poultry manure in a ratio of 4:1. A data logger measured temperature monthly. Residual concentrations of selected PAHs in the compost were determined monthly by gas chromatography/flame ionisation detection (GC/FID). Moisture, pH, ash content and C:N ratios were also monitored monthly. Microbial activity was measured by measuring CO(2) evolution. CONCLUSIONS: The results obtained in this experiment have shown that co-composting poultry manure with PAH-contaminated soil is capable of removing large concentrations of high molecular weight PAH from contaminated soil to levels below 1 mg kg(-1) in 19 months. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper adds to the body of knowledge necessary for the development of a cost effective technology for the remediation of soil contaminated with high molecular weight PAHs by providing information on the behaviour of selected PAHs and factors such as nutrient ratio, temperature and pH during composting.     

Bioremediation of PAH-contaminated soil with fungi – From laboratory to field scale

The purpose of this study was to develop a fungal bioremediation method that could be used for soils heavily contaminated with persistent organic compounds, such as polyaromatic hydrocarbons (PAHs). Sawmill soil, contaminated with PAHs, was mixed with composted green waste (1:1) and incubated with or without fungal inoculum. The treatments were performed at the laboratory and field scales. In the laboratory scale treatment (starting concentration 3500 mg kg⁻¹, sum of 16 PAH) the high molecular weight PAHs were degraded significantly more in the fungal-inoculated microcosms than in the uninoculated ones. In the microcosms inoculated with Phanerochaete velutina, 96% of 4-ring PAHs and 39% of 5- and 6-ring PAHs were removed in three months. In the uninoculated microcosms, 55% of 4-ring PAHs and only 7% of 5- and 6-ring PAHs were degraded. However, during the field scale (2 t) experiment at lower starting concentration (1400 mg kg⁻¹, sum of 16 PAH) the % degradation was similar in both the P. velutina-inoculated and the uninoculated treatments: 94% of the 16 PAHs were degraded in three months. In the field scale experiment the copy number of gram-positive bacteria PAH-ring hydroxylating dioxygenase genes was found to increase 1000 fold, indicating that bacterial PAH degradation also played an important role.     

Responses of nonstructural carbohydrates to shoot removal and soil moisture treatments in <Emphasis Type="Italic">Salix nigra</Emphasis>

Aboveground disturbances are common in dynamic riparian environments, and Salix nigra is well adapted with a vigorous resprouting response. Soil moisture stresses are also common, and S. nigra is flood tolerant and drought sensitive. The objective of this study was to quantify nonstructural carbohydrate (NSC) reserves in S. nigra following shoot removal and soil moisture treatments. NSC reserves provide energy for regeneration of shoot tissue until new functional leaves are developed. Three soil moisture treatments: well-watered (W), periodic flooding (F) and drought (D); and three shoot removal treatments: no shoots removed (R0), partial shoot removal (R1), and complete shoot removal (R2) were applied. Plants were harvested when new shoot development was observed (day 13). Statistical significance in the 3 × 3-factorial design was determined in two-factor ANOVA at P < 0.05. Both roots and cuttings were important reservoirs for NSC during resprouting response, with decreases in root (31%) and cutting (14%) biomass in R2 compared to R0. Rapid recovery of photosynthetic surface area (from 15 to 37% of R0) was found in R1. A clear pattern of starch mobilization was found in roots in R0, R1 and R2, with lowest root starch concentration in W, F higher than W, and D higher than F. Shoot starch concentration was lower in F and D compared to W in R0, however, in R1 shoot starch was reduced in W compared to F and D, possibly indicating reduced rates of translocation during soil moisture stress. Evidence of osmotic adjustment was found in roots and shoots with higher total ethanol-soluble carbohydrates (TESC) during soil moisture stress in F and D treatments. Total plant NSC pool was greater in F and D treatments compared to W, and progressively reduced from R0 to R1 to R2. Results indicated negative effects of drought, and to a lesser extent periodic flooding on resprouting response in S. nigra, with implications for reduced survival when exposed to combined stresses of aboveground disturbance and soil moisture.     

