Environ. Sci. Technol. 1997, 31, 2570-2576
Surfactant-Enhanced Mobilization and Biodegradation of Polycyclic Aromatic Hydrocarbons in Manufactured Gas Plant Soil A N D R E A S T I E H M , * ,†,‡ M I C H A E L S T I E B E R , §,| P E T E R W E R N E R , §,| A N D FRITZ H. FRIMMEL⊥ Lehrstuhl fu ¨ r Mikrobiologie, Technische Universita¨t Karlsruhe, Kaiserstrasse 12, 76128 Karlsruhe, Germany, DVGW-Forschungsstelle am Engler-Bunte-Institut, Technische Universita¨t Karlsruhe, Richard-Willsta¨tter-Allee 5, 76131 Karlsruhe, Germany, and Lehrstuhl fu ¨ r Wasserchemie, Technische Universita¨t Karlsruhe, P.O. Box 6980, 76128 Karlsruhe, Germany
The bioremediation of soil contaminated with polycyclic aromatic hydrocarbons (PAH) often is limited by a low bioavailability of the contaminants. The effect of two nonionic surfactants of the alkylphenolethoxylate type, Arkopal N-300 and Sapogenat T-300, on bioavailability of PAH in manufactured gas plant soil was evaluated in soil columns percolated by recirculating flushing water. Both surfactants enhanced the mass transfer rate of sorbed PAH into the aqueous phase due to solubilization. Solubilized PAH were available for biodegradation. Degradation of the surfactants themselves was monitored by counting cell numbers of surfactant degraders. It could be demonstrated that the rapid degradation of Arkopal N-300 resulted in a lack of oxygen and an inhibition of PAH degradation. Sapogenat T-300 was degraded more slowly, but a depletion of oxygen occurred after 54 d of incubation. Until then the surfactant-enhanced PAH mobilization resulted in an increased PAH degradation as compared to the treatment without surfactant. Therefore, biodegradability of the surfactants was shown to be one of the key functions for the use of surfactants in practice. Reduction of PAH content and toxicity of the contaminated soil was obtained in all cases. Decrease of soil toxicity as indicated by the bioluminescence test was most pronounced in case of the Sapogenat T-300-amended treatment. It is concluded that surfactants can be a useful tool for stimulating biodegradation of PAH in contaminated soil.
Introduction Contamination of soil with residues from town gas production is a problem in many industrial areas. Especially polycyclic * Author to whom correspondence should be addressed; telephone: 0049/40/7718-2984; fax 0049/40/7718-2684; e-mail address:
[email protected]. † Lehrstuhl fu ¨ r Mikrobiologie. ‡ Present address: Technische Universita ¨ t Hamburg-Harburg, Arbeitsbereich Gewa¨sserreinigungstechnik, Eissendorfer Strasse 42, 21073 Hamburg, Germany. § DVGW-Forschungsstelle am Engler-Bunte-Institut. | Present address: Technologiezentrum Wasser (TZW), Karlsruher Strasse 84, 76139 Karlsruhe, Germany. ⊥ Lehrstuhl fu ¨ r Wasserchemie.
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aromatic hydrocarbons (PAH) have been recognized as hazardous pollutants at manufactured gas plant sites. It has been demonstrated in laboratory studies that most PAH are biodegradable (1) but that their bioavailability is limited by low water dissolution rates (2-4). Sorption to solid matrices or incorporation in soil micropores are additional factors that hinder a rapid biodegradation of hydrophobic contaminants (5-7). In case of PAH-contaminated soil, sorption of the hydrocarbons can totally prevent a microbial attack (8, 9). Consequently, it has been concluded that mass transfer processes associated with contaminant release into the water phase limit the rate of removal of PAH rather than the explicit aqueous phase biodegradation kinetics. PAH mass transfer is at least one major limiting factor in achieving biotreatment of manufactured gas plant sites (10). The application of surfactants has become an interesting way to influence the mass transfer of PAH. Surfactants consist of a hydrophilic and a hydrophobic moiety and by this tend to concentrate at surfaces and interfaces thereby decreasing levels of surface tension and interfacial tension. Surfactant molecules form micellar aggregates in the water phase when a specific threshold, known as critical micellar concentration (cmc), is exceeded. Incorporation of hydrophobic compounds in the micelles is termed solubilization. In equilibrium, the amount of solubilized PAH linearly depends on the surfactant concentration above the cmc (11, 12). Solubilization and lowering of the surface and interface tension are thought to be the main reasons for a facilitated transport of pollutants adsorbed on solid phases to the surfactant containing aqueous phase. Washing with surfactant solutions has been shown to be effective for the removal of hydrophobic compounds such as hydrocarbons (13, 14), polychlorinated biphenyls (15), or tetrachloroethylene (16) from soil. Several attempts to enhance the bioavailability of hydrocarbons by the use of surfactants have been made. In studies done with artificially contaminated soil both positive and negative effects of the addition of synthetic surfactants or biosurfactants have been reported (17-23). Because of the contradictory results, especially the structure and the physicochemical properties of surfactants leading to toxicity are a question of intensive discussion (24). Nevertheless, it was reported recently that nonionic surfactants with a high hydrophilicity obtained by a long ethoxylate chain were nontoxic to PAH degrading bacteria. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene in liquid cultures (25). The aim of this study was to test the suitability of surfactants to enhance the bioremediation of PAH-contaminated soil from a former coal gasification site. The study was done with two nonionic surfactants of the alkylphenolethoxylate type known to be nontoxic. In-situ conditions were simulated in recirculating laboratory percolators. In order to get precise information about mass transfer and degradation processes, we monitored (i) the mobilization and biodegradation of PAH and (ii) the biodegradation of the surfactants themselves.
