Effect of a commercial alcohol ethoxylate surfactant (C<Subscript>11-15</Subscript>E<Subscript>7</Subscript>) on

Biodegradation of poorly soluble polycyclic aromatic hydrocarbons (PAHs) has been a challenge in bioremediation. In recent years, surfactant-enhanced bioremediation of PAH contaminants has attracted great attention in research. In this study, biodegradation of phenanthrene as a model PAHs solubilized in saline micellar solutions of a biodegradable commercial alcohol ethoxylate nonionic surfactant was investigated. The critical micelle concentration (CMC) of the surfactant and its solubilization capacity for phenanthrene were examined in an artificial saline water medium, and a type of marine bacteria, Neptunomonas naphthovorans, was studied for the biodegradation of phenanthrene solubilized in the surfactant micellar solutions of the saline medium. It is found that the solubility of phenanthrene in the surfactant micellar solutions increased linearly with the surfactant concentrations, but, at a fixed phenanthrene concentration, the biodegradability of phenanthrene in the micellar solutions decreased with the increase of the surfactant concentrations. This was attributed to the reduced bioavailability of phenanthrene, due to its increased solubilization extent in the micellar phase and possibly lowered mass transfer rate from the micellar phase into the aqueous phase or into the bacterial cells. In addition, an inhibitory effect of the surfactant on the bacterial growth at high surfactant concentrations may also play a role. It is concluded that the surfactant largely enhanced the solubilization of phenanthrene in the saline water medium, but excess existence of the surfactant in the medium should be minimized or avoided for the biodegradation of phenanthrene by Neptunomonas naphthovorans.     

Quantifying the biodegradation of phenanthrene by Pseudomonas stutzeri P16 in the presence of a nonionic surfactant.

The low water solubility of polycyclic aromatic hydrocarbons is believed to limit their availability to microorganisms, which is a potential problem for bioremediation of polycyclic aromatic hydrocarbon-contaminated sites. Surfactants have been suggested to enhance the bioavailability of hydrophobic compounds, but both negative and positive effects of surfactants on biodegradation have been reported in the literature. Earlier, we presented mechanistic models of the effects of surfactants on phenanthrene dissolution and on the biodegradation kinetics of phenanthrene solubilized in surfactant micelles. In this study, we combined the biodegradation and dissolution models to quantify the influence of the surfactant Tergitol NP-10 on biodegradation of solid-phase phenanthrene by Pseudomonas stutzeri P16. Although micellized phenanthrene does not appear to be available directly to the bacterium, the ability of the surfactant to increase the phenanthrene dissolution rate resulted in an overall increase in bacterial growth rate in the presence of the surfactant. Experimental observations could be predicted well by the derived model with measured biokinetic and dissolution parameters. The proposed model therefore can serve as a base case for understanding the physical-chemical effects of surfactants on nonaqueous hydrocarbon bioavailability.     

Enhanced biodegradation of phenanthrene by a marine bacterium in presence of a synthetic surfactant

The biodegradation of phenanthrene by the marine strain Sphingomonas sp. 2MPII (DSMZ 11572) was enhanced by the solubilizating properties of the nonionic surfactant Tween 80. After 197 h of incubation, 85 +/- 4% of the initial amount of phenanthrene (0.4 g l-1) was biodegraded in presence of Tween 80 (0.5 g l-1) as opposed to 52 +/- 5% without this synthetic surfactant. These results confirm that the activity of the strain 2MPII is limited by the bioavailability of the polycyclic aromatic hydrocarbon (PAH) substrate in the aqueous phase. Tween 80 appears to be efficient in increasing the bioavailability of hydrophobic compounds such as PAHs.     

Effect of calcium on the surfactant tolerance of a fluoranthene degrading bacterium

Surfactants are known to increase the apparent aqueous solubility of polycyclic aromatic hydrocarbons (PAHs) and may thus be used to enhance the bioavailability and thereby to stimulate the biodegradation of these hydrophobic compounds. However, surfactants may in some cases reduce or inhibit biodegradation because of toxicity to the bacteria. In this study, toxicity of surfactants on Sphingomonas paucimobilis strain EPA505 and the effect on fluoranthene mineralization were investigated using Triton X-100 as model surfactant. The data showed that amendment with 0.48 mM (0.3 g l-1) of Triton X-100 completely inhibited fluoranthene and glucose mineralization and reduced cell culturability by 100% in 24 h. Electron micrographs indicate that Triton X-100 adversely affects the functioning of the cytoplasmic membrane. However, in the presence of 4.13 mM Ca2+-ions, Triton X-100 more than doubled the maximum fluoranthene mineralization rate and cell culturability was reduced by only 10%. In liquid cultures divalent ions, Ca2+ in particular and Mg2+ to a lesser extent, were thus shown to be essential for the surfactant-enhanced biodegradation of fluoranthene. Most likely the Ca2+-ions stabilized the cell membrane, making the cell less sensitive to Triton X-100. This is the first report on a specific factor which is important for successful surfactant-enhanced biodegradation of PAHs.     

