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Published in J. Environ. Qual. 33:2015-2025 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Bioremediation and Biodegradation

Effect of Surfactants on Solubilization and Degradation of Phenanthrene under Thermophilic Conditions

Jonathan W. C. Wonga,*, Min Fanga,b, Zhenyong Zhaoa and Baoshan Xingb

a Department of Biology, Hong Kong Baptist University, Hong Kong SAR, P.R. China
b Department of Plant and Soil Sciences, University of Massachusetts, Amherst, MA 01002

* Corresponding author (jwcwong{at}hkbu.edu.hk)

Received for publication July 24, 2003.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The bioavailability and biodegradation of polycyclic aromatic hydrocarbons (PAHs) can be increased through the addition of surfactants. Previous studies of this nature have been conducted under mesophilic conditions. Hence, the aim of the present study was to investigate the effects of synthetic surfactants and biosurfactants on solubilization and degradation of phenanthrene (PHE) in a series of batch solution experiments under thermophilic conditions. Tween 80, Triton X-100, and biosurfactants produced from Pseudomonas aeruginosa strain P-CG3 (P-CG3) and Pseudomonas aeruginosa ATCC 9027 (P. 9027) were used in this study. Surfactants effectively enhanced the solubility of PHE at 50°C and the biosurfactant from P-CG3 was most effective with a 28-fold increase in apparent solubility of PHE at a concentration of 10 x critical micelle concentration (CMC) compared with the controls. However, addition of synthetic surfactants or biosurfactants inhibited the biodegradation of PHE in mineral salts medium by an isolate Bacillus sp. B-UM. Degradation of PHE diminished with increasing surfactant concentrations, and PHE degradation was completely inhibited for all the surfactants tested when the concentrations were greater than their respective CMC. The growth test suggested that Tween 80 and biosurfactants were degradable, but preferential utilization of these surfactants as substrates was not the mechanism for explaining the inhibition of PHE biodegradation. Because of the hydrophobic property of B-UM, degradation inhibition of PHE by surfactants was probably due to the reduction of direct contact between bacterial cells and PHE.

Abbreviations: B-UM, Bacillus sp. B-UM • CMC, critical micelle concentration • MSM, mineral salt medium • P. 9027, Pseudomonas aeruginosa ATCC 9027 • PAH, polycyclic aromatic hydrocarbon • P-CG3, Pseudomonas aeruginosa strain P-CG3 • PHE, phenanthrene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
POLYCYCLIC AROMATIC HYDROCARBONS are a group of ubiquitous contaminants with a high potential mutagenicity or carcinogenicity. The biodegradation of PAHs by microorganisms is one of the primary ways for eliminating PAHs from contaminated sites (Riser-Roberts, 1998; Alexander, 1999). However, biodegradation of PAHs is restricted by their limited bioavailability, which is mainly associated with PAH hydrophobic nature and strong adsorptive capacity in soil (Thomas et al., 1986; Volkering et al., 1998). It has been reported that the mass transfer rate of PAHs into the aqueous phase is the rate-limiting step in their degradation (Stucki and Alexander, 1987; Grimberg et al., 1996; Pignatello and Xing, 1996). Thus, it is essential to develop methods to desorb PAHs from soils, making them available to microorganisms.

Use of surfactants is a possible option for increasing bioavailability of PAHs. In general, surfactants could enhance the apparent solubility of PAHs by micellar formation, which commences at the critical micelle concentration (CMC) and then solubility is proportional to surfactant concentration (Edwards et al., 1991). However, biodegradation of PAHs is not always correspondingly enhanced by surfactants. Some research groups have found that addition of surfactants stimulated PAH biodegradation (Aronstein and Alexander, 1992; Bury and Miller, 1993; Tiehm, 1994; Volkering et al., 1995; Boonchan et al., 1998), whereas others reported no effect or inhibition by surfactants (Laha and Luthy, 1991; Tiehm, 1994; Guha and Jaffe, 1996; Boonchan et al., 1998). The contradictory results may be due to the varied interactions among PAH-degrading species, PAHs, and surfactants. Surfactants may be used as a growth substrate in preference to PAH compounds or toxic to some microorganisms, and hence PAH degradation would be reduced. If surfactants are neither toxic nor growth substrates, they can either enhance degradation of PAHs by forming micelles that are accessible to microorganisms, or decrease degradation by preventing cells from directly contacting PAHs (Guha and Jaffe, 1996; Stelmack et al., 1999).