Optimization of soil physical and chemical conditions for the bioremediation of creosote-contaminated soil

Mispah type soil (FAO : Lithosol) contaminated with >250 000 mg kg^-1 creosote was collected from the yard of a creosote treatment plant. The soils carbon, nitrogen and phosphorus contents were determined. Due to creosote contamination, thecarbon content of the soil was found to be 130,000 mg C kg^-1. This concentration was found to greatly affect the nitrogen content (0.08%). The phosphorus content was less affected (4.5%). It was estimated that a nutrient amendment to bring the soil to a C : N 10 : 1 would be adequate to stimulate microbial growth and creosote degradation. The soil was amended with a range of C : N ratios below and above the estimated ratio. In one of the treatments, the phosphorus content was amended. Sterile and natural controls were also set up. The soil was incubated at 30 °C on a rotaryshaker at 150 rpm in the dark for six weeks. Water content was maintained at 70% field capacity. The lowest nitrogen supplementation (C : N = 25 : 1) was more effective in enhancing microbial growth (3.12E + 05) and creosote removal (68.7%) from the soil. Additional phosphorus was not very effective in enhancing the growth of microorganisms and removal of creosote. The highest nitrogen supplementation(C : N = 5 : 1) did not enhance microbial growth and creosote removal.A relationship between mass loss and creosote removal was also observed. Phenolics and lower molecular mass polycyclic aromatic hydrocarbons (PAHs) were observed to be more susceptible to microbial degradation than higher molecular mass compounds. Nutrient concentration, moisture content and pH were thus observed to play very significant roles in the utilization of creosote in soil. These results are being used for the development of a bioremediation technology for the remediation of creosote contaminated soils in a treatment plant in South Africa.     

Enhanced Phytoremediation of Crude Oil-Polluted Soil by Four Plant Species: Effect of Inorganic and Organic Bioaugumentation

A field experiment investigating the removal and/or uptake of Polycyclic Aromatic Hydrocarbons (PAHs) and specific metals (As, Cd, Cr) from a crude oil polluted agricultural soil was performed during the 2013 wet season using four plant species: Fimbristylis littoralis, Hevea brasilensis (Rubber plants), Cymbopogom citratus (Lemon grass), and Vigna subterranea (Bambara nuts). Soil functional diversity and soil-enzyme interactions were also investigated. The diagnostic ratios and the correlation analysis identified mixed petrogenic and pyrogenic sources as the main contributors of PAHs at the study site. A total of 16 PAHs were identified, 6 of which were carcinogenic. Up to 42.4 mg kg^?1 total PAHs was recorded prior to the experiments. At 90 d, up to 92% total PAH reduction and 96% As removal were achieved using F. littoralis, the best performing species. The organic soil amendment (poultry dung) rendered most of the studied contaminants unavailable for uptake. However, the organic amendment accounted for over 70% of the increased dehydrogenase, phosphatase, and proteolytic enzymes activities in the study. Overall, the combined use of soil amendments and phytoremediation significantly improved the microbial community activity, thus promoting the restoration of the ecosystem.     

Humic substances change during the co-composting process of municipal solid wastes and sewage sludge

The change of the degree of stability of compost during the composting process was a kind of guideline for our study. This stability was estimated by monitoring the chemical fractionation (extraction of humic and fulvic acids, and humin) during two cycles of composting. Change of humin (H), humic-like acid carbon (CHA) and fulvic-like acid carbon (CFA) fractions during the composting process of municipal solid wastes were investigated using two windrows W1 (100% of municipal solid wastes) and W2 (60% of municipal solid wastes and 40% of dried sewage sludge). Humin and fulvic acid fractions in the two windrows decreased since the start of composting process and tend to stabilize. At the end of composting process, humic acid fraction is more important in the windrow without sludge (W1) than the one with sludge (W2). The humification indexes used in this study showed that the humic-like acid carbon fraction production takes place largely during the phase of temperature increase (thermophilic phase), and it appeared very active in the windrow W2. At the end of composting process, the E₄/E₆ ratio value indicated that the compost of W1 is more mature than the compost of W2. The humification ratio (HR) allowed a correct estimation of compost organic matter stabilization level.     