Biodegradation of Phenanthrene by Pseudomonas putida and a Bacterial Consortium in the Presence and in the Absence of a Surfactant

This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P. putida degraded phenanthrene to form 1-hydroxy-2-naphthoic acid (1H2Na) as the major metabolite. LC–MS analysis revealed the production of complementary intermediates in the presence of Brij 30, showing intense ions at mass-to-charge ratios (m/z) 97 and 195. Higher phenanthrene biodegradation rate was obtained in the presence of Brij 30. Conversely, in the case of Pyr01consortium, the addition of Brij 30 (0.5 g L⁻¹) had a negative effect on biodegradation: no phenanthrene biodegradation products were detected in the medium, whereas a production of several intermediates (m/z 97, 195 and 293) was obtained without surfactant. New results on phenanthrene metabolism by P. putida DSMZ 8368 and Pyr01 consortium in the presence and in the absence of Brij 30 we obtained. They confirm that the knowledge of the effect of a surfactant on bacterial cultures is crucial for the optimization of surfactant-enhanced PAHs biodegradation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-012-0265-z) contains supplementary material, which is available to authorized users.     

Influence of a nonaqueous phase liquid (NAPL) on biodegradation of phenanthrene

A series of batch reactor experiments was carried out to examine the effect of a nonaqueous phase liquid (NAPL) on the biodegradation of a hydrophobic solute. A mathematical program model that describes physical processes of solute solubilization and partitioning between the NAPL and aqueous phases as well as microbial degradation and oxygen utilization was used to analyze the test data. The model calculates the cumulative changes in concentration of substrate, cell mass, carbon dioxide, and dissolved oxygen as a function of time. The equations incorporate the effects of solute solubilization, partitioning, biodegradation, as well as oxygen availability. Hexadecane was used as the model NAPL and was not biodegraded in the timeframe of the experiments performed. The model solute was the polyaromatic hydrocarbon, phenanthrene. In agreement with several previous studies, experimental measurements showed that hexadecane increased rates of mineralization of 15 mg phenanthrene when present at low mass but decreased rates at high mass. Model results suggest that partitioning of the phenanthrene into the hexadecane phase limits bioavailability at high NAPL mass. Further the model suggests that mineralization rates were higher with the low NAPL mass because aqueous phenanthrene concentrations were higher in those treatments from ca. 20 to 40 h than in other treatments. Finally, experiments showed that the presence of hexadecane, at all masses tested, resulted in a lower cell yield, effectively increasing the amount of CO₂ produced during the experiment. Model results suggest that this is due to changes in phenanthrene metabolism that are induced by the presence of the hexadecane phase. Model studies aimed at increasing rates of biodegradation by modifying operating conditions are described along with practical approaches to implementing these modifications.     

Biodegradation of aged diesel in diverse soil matrixes: impact of environmental conditions and bioavailability on microbial remediation capacity

While bioremediation of total petroleum hydrocarbons (TPH) is in general a robust technique, heterogeneity in terms of contaminant and environmental characteristics can impact the extent of biodegradation. The current study investigates the implications of different soil matrix types (anthropogenic fill layer, peat, clay, and sand) and bioavailability on bioremediation of an aged diesel contamination from a heterogeneous site. In addition to an uncontaminated sample for each soil type, samples representing two levels of contamination (high and low) were also used; initial TPH concentrations varied between 1.6 and 26.6 g TPH/kg and bioavailability between 36 and 100 %. While significant biodegradation occurred during 100 days of incubation under biostimulating conditions (64.4–100 % remediation efficiency), low bioavailability restricted full biodegradation, yielding a residual TPH concentration. Respiration levels, as well as the abundance of alkB, encoding mono-oxygenases pivotal for hydrocarbon metabolism, were positively correlated with TPH degradation, demonstrating their usefulness as a proxy for hydrocarbon biodegradation. However, absolute respiration and alkB presence were dependent on soil matrix type, indicating the sensitivity of results to initial environmental conditions. Through investigating biodegradation potential across a heterogeneous site, this research illuminates the interplay between soil matrix type, bioavailability, and bioremediation and the implications of these parameters for the effectiveness of an in situ treatment.     

Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant).

A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.     