To date, most studies on the effect of surfactants on PAH degradation have been performed under mesophilic conditions. Since elevated temperatures can increase the enzyme activities involved in the degradation, solubility, and mass transfer rates of PAHs, thermophilic conditions are expected to achieve a high removal rate of PAH compounds from contaminated soils (Margesin and Schinner, 2001). Substrate utilization rates of thermophilic bacteria have been reported to be 3 to 10 times greater than those observed with the mesophilic bacteria (Lapara and Alleman, 1999). Thermophilic bioremediation of PAH-contaminated soils or municipal solid waste has been proven to be a beneficial approach for PAH degradation (Potter et al., 1999; Wong et al., 2002b). One of our previous studies (Fang et al., 2002) also demonstrated the enhanced removal rate of three- and four-ringed PAHs in PAH-contaminated soil by Tween 80 during thermophilic composting. However, the reasons for the enhancement are unknown. Therefore, the objective of this study is to determine the effect of different surfactants on solubilization and degradation of PHE under thermophilic conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Surfactants
Tween 80 and Triton X-100 were used as synthetic surfactants, because it is reported that both of them are not toxic and able to enhance solubilization of organic compounds (Edwards et al., 1991; Laha and Luthy, 1992). Similarly, biosurfactants that can enhance bioavailability and have a potential to stimulate degradation of hydrocarbons (Zhang and Miller, 1992; Schippers et al., 2000) were also used. Because thermo-tolerant microorganism produced biosurfactants are not available commercially yet, one such biosurfactant was isolated in our laboratory according to the modified method outlined by Deziel et al. (1996). The strain named as P-CG3 was identified as Pseudomonas aeruginosa, which used hexadecane as a carbon source, but not PHE at 50°C. The properties of each surfactant are shown in Table 1.


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  		Table 1. Properties of selected synthetic surfactants and biosurfactants.

 
Phenanthrene Degradation by Strain B-UM
Phenanthrene (purity > 98%; Aldrich Chemical Co., St. Louis, MO) was used in this study as a model PAH. We used a PHE-degrading bacterium (Bacillus sp. B-UM), which was enriched and isolated from a PAH-contaminated soil compost in our lab at 50°C as described by Wong et al. (2002a). Cell hydrophobicity of B-UM was measured by a method described by Yuste et al. (2000) for understanding its affinity for an organic phase. Cells are considered to be hydrophobic when the colony-forming units (cfu) ratio on Luria–Bertani (LB) medium of cell suspension after treatment with n-hexadecane over the initial cell suspension was 10–1 or lower.

A degradation test was performed at various concentrations of PHE for estimating PHE degradation rates by B-UM in mineral salts medium (MSM) at 50°C. All experiments in the later sections were preformed at the same temperature (50°C), unless noted otherwise. The MSM contained (per liter of deionized water): 200 mg (NH4)2SO4, 350 mg KH2PO4, 775 mg K2HPO4, 100 mg MgSO4, 70 mg CaCl2, and 1 mL of trace element solution (200 mg FeSO4, 2 mg CuSO4, 2 mg CoCl2, 20 mg MnCl2, 10 mg NaMoO4, and 10 mg ZnSO4 per 100 mL). The pH was adjusted to 7.0. After growth of B-UM in MSM, the cells were centrifuged, washed, and resuspended in the MSM to give a final cell density of 1 x 107 cells mL–1. Phenanthrene dissolved in chloroform at various concentrations was carefully added to the bottom of 40-mL tubes. The chloroform was allowed to evaporate before 20 mL of MSM with the cultured bacteria were added to the tubes with the PHE concentrations of 50, 100, 250, and 500 mg L–1. The tubes were placed on a gyratory shaker (150 rpm) and incubated for 10 d. Periodically, duplicate tubes were sacrificed to determine the residual PHE and soluble protein concentrations as indicators for bacterial growth (Lowry et al., 1951). To distinguish between abiotic losses and microbial degradation, the control with 200 mg L–1 HgCl2 was prepared and incubated following the same procedures as described above. Residual PHE was extracted with 10 mL dichloromethane three times. The extracts were combined and analyzed by high-performance liquid chromatography (HPLC), equipped with a UV detector (254 nm). A 10-µL injection of the extract was separated on a Hewlett-Packard (Palo Alto, CA) reverse-phase C18 column (4.6 mm x 25 cm). The eluent was 100% acetonitrile with a flow rate of 1.5 mL min–1.

