Use of surfactants and slurrying to enhance the biodegradation in soil of compounds initially dissolved in nonaqueous-phase liquids

A study was conducted to find means of enhancing the biodegradation of hydrophobic organic compounds in nonaqueous-phase liquids (NAPLs). The effects of surfactants, identity of the NAPL and agitation was investigated. When present in NAPLs, phenanthrene, di-(2-ethylhexyl) phthalate (DEHP) and biphenyl were mineralized slowly in soil. Addition of Triton X-100 or Alfonic 810-60 did not enhance the degradation of phenanthrene initially in hexadecane or dibutyl phthalate. Slurrying the soil increased the rate and extent of mineralization of phenanthrene initially in hexadecane but not in dibutyl phthalate. Addition of either of the two surfactants to the slurries did not promote the transformation. Triton X-100, Alfonic 810-60 and Tergitol 15-S-9 below their critical micelle concentrations increased the rate and sometimes the extent of mineralization in soil slurries of phenanthrene initially in 2,2,4,4,6,8,8-heptamethylnonane, but other surfactants were not stimulatory. Slurrying the soil promoted the initial mineralization of DEHP initially in dibutyl phthalate, and Alfonic 810-60 and Triton X-100 further stimulated the rate and extent of degradation in the slurries. Alfonic 810-60 increased the extent of mineralization in slurries of biphenyl in hexadecane but not in dibutyl phthalate, cyclohexane, kerosene or two oils. Little mineralization of biphenyl or DEHP initially in dibutyl phthalate occurred in soil slurries, but Tween 80, Tergitol 15-S-40 and Tergitol 15-S-9 increased the extent of mineralization. However, vigorous agitation of the slurries of soil acclimated to DEHP or the use of small volumes of the NAPL resulted in marked enhancement of the degradation. Thus, biodegradation of constituents of NAPLs in soil can be increased by the use of some surfactants, slurrying or intense agitation, but the effect will vary with the NAPL and the constituents.     

Bioavailability of solid and non-aqueous phase liquid (NAPL)-dissolved phenanthrene to the biosurfactant-producing bacterium Pseudomonas aeruginosa 19SJ

The biodegradation of phenanthrene by the biosurfactant-producing strain Pseudomonas aeruginosa 19SJ was investigated in experiments with the compound present either as crystals or dissolved in non-aqueous phase liquids (NAPLs). Growth on solid phenanthrene exhibited an initial phase not limited by dissolution rate and a subsequent, carbon-limited phase caused by exhaustion of the carbon source. Rhamnolipid biosurfactants were produced from solid phenanthrene and appeared in solution and particulate material (cells and phenanthrene crystals). During the carbon-limited phase, the concentration of rhamnolipids detected in culture exceeded the critical micelle concentration (CMC) determined with purified rhamnolipids. The biosurfactants caused a significant increase in dissolution rate and pseudosolubility of phenanthrene, but only at concentrations above the CMC. Externally added rhamnolipids at a concentration higher than the CMC increased the biodegradation rate of solid phenanthrene. Mineralization curves of low concentrations of phenanthrene initially dissolved in two NAPLs [2,2,4,4,6,8,8-heptamethylnonane and di(2-ethylhexyl)phthalate] were S-shaped, although no growth was observed in the population of suspended bacteria. Biosurfactants were not detected in solution under these conditions. The observed mineralization was attributed not only to suspended bacteria, but also to bacterial populations growing at the NAPL–water interface, mineralizing the compound at higher rates than predicted by abiotic partitioning. We suggest that rhamnolipid production and attachment increased the bioavailability of phenanthrene, so promoting biodegradation activity.     

Effect of inorganic nutrients on the acclimation period preceding mineralization of organic chemicals in lake water.