Microbial Factors Rather Than Bioavailability Limit the Rate and Extent of PAH Biodegradation in Aged Crude Oil Contaminated Model Soils

The rate and extent of polynuclear aromatic hydrocarbons (PAH) biodegradation in a set of aged model soils that had been contaminated with crude oil at the high concentrations (i.e.,>20,000?mg/kg) normally found in the environment were measured in 90-week slurry bioremediation experiments. Soil properties such as organic matter content, mineral type, particle diameter, surface area, and porosity did not significantly influence the PAH biodegradation kinetics among the 10 different model soils. A comparison of aged and freshly spiked soils indicates that aging affects the biodegradation rate and extent only for higher-molecular-weight PAHs, while the effects of aging are insignificant for 4-ring PAHs and total PAHs. In all model soils with the exception of kaolinite clay, the rate of abiotic desorption was faster than the rate of biodegradation during the initial phase of bioremediation treatment, indicating that PAH biodegradation was limited by microbial factors. Similarly, any of the higher-molecular-weight PAHs that were still present after 90 weeks of treatment were released rapidly during abiotic desorption tests, which demonstrates that bioavailability limitations were not responsible for the recalcitrance of these hydrocarbons. Indeed, an analysis of microbial counts indicates that a severe reduction in hydrocarbon degrader populations may be responsible for the observed incomplete PAH biodegradation. Therefore, it can be concluded that the recalcitrance of PAHs during bioremediation is not necessarily due to bioavailability limitations and that these residual contaminants therefore might pose a greater risk to environmental receptors than previously thought.     

Optimizing biodegradation of phenanthrene dissolved in nonaqueous-phase liquids

 A study was conducted to optimize the biodegradation in soil slurries of phenanthrene initially dissolved in nonaqueous-phase liquids (NAPLs). The slow rate of degradation of phenanthrene in dibutyl phthalate was increased by addition of phenanthrene-degrading microorganisms to soil slurries containing the NAPL. The rate was further increased and the acclimation phase was shortened if the inoculum was grown in a medium containing the hydrocarbon and the phthalate before addition to the slurries. Composition of the growth medium only shortened the acclimation but had no effect on the rate. Vigorous agitation increased the rate and extent of mineralization of phenanthrene in dibutyl phthalate. The effect of temperature was affected by the presence and identity of the inoculum. Rapid and extensive mineralization of phenanthrene initially present in hexadecane and diesel oil were attained by use of intense agitation of the NAPL/soil slurry and inoculation with microorganisms grown in the presence of the NAPLs, but the influence of these variables was less with other NAPLs. Vigorous agitation and addition of an inoculum 24 h after introduction of a nonionic surfactant enhanced biodegradation of phenanthrene initially in 150 Bright stock oil and dibutyl phthalate. The results suggest improved means for the bioremediation of sites contaminated with NAPLs. Received: 17 May 1995/Received revision: 1 August 1995/Accepted: 22 August 1995     

Modification of PAHs Biodegradation with Humic Compounds

The effect of extractable humic substances (EHS) on the bioremediation of phenanthrene in a slurry phase was investigated using adapted microorganisms with polycyclic aromatic hydrocarbons (PAHs). Two concentrations of EHS were used: 150 and 30 mg/kg soil. The phenanthrene concentration was 500 mg/kg soil. The results showed that the trend of biodegradation was increased after four weeks retardation. These tests showed that humic compounds could overcome the bond between the soil and phenanthrene in the presence of the bacterial consortium. The bacterial density in the medium with EHS was about six-fold greater in magnitude than in the medium without the humic compounds. The chemical relationship between phenanthrene and the humic substances in the form of a phenanthrene-humic-soil complex or phenanthrene-humic is loosely associated and reversible. Therefore, after the initial inhibition by humic substances, the bioavailability of phenanthrene increases.     

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.     

Biodegradation and bioremediation of hydrocarbons in extreme environments

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure, Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.     

Basic examinations on chemical pre-oxidation by ozone for enhancing bioremediation of phenanthrene contaminated soils

Biological treatment of polycyclic aromatic hydrocarbons (PAH) has been demonstrated to be a feasible and common remediation technology which has been successfully applied to the clean-up of contaminated soils. Because bioavailability of the contaminants is of great importance for a successful bioremediation, a chemical pre-oxidation step by ozone was tested to enhance the subsequent biodegradation steps. Oxidation of PAH by ozone should result in reaction products that have a better solubility in water and thus a better bioavailability. A major part of this work was done by examinations of the model substance phenanthrene as a typical compound of PAH. After initial ozonation of phenanthrene, analysis by GC-MS showed at least seven identified conversion-products of phenanthrene. In comparison with phenanthrene these conversion products were more efficiently biodegraded by Sphingomonas yanoikuyae or mixed cultures when the ozonation process resulted in monoaromatic compounds. Primary ozonation products with biphenylic structures were found not to be biodegradable. Investigations into the toxicity of contaminated and ozonated soils were carried out by well-established toxicity assays using Bacillus subtilis and garden cress. The ozonated soils surprisingly showed higher toxic or inhibitory effects towards different organisms than the phenanthrene or PAH itself. The microbial degradation of phenanthrene in slurry reactors by S. yanoikuyae was not enhanced significantly by preozonation of the contaminated soil.     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