Biosurfactant Production and Extraction
Two strains with the ability to produce biosurfactant were used in this work. One was the isolated strain, P-CG3, and another was Pseudomonas aeruginosa ATCC 9027 (P. 9027) purchased from the American Type Culture Collection (Rockville, MD). Both strains were cultured in 2-L flasks with proteose peptone glucose ammonium salts (PPGAS) medium as described by Zhang and Miller (1992). After 72 h of culture on a gyratory shaker (150 rpm) at 37°C for P. 9027 and 50°C for P-CG3, biosurfactants were harvested followed by extractions (Zhang and Miller, 1992) and surface tension measurements. Our preliminary tests showed that P. 9027 did not produce a significant amount of biosurfactant at 50°C.

Bacterial cells were removed from the culture broth by centrifugation (8000 x g, 20 min). The cell-free supernatant was subjected to acid precipitation by adding 1 M HCl to achieve a final pH of 2.0, and then was allowed to precipitate at 4°C over night. The precipitate was centrifuged at 8000 x g for 20 min and freeze-dried. The dried surfactant was extracted with 10 mL of solvent (chloroform to ethanol = 2:1, v/v) three times at 45°C. After the organic solvents were evaporated on a rotary evaporator, the surfactants were weighed, and then dissolved in deionized water. The biosurfactant concentration was estimated independently by surface tension measurements using a DuNouy ring digital tensiometer (KSV Instrument Ltd., Monroe, CT).

Phenanthrene-Degrading Microorganism Growth on Surfactants
The cells of B-UM prepared in the PHE degradation test described above were inoculated into MSM containing each surfactant as a sole carbon source at various concentrations (0, 0.25, 0.5, 1.0, 3.0 x CMC). After a 10-d incubation, growth was determined by soluble protein concentration in the culture broth. The control without any surfactant was also prepared to detect the effect of any other possible factors on bacterial growth.

Phenanthrene Solubilization
The solubility of PHE was evaluated in the presence and the absence of four selected surfactants. For each treatment, 100 µL of PHE (5.0 mg) dissolved in chloroform was carefully added to the bottom of 40-mL tubes. The amount of added PHE was well in excess of its aqueous saturation even at concentrations of 10 x CMC. After the chloroform evaporated, mineral salts medium with various concentrations of surfactants (0, 0.25, 0.5, 1.0, 3.0, 10.0 x CMC) was added up to minimal headspace. The tubes were placed in a shaker (150 rpm) for an equilibration period of 24 h. After equilibration, a 5-mL sample was removed from each tube using a pipette and each sample was filtered through a 10-mL glass syringe packed with glass wool to remove any solid PHE particles. For the biosurfactant treatments, 0.5 mL of concentrated HCl was added to each sample to precipitate biosurfactants, thus reducing emulsification during solvent extraction. The concentration of PHE in each sample was measured by the same method described earlier.