The addition of phosphate, nitrate, or sulfate (each at 10 mM) decreased the acclimation period for the mineralization of low concentrations of p-nitrophenol (PNP) in lake water. Added phosphate shortened the acclimation period for biodegradation of 2 ng to 2 micrograms of PNP per ml in various lake water samples and of 2,4-dichlorophenoxyacetate at 100 ng/ml. Added P enhanced the rate of growth of PNP-mineralizing microorganisms in waters containing 200 ng or 2 micrograms of PNP per ml. We suggest that the effect of P on the acclimation period results from an increase in the growth rate of the initially small population of microorganisms able to mineralize the synthetic chemicals.     

Effect of inorganic nutrients on the acclimation period preceding mineralization of organic chemicals in lake water

The addition of phosphate, nitrate, or sulfate (each at 10 mM) decreased the acclimation period for the mineralization of low concentrations of p-nitrophenol (PNP) in lake water. Added phosphate shortened the acclimation period for biodegradation of 2 ng to 2 micrograms of PNP per ml in various lake water samples and of 2,4-dichlorophenoxyacetate at 100 ng/ml. Added P enhanced the rate of growth of PNP-mineralizing microorganisms in waters containing 200 ng or 2 micrograms of PNP per ml. We suggest that the effect of P on the acclimation period results from an increase in the growth rate of the initially small population of microorganisms able to mineralize the synthetic chemicals.     

Biodegradation kinetics of naphthalene in nonaqueous phase liquid-water mixed batch systems: comparison of model predictions and experimental results

A model is formulated to describe dissolution of naphthalene from an insoluble nonaqueous phase liquid (NAPL) and its subsequent biodegradation in the aqueous phase in completely mixed batch reactors. The physicochemical processes of equilibrium partitioning and mass transfer of naphthalene between the NAPL and aqueous phases were incorporated into the model. Biodegradation kinetics were described by Monod's microbial growth kinetic model, modified to account for the inhibitory effects of 1,2-naphthoquinone formed during naphthalene degradation under certain conditions. System parameters and biokinetic coefficients pertinent to the NAPL-water systems were determined either by direct measurement or from nonlinear regression of the naphthalene mineralization profiles obtained from batch reactor tests with two-component NAPLs comprised of naphthalene and heptamethylnonane. The NAPLs contained substantial mass of naphthalene, and naphthalene biodegradation kinetics were evaluated over the time required for near complete depletion of naphthalene from the NAPL. Model predictions of naphthalene mineralization time profiles compared favorably to the general trends observed in the data obtained from laboratory experiments with the two-component NAPL, as well as with two coal tars obtained from the subsurface at contaminated sites and composed of many different PAHs (polycyclic aromatic hydrocarbon compounds). The effects of varying the NAPL mass and the naphthalene mole fractions in the NAPL are discussed. It was observed that the time to achieve a given percent removal of naphthalene does not change significantly with the initial mass of naphthalene in a fixed volume of the NAPL. Significant changes in the mineralization profiles are observed when the volume (and mass) of NAPL in the system is changed.     

Plasmid transfer between strains of Pseudomonas putida, and their survival, within a pilot scale percolating-filter sewage treatment system

Abstract: The effect of Pseudomonas aeruginosa UG2 biosurfactants or UG2 inocula on phenanthrene mineralization in uninoculated nonsterile soil slurries and slurries inoculated with the phenanthrene-mineralizing Pseudomonas sp. UG14r was investigated. In sandy loam and silt loam slurries amended with phenanthrene, inoculation with UG14r alone or in co-culture with UG2Lr reduced the lag period before onset of phenanthrene mineralization by 1 week. The total amount mineralized after 5 weeks was lower or not significantly different from the uninoculated control slurries. Inoculation with P. aeruginosa UG2Lr alone did not improve phenanthrene mineralization. In creosote-contaminated soil slurries, no lag period in phenanthrene mineralization was observed in any treatment. After 4 weeks, the greatest extent of mineralization was observed in creosote-contaminated soil slurries inoculated with the UG14r-UG2Lr co-culture and UG14r alone. In sandy loam and silt loam soil slurries inoculated with Pseudomonas sp. UG14r, addition of UG2 rhamnolipid biosurfactants (100 to 400 mg rhamnose equivalents (RE) · l⁻¹ slurry) inhibited phenanthrene mineralization by 10 to 15%. Mineralization was also inhibited in uninoculated sandy loam slurries. In creosote-contaminated soil slurries inoculated with Pseudomonas sp. UG14r, biosurfactants at 250 mg RE · l⁻¹ slurry enhanced mineralization whereas 400 mg RE · l⁻¹ had no effect, compared to unamended slurries. In uninoculated creosote-contaminated soil slurries, UG2 biosurfactants at 250 and 400 mg RE · l⁻¹ slurry enhanced mineralization, compared to unamended slurries.     