Phosphorus Effects on the Mycelium and Storage Structures of an Arbuscular Mycorrhizal Fungus as Studied in the Soil and Roots by Analysis of Fatty Acid Signatures

The distribution of an arbuscular mycorrhizal (AM) fungus between soil and roots, and between mycelial and storage structures, was studied by use of the fatty acid signature 16:1(omega)5. Increasing the soil phosphorus level resulted in a decrease in the level of the fatty acid 16:1(omega)5 in the soil and roots. A similar decrease was detected by microscopic measurements of root colonization and of the length of AM fungal hyphae in the soil. The fatty acid 16:1(omega)5 was estimated from two types of lipids, phospholipids and neutral lipids, which mainly represent membrane lipids and storage lipids, respectively. The numbers of spores of the AM fungus formed in the soil correlated most closely with neutral lipid fatty acid 16:1(omega)5, whereas the hyphal length in the soil correlated most closely with phospholipid fatty acid 16:1(omega)5. The fungal neutral lipid/phospholipid ratio in the extraradical mycelium was positively correlated with the level of root infection and thus decreased with increasing applications of P. The neutral lipid/phospholipid ratio indicated that at high P levels, less carbon was allocated to storage structures. At all levels of P applied, the major part of the AM fungus was found to be present outside the roots, as estimated from phospholipid fatty acid 16:1(omega)5. The ratio of extraradical biomass/intraradical biomass was not affected by the application of P, except for a decrease at the highest level of P applied.     

Optimization and enhancement of soil bioremediation by composting using the experimental design technique

The objective of this study was the application of the experimental design technique to optimize the conditions for the bioremediation of contaminated soil by means of composting. A low-cost material such as compost from the Organic Fraction of Municipal Solid Waste as amendment and pyrene as model pollutant were used. The effect of three factors was considered: pollutant concentration (0.1–2 g/kg), soil:compost mixing ratio (1:0.5–1:2 w/w) and compost stability measured as respiration index (0.78, 2.69 and 4.52 mg O₂ g⁻¹ Organic Matter h⁻¹). Stable compost permitted to achieve an almost complete degradation of pyrene in a short time (10 days). Results indicated that compost stability is a key parameter to optimize PAHs biodegradation. A factor analysis indicated that the optimal conditions for bioremediation after 10, 20 and 30 days of process were (1.4, 0.78, 1:1.4), (1.4, 2.18. 1:1.3) and (1.3, 2.18, 1:1.3) for concentration (g/kg), compost stability (mg O₂ g⁻¹ Organic Matter h⁻¹) and soil:compost mixing ratio, respectively.     

Enhanced Sono-Fenton-Like Oxidation of PAH-Contaminated Soil Using Nano-Sized Magnetite as Catalyst: Optimization with Response Surface Methodology

In the current study, Fe₃O₄ NPs were synthesized and used as catalysts in a sono-Fenton-like process for remediation of polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The effects of operational variables were studied using central composite design (CCD) optimization approach. Results indicated that the effects of H₂O₂ concentration, Fe₃O₄ NPs dosage, ultrasonic power and pH were significant for pyrene removal as a contaminant model. In optimum experimental conditions, including H₂O₂ concentration of 78 mM, Fe₃O₄ NPs dosage of 18 mM, ultrasonic power of 313 W and pH value of 3.46, the observed pyrene removal was obtained 98.37%, which was verified through the additional experimental tests (99.33%). Pseudo first-order kinetic model was well fitted with the experimental data of pyrene removal with significant coefficient of correlation (R²: 0.9672). Accordingly, an unwashed real soil sample containing diffident PAHs (pyrene, flurene, acenaphthylene, phenenthrene, chrysene, etc) was subjected to sono-Fenton-like process based on optimized conditions. The obtained findings revealed that the removal (%) ranged between 37.7% and 85.19% for different PAHs.     

Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost

AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.     

Biostimulation of micro-organisms from sugarcane bagasse pith for the removal of weathered hydrocarbon from soil

AIMS: In this study we studied the biostimulation of micro-organisms associated with sugarcane bagasse pith for the removal of total petroleum hydrocarbon from a soil contaminated with weathered hydrocarbon. METHODS AND RESULTS: Carbon, nitrogen and phosphorus were added at a ratio of 100 : 10 : 1, water content of 40%, and soil : bagasse ratio of 49 : 1. A significant positive difference (P < 0.05) was observed in total petroleum hydrocarbon removal (38 and 48%) by micro-organisms associated with bagasse and native soil micro-organisms, respectively. In addition, total petroleum hydrocarbon removal increased to 60% in a system where both autochthonous soil and bagasse micro-organisms were present. CONCLUSIONS: Micro-organisms from sugarcane bagasse pith can be stimulated for removal of weathered hydrocarbon from contaminated tropical soils, without they being inhibited by indigenous soil micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil of with hydrocarbons can be diminished by stimulation of autochthonous microflora present in soil and agricultural residues. This work contributes to the microbiology of composting, as low amounts of bulking agents for hydrocarbon removal from soil, can be used.     