Effect of Surfactants on Phenanthrene Degradation
Phenanthrene biodegradation by B-UM was studied in 40-mL tubes with or without surfactants, as described in the degradation test of B-UM. The final mass of PHE added was 5 mg tube–1. Surfactant solutions were sterilized by passing through a 0.45-µm pore-size membrane filter before adding to the tubes. Each tube contained 20 mL of MSM with 0, 0.25, 0.5, 1.0, or 3.0 x CMC of these four surfactants, respectively. The tubes were incubated on a gyratory shaker for 10 d. Periodically, duplicate tubes were sacrificed for soluble protein and residual PHE concentration determination. For determination of abiotic loss, phenanthrene was coated, inoculated, and incubated as described above except for the addition of 200 mg L–1 HgCl2 in the control tubes.

One-way ANOVA was used to determine statistical difference between duplicate treatments in this study. Pearson correlation coefficients and p values were determined for all possible variable pairs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Phenanthrene Degradation by Strain B-UM
The strain B-UM was able to use PHE as a sole carbon source and PHE degradation was accompanied with cell growth as indicated by the increase in protein concentrations during the culture period (Fig. 1) . High concentrations of PHE were beneficial to the B-UM growth. More than 95% of PHE remained after 10 d of incubation in the control samples with addition of HgCl2 to suppress microbial activities, indicating that the disappearance of PHE was mainly attributed to microbial degradation. Cell hydrophobicity of B-UM was evaluated by measuring the proportion of cells adhered to hexadecane. This test revealed that this strain clearly had an affinity for the organic phase. Hydrophobic cell surfaces may be able to facilitate cells to get into physical contact with the oil phase by specific adhesion mechanisms.



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  		Fig. 1. (A) Phenanthrene (PHE) degradation and (B) soluble protein accumulation during PHE degradation by isolated strain Bacillus sp. B-UM at various PHE concentrations.

 
Solubilization of Phenanthrene in the Presence of Surfactants
Solubilization of PHE varied considerably in the presence of different surfactants (Fig. 2) . The aqueous solubility of PHE at 50°C in the absence of surfactants was approximately 1.8 mg L–1. When the concentrations of biosurfactants were below the CMCs, the solubility of PHE slightly increased with increasing surfactant concentrations. When concentrations were greater than or equal to their respective CMCs, solubility of PHE was enhanced remarkably, up to 50 and 28 mg L–1 for P-CG3 and P. 9027 biosurfactants, respectively, at 10 x CMC concentrations. For synthetic surfactants, a sharp increase of PHE solubility was also noted at the concentrations above the CMCs and the solubility of PHE was 16 and 11 mg L–1 for Tween 80 and Triton X-100, respectively, at their 10 x CMC. The biosurfactants from P-CG3 had the highest solubilizing power (28-fold increase) among the four surfactants. The solubility enhancement was observed to be proportional to the concentrations of all four surfactants at the supra-CMC concentrations.



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  		Fig. 2. Enhancement of phenanthrene solubilization with increasing concentrations of synthetic surfactants and biosurfactants. P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 
Degradation of Phenanthrene in the Presence of Surfactants
Biological degradation of PHE in the presence or absence of surfactants at 50°C was monitored by determining the change in PHE concentration in solution phase (Fig. 3) . Over a 10-d incubation period of B-UM with PHE as the sole carbon source, 99% of the initial PHE was removed in the absence of surfactants. However, with each surfactant, a significant inhibition was observed and this inhibition increased with increasing surfactant concentrations. At the supra-CMC surfactant concentrations, degradation of PHE was completely inhibited. Phenanthrene losses through volatilization or abiotic degradation were assessed by the sterile control with 200 mg L–1 HgCl2. The controls revealed that abiotic processes were not the major factor for disappearance of PHE. To have a clear understanding of the extent of PHE removal in the presence of each surfactant, the kinetic rate constant (k) of PHE was calculated (Table 2). The degradation of PHE was approximately described by the first-order kinetics, which is expected for a substrate, like PHE, with low water solubility and a limited rate of mass transfer (Alexander, 1999). The low k values indicate that degradation of PHE was reduced in the presence of either biosurfactants or synthetic surfactants. A minor abiotic loss of PHE was noted by k values in the treatments with addition of surfactants at ≥1.0 x CMCs.