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.     

Effect of Humic Fractions and Clay on Biodegradation of Phenanthrene by a Pseudomonas fluorescens Strain Isolated from Soil

The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.     

Effects of enrichment with phthalate on polycyclic aromatic hydrocarbon biodegradation in contaminated soil

The effect of enrichment with phthalate on the biodegradation of polycyclic aromatic hydrocarbons (PAH) was tested with bioreactor-treated and untreated contaminated soil from a former manufactured gas plant (MGP) site. Soil samples that had been treated in a bioreactor and enriched with phthalate mineralized (14)C-labeled phenanthrene and pyrene to a greater extent than unenriched samples over a 22.5-h incubation, but did not stimulate benzo[a]pyrene mineralization. In contrast to the positive effects on (14)C-labeled phenanthrene and pyrene, no significant differences were found in the extent of biodegradation of native PAH when untreated contaminated soil was incubated with and without phthalate amendment. Denaturing-gradient gel electrophoresis (DGGE) profiles of bacterial 16S rRNA genes from unenriched and phthalate-enriched soil samples were substantially different, and clonal sequences matched to prominent DGGE bands revealed that beta-Proteobacteria related to Ralstonia were most highly enriched by phthalate addition. Quantitative real-time PCR analyses confirmed that, of previously determined PAH-degraders in the bioreactor, only Ralstonia-type organisms increased in response to enrichment, accounting for 89% of the additional bacterial 16S rRNA genes resulting from phthalate enrichment. These findings indicate that phthalate amendment of this particular PAH-contaminated soil did not significantly enrich for organisms associated with high molecular weight PAH degradation or have any significant effect on overall degradation of native PAH in the soil.     

Effects of Heterogeneity and Experimental Scale on the Biodegradation of Diesel

Biodegradation of petroleum hydrocarbon contamination is a common method forremediating soils and groundwater. Due to complexities with field-scale studies,biodegradation rates are typically evaluated at the bench-scale in laboratory studies.However, important field conditions can be difficult to mimic in the laboratory. Thisstudy investigates three scaling factors that can impact laboratory biodegradation ratesand that are frequently unaccounted for in typical laboratory experimental procedures.These factors are soil heterogeneity, morphology of petroleum hydrocarbon non-aqueous phase liquids (NAPLs) and soil moisture distribution. The effects of these factors on the biodegradation rate of diesel NAPL is tested under a variety of experimental procedures from well-mixed batch studies to four-foot static soil columns. The results indicate that a high degree of variability results from even small-scale heterogeneities. In addition, it appears that as the experimental scale increases, the measured biodegradation rates slow. The results indicate that diesel biodegradation rates derived from small-scale experiments are not necessarily representative of field-scale biodegradation rates.     

Nutrient-enhanced survival of and phenanthrene mineralization by alginate-encapsulated and freePseudomonas sp. UG14Lr cells in creosote-contaminated soil slurries

The effects of nutrient amendment and alginate encapsulation on survival of and phenanthrene mineralization by the bioluminescentPseudomonas sp. UG14Lr in creosote-contaminated soil slurries were examined. UG14Lr was inoculated into creosote-contaminated soil slurries either as a free cell suspension or encapsulated in alginate beads prepared with montmorillonite clay and skim milk. Additional treatments were free-cell-inoculated slurries amended with sterile alginate beads, free-cell-inoculated and uninoculated slurries amended with skim milk only, and uninoculated, unamended slurries. Mineralization was determined by measuring¹⁴CO₂ released from radiolabelled phenanthrene. Survival was measured by selective plating and bioluminescence. Inclusion of skim milk was found to enhance both survival of and phenanthrene mineralization by free and encapsulated UG14Lr cells.     