Changes in physical,chemical and microbial parameters during the composting of municipal sewage sludge

Changes in physical, chemical and microbial parameters were investigated during the composting of municipal sewage sludge. Raw sewage sludge (30% dry matter) was mixed with compost from sewage sludge (85% dry matter) in 3:1 ratio (v/v). The mixture was divided into 4 windrows which were composted under the same conditions except the turning factor. The turning was every 7, 10, 15 days and according to the temperature which must be (55–65°C) for windrow 1 (W1), windrow 2 (W2), windrow 3 (W3) and windrow 4 (W4), respectively. Water was added to adjust the moisture content (40–60%). The composting process consisted of 2 periods; fermentation (12 weeks) and maturation (4 weeks). The results showed that the temperature reached the maximum after 12 weeks for W1 and 11 weeks for W2, W3 and W4 and then decreased. The final compost was nearly odourless and black, especially in case of W4. The general trend indicates a decrease in organic matter, organic carbon and nitrogen (N), whereas ash, potassium (K) and phosphorus (P) increased and consequently C/K and C/P ratios decreased. There was a slight increase in C/N ratio. The pH increased and then decreased to near neutrality at the end. The mesophilic bacteria increased during the fermentation period and decreased after that, whereas the thermophilic ones increased with increasing of temperature, decreased after 2 weeks and increased again during the fermentation period and then decreased. The mesophilic and thermophilic fungi were present during the first week and disappeared after that. The final compost was pathogens-free as indicated by the counts of coliforms and Salmonella.     

Modelling of parameters for optimization of maturity in composting trimming residues

In this paper, changes in physico-chemical parameters during trimmings residue composting (cation exchange capacity, germination index, self-heated, NH₄/NO₃ ratio and C_FA/C_HA ratio) in relation to environmental composting parameters (time, aeration, moisture and particle size) of the composting process were studied. A central composite experimental design was used to obtain the polynomial model for each dependent variable. Results of the modelling showed that among the studied range, moisture was the highest influenced parameter in maturity evaluation, with respect to aeration and particle size. An exception was found for CEC evolution. In this parameter, the highest influence was found for particle size. Moreover, a product with acceptable chemical properties entails operating at medium moisture content (55%) and medium-to-high particle size (3–5 cm). Moderate to low aeration (0.2 m³ air kg⁻¹ d⁻¹) would be the best compromise to composting this residue, due to the scarce statistical influence of this independent variable.     

The effect of physico-chemically immobilized methylene blue and neutral red on the anode of microbial fuel cell

A fast and cost effective immobilization of electron carriers, methylene blue (MB) and neutral red (NR) by pH shift was proposed to improve bioanodic performance. The adsorption of mediators onto the carbon cloth anode was verified using cyclic voltammogram (CV) and the effect of the immobilized mediators on acclimation, power density, and acetate removal of MFCs was investigated. A peak power density of P _max(MB) = 11.3 W/m³ was achieved over days 110 ∼ 120, as compared to P _max(Control) = 5.4 W/m³ and P _max(NR) = 3.1 W/m³ for the treated anode after 15 sequential fed-batch operations. The VFA removal rates however were similar for all MFC systems, ranging from 82 to 87%. It could be suggested that the increase in power density for the MB treated electrode resulted from an enhanced electron transport from exo-electrogenic bacteria. MB may also have a selective effect on the bacterial community during the start-up stage, increasing the voltage production and acetate removal from day 1 to 16. However, MFC with NR treated anode produced an initial voltage under 100 mV, with lower coulombic efficiency (CE). NR exhibited less favourable mediator molecule binding to the electrode surface, when subject to pH driven physico-chemical immobilization.     

Enhanced lindane removal from soil slurry by immobilized Streptomyces consortium

The aim of this work was to assess lindane removal from soil slurry by a Streptomyces consortium immobilized in cloth sachets, at different inoculum, lindane and slurry concentrations. In concentrated slurry (soil/water ratio of 2:3), the higher lindane removal (35.3 mg Kg⁻¹) was obtained with the medium inoculum (10⁷ CFU g⁻¹) and the highest lindane concentration tested, at 7 days of incubation. Although, lindane removal was also detected in abiotic controls, probably caused by pesticide adsorption to soil particles. Thus, these parameters were selected for evaluating the pesticide removal in diluted slurry (soil/water ratio of 1:4). After 14 days of incubation, 28.7 mg Kg⁻¹ of lindane were removed. Also, a phytotoxicity assay demonstrated that seeds growing on diluted slurries bioremediated during 7 and 14 days, showed an improvement in biological parameters, compared to those growing on non-bioremediated slurries. Thus, bioremediated slurries would not have toxic effects on lettuce seeds.     