Determination of phenanthrene bioavailability by using a self-dying reporter bacterium: test with model solids and soil

The present study was conducted to investigate the performance and feasibility of a self-dying reporter bacterium to visualize and quantify phenanthrene bioavailability in soil. The self-dying reporter bacterium was designed to die on the initiation of phenanthrene biodegradation. The viability of the reporter bacterium was determined by a fluorescence live/dead cell staining method and visualized by confocal laser scanning microscopic observation. Phenanthrene was spiked into four types of model solids and a sandy loam. The bioavailability of phenanthrene to the reporter bacterium was remarkably declined with the hydrophobicity of the model solids: essentially no phenanthrene was biodegraded in the presence of 9-nm pores and about 35.8% of initial phenanthrene was biodegraded without pores. Decrease in bioavailability was not evident in the nonporous hydrophilic bead, but a small decrease was observed in the porous hydrophilic bead at 1000 mg/kg of phenanthrene. The fluorescence intensity was commensurate with the extent of phenanthrene biodegradation by the reporter bacterium at the concentration range from 50 to 500 mg/kg. Such a quantitative relationship was also confirmed with a sandy loam spiked up to 1000 mg/kg of phenanthrene. This reporter bacterium may be a useful means to determine phenanthrene bioavailability in soil.     

Effect of surfactant on the estimation by solid phase microextraction of bioavailable pyrene in soil samples

This study was focused on the effect of the presence of surfactant on the bioremediation efficacy and sensitivity of solid phase microextraction (SPME) in the pyrene-contaminated soil. Soils with 1.3 and 7.6% soil organic matter (SOM) were tested for biodegradation by microorganisms and extracted by aqueous solutions of the matrix used for SPME. For the biodegradation test, the presence of Triton X-100 at 5× CMC (critical micelle concentration) significantly enhanced pyrene removal for soil with lower SOM content (1.3%). However, this removal was insignificant for soil with higher SOM content (7.6%). The results may suggest that 5× CMC was not sufficient to improve significantly pyrene desorption for soil with higher SOM content. For the bioavailability test, in the absence of Triton X-100, SPME estimation of bioavailability in soils with indigenous or seeded microorganisms had an error range within 15%. However, with addition of Triton X-100, SPME estimations showed a significant decline (41 and 77%), in relation to their predicted values, for soil samples with SOM of 1.3 and 7.6%, respectively. The main reason for this underestimation is that micelle formation from the application of surfactant impacted the concentration of dissolved pyrene, rather than competitive site occupation between pyrene and surfactant molecules for SPME fiber. Thus, if soil samples contain surfactant, SPME would significantly underestimate bioavailability and risk level of PAH-contaminated sites.     

Release Of Petroleum Hydrocarbons From Bioremediated Soils

This article presents a qualitative evaluation of the extent to which the bioavailability (release) of a chemical is related to the biodegradation of hydrocarbons in a field bioremediation unit. The objectives of this research were to (1) quantify the rate of release of petroleum hydrocarbons from two soils that were bioremediated, (2) explore hydrocarbon release as a process affecting bioremediation; and (3) investigate the impact of bioremediation on chemical release in the two soils. An experimental protocol was used to quantify the rate of release of these hydrocarbons from two soils that had been bioremediated in a field-scale prepared bed land treatment unit. One soil showed little change in hydrocarbon concentration during 55 weeks of prepared bed bioremediation. The field study results indicated that, prior to the bioremediation, this soil had reached an environmentally acceptable endpoint. The second soil showed considerable hydrocarbon loss as a result of the bioremediation. The rate of hydrocarbon release was determined for the first soil and for the second soil at time zero and after 1, 2, and 7 months of prepared bed bioremediation. The results indicated: (1) the fraction (F) of the specific hydrocarbons that were released rapidly from the soil and the rates of release (k₂) of the residual hydrocarbons that were released slowly, (2) that the mass of each chemical of concern that was released from the first soil was very low; and (3) that the hydrocarbon released rapidly from the second soil decreased as treatment progressed. The experiments also verified, qualitatively, that some portion of each chemical evaluated was not able to be released, and thus was unavailable for bioremediation in the prepared bed land treatment unit.