Substrate availability in phenanthrene biodegradation: Transfer mechanism and influence on metabolism

 The mechanism of phenanthrene transfer to the bacteria during biodegradation by a Pseudomonas strain was investigated using a sensitive respirometric technique (Sapromat equipment) allowing the quasi-continuous acquisition of data on oxygen consumption. Several systems of phenanthrene supply, crystalline solid and solutions in non-water-miscible solvents (silicone oil and 2,2,4,4,6,8,8-heptamethylnonane) were studied. In all cases, analysis of the kinetics of oxygen consumption demonstrated an initial phase of exponential growth with the same specific growth rate. In order to analyze the second phase of growth and phenanthrene degradation, a study of the kinetics of phenanthrene transfer to the aqueous phase was conducted by direct experimentation, with the crystal and silicone oil systems, in abiotic conditions. The data allowed the validation of a model based on phase-transfer laws, describing the variations, with substrate concentrations, of rates of phenanthrene transfer to the aqueous phase. Analysis of the biodegradation curves then showed that exponential growth ended in all cases when the rates of phenanthrene consumption reached the maximal transfer rates. Thereafter, the biodegradation rates closely obeyed, for all systems, the transfer rate values given by the model. These results unambiguously demonstrated that, in the present case, phenanthrene biodegradation required prior transfer to the aqueous phase. With the silicone oil system, which allowed high transfer and biodegradation rates, phenanthrene was directed towards higher metabolite production and lower mineralization, as shown by oxygen consumption and carbon balance determinations. Received: 30 November 1994/Accepted: 11 January 1995     

Effects of Soil Water Content on Biodegradation of Phenanthrene in a Mixture of Organic Contaminants

Phenanthrene biodegradation was investigated at different soil water contents [0.11, 0.22, 0.33, 0.44 g H₂O (g soil)^?1] to determine the effects of water availability on biodegradation rate. A subsurface horizon of Kennebec silty loam soil was used in this study. [9-¹⁴C] phenanthrene was dissolved in a mixture of organic contaminants that consisted of 76% decane, 6% ρ-xylene, 6% phenanthrene, 6% pristane, and 6% naphthalene, and then added to the soil. The highest rate of mineralization, in which 0.23% of the [9-¹⁴C] phenanthrene degraded to ¹⁴CO₂ after 66 days of incubation, was observed at the soil water content of 0.44 g H₂O/g dry soil. Most of the ¹⁴C remained in the soil as the parent compound or as nonextractable compounds by acetonitrile at the highest water content. Concentrations of nonextractable compounds increased with water content, but residual extractable phenanthrene decreased significantly with increasing water content, which presumably indicates that bio-transformation occurred. The mineralization analysis of radiolabeled 9th carbon in phenanthrene underestimated phenanthrene biodegradation. The strong adsorption and low solubility of phenanthrene contributed to the low mineralization of phenanthrene 9th carbon. The other components were subject to higher biological and abiotic dissipation processes with increasing soil water content.     