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  		Fig. 3. Effect of synthetic surfactants and biosurfactants at various concentrations on phenanthrene degradation with time. CMC, critical micelle concentration; P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 

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  		Table 2. Kinetic rate constants (k) of polycyclic aromatic hydrocarbon (PAH) degradation by Bacillus sp. B-UM in mineral salts medium.{dagger}

 
Changes in Surface Tension in Degrading Test
The changes in surface tension before and after degradation test were determined to understand their relationship with degradation (Fig. 4) . The surface tension was significantly reduced with the addition of each surfactant. After a 10-d incubation, the surface tension in the control without surfactants increased slightly. The variation in composition of medium or growth of bacteria might contribute to this change. This indicates that the strain B-UM does not have an ability to produce surface-active substances in MSM with PHE as the sole carbon source. There was no significant change in surface tension for all surfactant treatments, indicating that these four surfactants retained their activity at 50°C after 10 d of degradation test. Degradation rates were linearly correlated with surface tension (r > 0.98, p < 0.05), that is, inhibition on degradation was accompanied with a surface tension decline (Fig. 5) .



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  		Fig. 4. Changes in surface tension before and after degradation of phenanthrene in the presence of synthetic surfactants or biosurfactants. CMC, critical micelle concentration; P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 


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  		Fig. 5. A positive correlation between surface tension of mineral salts medium and degradation rates of phenanthrene during degradation tests with synthetic surfactant and biosurfactant addition. r, Pearson correlation coefficient. P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 
Strain B-UM Growth on Surfactants
The ability of surfactant utilization by strain B-UM was evaluated by a growth test with each surfactant as a sole carbon source in MSM. The growth of bacterial cells was observed in the presence of surfactants (Fig. 6) . Except for Triton X-100, growth of bacterial cells increased with increasing surfactant concentrations, indicating that these surfactants can be used as growth substrates and are not toxic to B-UM. For Triton X-100, it did not appear to be a growth substrate, but no growth inhibition was observed within the tested concentration range as compared with the control without surfactants.



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  		Fig. 6. Growth test of phenanthrene-degrading strain Bacillus sp. B-UM in the presence of individual synthetic surfactants and biosurfactants. CMC, critical micelle concentration; P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 
To understand the effect of biodegradable surfactants on degradation of PHE, the bacterial growth (represented by soluble protein concentrations) from assimilating surfactants and PHE is shown in Fig. 7 . The bacterial growth from PHE was estimated by subtracting the surfactant-produced protein concentrations (Fig. 6) from the total soluble protein concentrations in the mixed system. The soluble protein concentration in the control without surfactants reached 180 mg L–1. In the tubes with surfactant addition, the low soluble protein concentrations were observed due to the inhibitory effect of surfactants on PHE degradation. With addition of surfactants at a concentration < CMC, soluble protein concentrations were between 105 and 170 mg L–1, in which less than 5 mg L–1 of soluble protein was produced by metabolizing surfactants. Hence, although strain B-UM was able to use surfactants as growth substrates, the portion of bacteria grown on surfactants was negligible compared with that grown on PHE. These results suggest that inhibition of PHE degradation was not caused by preferential use of the surfactants. The preferential use of PHE by B-UM is not clearly understood. We assumed that acclimatization of this strain to PHE as a specific substrate during enrichment and isolation may partly explain this result.



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  		Fig. 7. Growth balance of strain Bacillus sp. B-UM during a degradation test. Hatched and crosshatched bars represent bacterial growth from metabolizing phenanthrene (PHE) and each surfactant, respectively. CMC, critical micelle concentration; P. 9027, Pseudomonas aeruginosa ATCC 9027; P-CG3, Pseudomonas aeruginosa strain P-CG3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
All four surfactants enhanced PHE solubility at 50°C. These results prove that the use of surfactants to enhance solubilization of PAHs under thermophilic conditions can be as effective as in mesophilic conditions (Zhang and Miller, 1992, 1994; Zhang et al., 1997; Kanga et al., 1997). Among the four surfactants, the biosurfactant produced from P-CG3 had the highest solubility enhancement potential for PHE. Solubilization capacity of the surfactants is dependent on surfactant structures and concentrations. It has generally been shown that the amount of solubilized hydrocarbons increases as the size of the micelle increases (Myers, 1988; Kanga et al., 1997). The P-CG3 biosurfactant may be able to form large micelles due to its favorable structures, which warrants further investigation.