Composting of de-inking paper sludge with poultry manure at three nitrogen levels using mechanical turning: behavior of physico-chemical parameters.

De-inking paper sludge (DPS) is rich in carbon (C) but poor in nitrogen (N). Thus, it has a high C:N ratio which limits the composting process. Accordingly, the goal of this study was to investigate the effect of three N treatments on DPS composting. Compost piles of 100 m3 were formed by mixing raw DPS with poultry manure and chicken broiler floor litter, giving on average 0.6%, 0.7% and 0.9% total N. The changes in physico-chemical parameters, total weight and fiber losses, and maturity of composting piles of DPS were monitored during 24 weeks. The compost piles had a neutral to alkaline pH throughout the study. Inorganic N decreased whereas organic N increased over time for all treatments. These changes in magnitude were different among N treatments resulting in a final total N content of 0.9% for the 0.6% N treatment whereas final total N contents of 0.7% and 0.9% N were measured for the 0.7% and 0.9% N treatments. The total weight, cellulose and hemicellulose losses were higher in 0.6% N treatment giving the lowest C:N ratio after 24 weeks of composting. However, none of the 24 week-old composts of DPS were mature based on their final C:N ratio and colorimetric test of maturity. Except for copper, their final total trace element contents meet most known standards or guidelines for organic soil conditioners. Overall, 0.6% N treatment was the best to enhance DPS composting using mechanical turning, but a period of more than 24 weeks was required to reach compost maturity.     

3株多环芳烃高效降解菌株的分离鉴定及降解特性

筛选分离降解多环芳烃(PAHs)的优势菌种对开展多环芳烃污染生态系统修复具有重要的现实意义。本研究以焦化厂周围受多环芳烃污染的土壤为菌源,经过富集培养驯化和平板分离,获得11株能降解多环芳烃的菌株。通过形态观察、生理生化特征及16S rRNA序列比对对菌株进行鉴定,筛选出3株PAHs高效降解菌,分别命名为DJ-3、DJ-8、DJ-10。经16S rRNA序列分析鉴定,DJ-3为假单胞菌属、DJ-8为克雷伯氏菌属、DJ-10为芽孢杆菌属。对菌株降解能力的研究表明,3株菌(DJ-3、DJ-8、DJ-10)培养7 d后对混合多环芳烃中菲(200 mg·L^-1)、芘(200 mg·L^-1)和萘(160 mg·L^-1)的降解率分别为48.9%～65.9%、38.9%～43.1%和57.6%～64.9%。3株菌对多环芳烃混合样品(1200 mg·L^-1)的降解率分别为49.1%、44.5%、53.9%,远高于其他8株筛选菌,为PAHs高效降解菌株。3种菌株两两之间和三者组合均无拮抗关系。研究结果将为构建高效的多环芳烃降解菌群、提高多环芳烃原位污染土壤的生物修复效果奠定基础。     

Application of Biosurfactants Produced by Pseudomonas aeruginosa SP4 for Bioremediation of Soils Contaminated by Pyrene

Biosurfactants are considered to facilitate PAHs dissolution in soil slurries for bioremediation applications. In this work, the carbon and nitrogen sources, pH, C/N ratio, and salinity, were considered for optimization of biosurfactant production by Pseudomonas aeruginosa SP4 isolate to enhance pyrene removal from the contaminated soil. Analysis of ANOVA indicated that the carbon source was the most effective factor, followed by pH, nitrogen source, C/N ratio, and salinity. Taguchi experimental design proposed the optimum operating conditions of olive oil, NH₄NO₃, C/N ratio of 5, salinity of 0.5%, and pH 7. Applying the conditions determined by Taguchi design led to a production yield of 452 mg L^?1 (13% improvement) at the optimum conditions. The main characteristics of produced biosurfactant included the critical micelle concentration (CMC) of 60 mg L^?1 and liquid medium surface tension of 29.5 mN m^?1. Produced biosurfactant was used for bioremediation of soil artificially contaminated with 500 mg kg^?1 of pyrene. Following the addition of 250 mg L^?1 biosurfactant, the pyrene removal of 84.6% was obtained compared to 59.8% for control sample without any surfactant.