Accelerated Biodegradation of High and Low Concentrations of p-Nitrophenol (PNP) by Bacterial Inoculation in Industrial Wastewater: The Role of Inoculum Size on Acclimation Period

The effect of inoculum size on the acclimation period and rate and extent of p-nitrophenol (PNP) degradation at high (1–10 mg/L) and low (26 μg/L) concentrations for two bacteria was determined in defined media as well as industrial wastewater. Increased inoculum size did not affect the acclimation period of either bacterium at high (1–10 mg/L) PNP concentrations. At low PNP concentrations (26 μg/L), the two bacteria behaved differently. The acclimation period was shortened and both the rate and extent of mineralization of PNP were enhanced by increasing the Corynebacterium sp. inoculum size from 3 × 10⁵ to 3 × 10⁶ cells/ml. Addition of phosphate or elimination of predators also reduced the acclimation period. Conversely, increasing the inoculum size from 3 × 10⁵ to 5 × 10⁶ cells/ml of Pseudomonas putida lengthened the acclimation period and reduced both the rate and extent of mineralization. It is suggested that, in a given environment, the success of an introduced species to enhance the degradation of a chemical depends upon (i) concentration of the chemical, (ii) selection of an appropriate microorganism, and (iii) utilization of a suitable inoculum size. Received: 1 April 1996 / Accepted: 6 May 1996     

Microemulsion‐mediated removal of residual gasoline from soil columns

In situ pumping of micellular solutions of surfactant (S) and cosurfactant (CoS) in water (W) through contaminated soils or aquifers offers potential for enhanced remediation of residual nonaqueous‐phase liquids (NAPLs). Extremely low interfacial tension generated between a W/S/CoS mixture and residual NAPL in soil pores may initially mobilize the NAPL, which is then transported temporarily as a separate phase by immiscible displacement. The NAPL is then solubilized by micro‐emulsification as the W/S/CoS mixture forms a stable W/S/CoS/NAPL micro‐emulsion that undergoes miscible displacement through the pore space. This remediation technique was tested under laboratory conditions by sequentially flushing a saline solution and a W/S/CoS mixture through columns of a sandy soil recently contaminated with residual leaded gasoline (LG). Prior to the flushings, the soil was initially contaminated by applying a W/S/CoS/LG microemulsion. A simple conceptual transport model with kinetic clogging of soil pores adequately described breakthrough curves for gasoline and organolead in the soil columns.     

Utilization of sorbed compounds by microorganisms specifically isolated for that purpose

A bacterium obtained by enrichment on nonsorbed phenanthrene was unable to degrade phenanthrene sorbed to polyacrylic beads and had little activity on phenanthrene sorbed to lake-bottom sediment. A bacterium obtained by enrichment on phenanthrene sorbed to polyacrylic beads readily mineralized the compound sorbed to the beads or the sediment. Degradation by the second bacterium of phenanthrene sorbed to beads 38–63 μm or 63–150 μm in diameter was more rapid than the rate of desorption of the hydrocarbon in the absence of the bacterium. Little degradation of sorbed, nonleachable phenanthrene in soil was effected by another isolate obtained by enrichment with the nonsorbed hydrocarbon, but a mixed culture and the bacterium obtained by enrichment on the sorbed compound extensively degraded phenanthrene. Because microorganisms specifically obtained for their capacity to degrade sorbed phenanthrene are more active than species not specialized for use of the bound compound, we suggest that microorganisms enriched on nonsorbed compounds may not be appropriate for evaluation of biodegradation and bioremediation of sorbed compounds. Received: 3 June 1997 / Received revision: 2 September 1997 / Accepted: 15 September 1997     

Repeated inoculation as a strategy for the remediation of low concentrations of phenanthrene in soil

Phenanthrene, a polycyclic aromatic hydrocarbon, becomes increasingly unavailable to microorganisms for degradation as it ages in soil. Consequently, many bioaugmentation efforts to remediate polycyclic aromatic hydrocarbons in soil have failed. We studied theeffect of repeatedly inoculating a soil with a phenanthrene-degrading Arthrobacter sp. on the mineralization kinetics of low concentrations of phenanthrene. After the first inoculation, the initial mineralization rate of 50 ng/g phenanthrene declined in a biphasicexponential pattern. By three hundred hours after inoculation, there was no difference in mineralization rates between the inoculated and uninoculated treatments even though a large fraction of the phenanthrene had not yet been mineralized. A second and third inoculation significantly increased the mineralization rate, suggesting that, though themineralization rate declined, phenanthrene remained bioavailable. Restirring the soil, without inoculation, did not produce similar increases in mineralization rates, suggesting absence of contact between cells and phenanthrene on a larger spatial scale (>mm) is not the cause of the mineralization decline. Bacteria inoculated into soil 280 hours beforethe phenanthrene was added could not maintain phenanthrene degradation activity. We suggest sorption lowered bioavailability of phenanthrene below an induction threshold concentration for metabolic activity of phenanthrene-degrading bacteria.     