The solubility curve demonstrated the different behaviors of synthetic surfactants and biosurfactants on solubility of PHE. At surfactant concentrations below their CMCs, the solubility of PHE gradually increased with an increase in biosurfactant concentrations, whereas synthetic surfactant did not show any enhancement of PHE solubility. A gradual rather than sharp inflection of the solubility curve in the vicinity of the CMC of biosurfactants may be due to the mixture of biosurfactant compounds (Edwards et al., 1991). Zhang and Miller (1994) reported that biosurfactants from P. 9027 are a mixture for four monorhamnolipids. Although the structure and component of the biosurfactants are unknown in this study, the different rhamnolipid homologues were expected because they were produced from the same bacterial species. Additionally, synthetic surfactants have a non-ionic nature and they did not respond to pH variations, whereas molecular structures of biosurfactants are sensitive to pH in the range of 6.0 to 7.0 (Ishigami et al., 1987). The pH in the solubilization test was approximately 6.8, which may partly account for the different behaviors between the biosurfactants and synthetic surfactants on PHE solubility.

Although the four surfactants enhanced the PHE solubility, PHE biodegradation did not correspondingly increase. On the contrary, the four surfactants inhibited the degradation of PHE under thermophilic conditions. When surfactant concentrations were below their respective CMCs, the inhibition increased proportionally with increasing surfactant concentrations; at the supra-CMC concentrations, degradation was completely inhibited. The likely causes for this inhibition are: (i) use of surfactants as preferential growth substrates, (ii) toxicity effect, and/or (iii) lower uptake of PHE by microorganisms (Laha and Luthy, 1991; Stelmack et al., 1999). Our growth tests revealed that Tween 80 and two types of biosurfactants were biodegradable, but within the tested concentration ranges of these surfactants, their contribution to bacterial growth as substrates was negligible compared with the growth on PHE (Fig. 7). These results suggest that neither toxicity nor preferential use of surfactants as substrates were the major factors responsible for the inhibition of PHE degradation. Therefore, uptake reduction of PHE by microorganisms in the presence of surfactants was probably the major cause for inhibition. There are two possible pathways for bacteria to take up solid hydrocarbons, that is, either by dissolution of the target molecules in the aqueous phase, or by adhesion of bacteria directly to the solid–water interface (Volkering et al., 1998). For very low-solubility hydrocarbons, microbial cells may secrete surface-active substances to increase the mass transfer and result in increasing uptake. The strain B-UM did not show any ability to produce surfactants for increasing dissolution of PHE in MSM. Therefore, the direct adhesion of cells to PHE surface would be the major pathway for B-UM to take up PHE. The hydrophobic property of B-UM supports this hypothesis.

It has been shown that surfactants can inhibit biodegradation of hydrocarbons by de-adhesion of cells from the liquid/solid–water interface (Neu, 1996; Volkering et al., 1998). Triton X-100 has been reported to inhibit the growth of an Arthrobacter sp. on n-hexadecane, which was probably due to diminishing bacterial adhesion to the liquid–liquid interface (Efroymson and Alexander, 1994). It has been reported that degradation of anthracene by two bacterial strains, Mycobacterium sp. and Pseudomonas sp., which were able to adhere to solid surfaces of anthracene for direct uptake, was inhibited by Triton X-100 and Dowfax 8390 (Stelmack et al., 1999). Theoretically, microbes can attach to or detach from a solid surface depending on the surfactant nature and the surface energies of the cell and substrate (Gerson, 1993). Because all surfactants had an inhibitory result for PAH degradation in this study, free energy of adhesion was expected to increase by surfactants, which diminished bacterial adhesion to solid surfaces. To date, most research on interactions of cells, surfactants, and PAHs has been performed under mesophilic conditions. Our study, for the first time, presented assessment of the effect of surfactants on PAH degradation under thermophilic conditions. This finding will be used to improve the composting strategy in a PAH-contaminated soil treatment in our further study.