Factors affecting the microbial degradation of phenanthrene in soil

Summary Because phenanthrene was mineralized more slowly in soils than in liquid media, a study was conducted to determine the environmental factors that may account for the slow biodegradation in soil. Mineralization was enhanced by additions of phosphate but not potassium, and it was reduced by additions of nitrate. Aeration or amending the soil with glucose affected the rate of mineralization, although not markedly. Phenanthrene was sorbed to soil constituents, the extent of sorption being directly related to the percentage of organic matter in the soil. Soluble phenanthrene was not detected after addition of the compound to a muck soil. The rate of mineralization was slow in the organic soil and higher in mineral soils with lower percentages of organic matter. We suggest that sorption by soil organic matter slows the biodegradation of polycyclic aromatic hydrocarbons that are otherwise readily metabolized. Offprint requests to: M. Alexander     

Bacterial Adhesion to Soil Contaminants in the Presence of Surfactants

It has been proposed that addition of surfactants to contaminated soil enhances the solubility of target compounds; however, surfactants may simultaneously reduce the adhesion of bacteria to hydrophobic surfaces. If the latter mechanism is important for the biodegradation of virtually insoluble contaminants, then the use of surfactants may not be beneficial. The adhesion of a Mycobacterium strain and a Pseudomonas strain, isolated from a creosote-contaminated soil, to the surfaces of highly viscous non-aqueous-phase liquids (NAPLs) was measured. The NAPLs were organic material extracted from soils from two creosote-contaminated sites and two petroleum-contaminated sites. Cells suspended in media with and without surfactant were placed in test tubes coated with an NAPL, and the percentages of cells that adhered to the surface of the NAPL in the presence and absence of surfactant were compared by measuring optical density. Test tubes without NAPLs were used as controls. The presence of either Triton X-100 or Dowfax 8390 at a concentration that was one-half the critical micelle concentration (CMC) inhibited adhesion of both species of bacteria to the NAPLs. Both surfactants, when added at concentrations that were one-half the CMCs to test tubes containing previously adhered bacteria, also promoted the removal of the cells from the surfaces of the NAPL-coated test tubes. Neither surfactant was toxic to the bacteria. Further investigation showed that a low concentration of surfactant also inhibited the growth of both species on anthracene, indicating that the presence of a surfactant resulted in a reduction in the uptake of the solid carbon source.     

Degradation of xenobiotic compounds in situ: Capabilities and limits

Abstract: Exploiting microorganisms for remediation of waste sites is a promising alternative to groundwater pumping and above ground treatment. The objective of in situ bioremediation is to stimulate the growth of indigenous or introduced microorganisms in regions of subsurface contamination, and thus to provide direct contact between microorganisms and the dissolved and sorbed contaminants for biotransformation. Subsurface microorganisms detected at a former manufactured gas plant site contaminated with coal tars mineralized significant amounts of naphthalene (8–43%) and phenanthrene (3–31%) in sediment-water microcosms incubated for 4 weeks under aerobic conditions. Evidence was obtained for naphthalene mineralization (8–13%) in the absence of oxygen in field samples. These data suggest that biodegradation of these compounds is occurring at the site, and the prospects are good for enhancing this biodegradation. Additional batch studies demonstrated that sorption of naphthalene onto aquifer materials reduced the extent and rate of biodegradation, indicating that desorption rate was controlling the biodegradation performance.