The inhibition of surfactants on PHE degradation due to lower availability of interfaces can be further verified by the change in surface tension. Adsorption of surfactants onto nonpolar, hydrophobic surfaces is primarily by dispersion force interaction and results in aggregation of surfactant molecules at solid–liquid interfaces in aqueous solution. A decrease in surface tension coincided with a higher aggregation of surfactant molecules at the interface (Kosaric, 1993), which implied less available interface for PAH-degrading bacterial cells. When the concentrations of surfactant were near or above the CMCs, the surfactant molecules at the solid–water interface reached saturation, and micelle-accommodated PHE began to form at this point (Myers, 1988). Surfactants could entirely prevent the cells from contacting the surface of PHE and the PHE in micelle; hence, the degradation did not occur any more. This is in agreement with the observation that the degradation of PHE was negatively correlated with the aggregation of surfactants on the PHE–water interface (Fig. 6). Our results demonstrated that although surfactants increased the aqueous PHE solubility, PHE present in the micellar phase was not freely available to the hydrophobic strain, B-UM.

The four surfactants showed an identical effect on PHE degradation under thermophilic conditions. The hydrophobic property of strain B-UM may be responsible for this inhibition. It is reported that the Triton X-100 inhibited the adhesion of marine bacteria to hydrophobic but not to hydrophilic surfaces (Paul and Jeffrey, 1985). The positive effect of Tween 80 on PAH degradation during thermophilic composting in our previous study (Fang et al., 2002) seemed contradictory with the present results. The complexity of indigenous microbial communities and their interactions in the composting mass may account for the different effects of surfactants on PAH degradation. Because microorganisms are able to change their cell surface hydrophobicity under different growth conditions and in different growth phases (Neu, 1996; Al-Tahhan et al., 2000), the effect of various surfactants on hydrocarbon biodegradation is unpredictable. This may be the reason why inconsistent results have been obtained in the literature. Nevertheless, we observed inhibitory results by the four surfactants for one hydrophobic strain, B-UM, that was capable of using PHE through direct uptake. Presumably, an enhanced degradation by surfactants may occur for hydrophilic PAH-degrading microorganisms or those that degrade hydrocarbons not via direct uptake from interface. Therefore, the effect of surfactants on PHE degradation is not only dependent on surfactant structure and concentration, but also on surface property of cells and uptake pathways of hydrocarbons.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In the present study, the addition of synthetic surfactants or biosurfactants effectively enhanced the solubility of PHE under thermophilic conditions. However, the biodegradation of PHE in mineral salts medium by an isolate Bacillus sp. B-UM under thermophilic conditions was inhibited by surfactant addition. Because of the hydrophobic surface property of Bacillus sp. B-UM, inhibition of PHE degradation by surfactants was probably due to the reduction of direct contact between bacterial cells and PHE. Additional studies are required to provide a thorough assessment of the effect of surfactants on the biodegradation of PAHs using other PAH-degrading microorganisms, which may have lower hydrophobic surface properties. The results of this study indicated that the biosurfactants produced by the isolated Pseudomonas aeruginosa P-CG3 have a potential to be applied for improving remediation efficiency under thermophilic composting conditions, especially for contaminated sites where the biodegradation of PAHs is limited by their low aqueous solubility.


   ACKNOWLEDGMENTS
 
The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, PR China (Project no. HKBU 2082/01M). We would like to thank Dr. Bill Coli for excellent technical help and Dr. D. Julian McClements (Food Science, University of Massachusetts) for letting us use the digital tensiometer.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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JEQ 2004 33: 1947-1953. [Full Text]  




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