Materials and Methods Chemicals. Arkopal N-300 [C9H19C6H4O(CH2CH2O)30H; cmc of 0.50 mM (25)] and Sapogenat T-300 [(C4H9)3C6H2O(CH2CH2O)30H; cmc of 0.15 mM (25)] were supplied by Hoechst AG, Frankfurt, Germany. Both chemicals are nonionic surfactants of the alkylphenolethoxylate type. All other chemicals were commercial products of the highest purity available from Sigma (Deisenhofen, Germany), Merck (Darmstadt, Germany), or Fluka (Neu-Ulm, Germany).
S0013-936X(96)00996-0 CCC: $14.00
1997 American Chemical Society
FIGURE 1. Scheme of a laboratory percolator. Percolation System and Culture Conditions. The percolation experiments were done with a mineral medium that has been described previously (26). Laboratory percolators are shown in Figure 1. The soil column, the reservoir, and the electrode vessel were made of glass. Further materials used were stainless steel and Teflon. Soil columns had a diameter of 77 mm and a volume of 2 L. The columns contained 2.7 kg of contaminated soil. The water reservoir had a volume of 30 L, and the additional vessel containing the electrodes had a volume of 2 L. The whole system was filled with 27 L of medium and was kept at 20 °C. Electrodes and meters were coupled with a computercontrolled data acquisition system. Factors like rate of aeration, water flow, and pH values were automatically controlled and registered. Synthetic air consisting of 80% N2 and 20% O2 (Messer-Griesheim, Krefeld, Germany) was blown into the medium reservoir through glass sinter plates. Aeration was regulated in a way that the foam caused by the surfactant did not pass the upper border of the reservoir. The
exhaust air was cooled to 2 °C to minimize losses of volatile contaminants and water. O2 consumption and CO2 production were measured continuously in the exhaust air by gas analysers from Rosemount (Hanau, Germany). The mobile phase was recirculated through the columns with a pump rate of 12 L/h. The pH of the aqueous medium was automatically held between pH 6.9 and pH 7.1 by the addition of NaOH or HCl, respectively. Inoculation was done with mixed cultures enriched from the contaminated soil. One set of mixed cultures was enriched on fluorene, phenanthrene, anthracene, fluoranthene, or pyrene as single carbon sources as described previously (25). Additionally, inoculation was done with a mixed culture grown on a mixture of naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, fluoranthene, pyrene, and benz[a]anthracene. Soil. The soil contaminated with PAH (Table 1) was excavated in a depth of 6.0-7.0 m on a former coal gasification site at Karlsruhe, Germany. The gravel-sandy sample was homogenized and sieved through a 10-mm sieve. Particle size analysis revealed that about 65% of the sieved soil was smaller than 2 mm. Permeability of the soil sample was about Kf ) 7 × 10-4 m/s with an effective pore volume of 15% (27). The homogenized soil was stored at 4 °C before further treatment. Analytical Procedures. Samples of flushing water were taken at the outlet of the soil columns (Figure 1) with exception of the samples taken in the reservoir directly before the start of soil flushing (time 0 h). Sample volumes were between 200 and 275 mL. PAH taken out by the sampling procedure are given in Table 2. The concentration of particulate PAH in the aqueous phase was determined after extraction on a rotator for 3 h. Extraction of 100 mL of sample without surfactant was done with 1 mL of cyclohexane; 2.5 mL of cyclohexane was used for the extraction of 50 mL of sample containing surfactants. Emulsions being formed during extraction in the presence of surfactant were broken by centrifugation. The concentration of particulate PAH in the soil was determined after extraction of 20-g samples with 30 mL of toluene. Prior to extraction, samples were dried with Na2SO4. Extraction occurred in closed bottles for 40 h on a rotary shaker. Under these conditions equilibrium was reached. Homogeneity of the soil was verified by parallel extractions. Standard deviations of the concentrations of particulate PAH in the soil were between 5% and 17% (Table 1). Both the cyclohexane and the toluene extracts were analyzed with a gas chromatograph equipped with a flame ionization detector and a 25-m coated glass capillary column (HP-Ultra 2, Hewlett-Packard). Operating conditions were as follows: injector temperature, 275 °C; detector temperature,
TABLE 1. Physicochemical Characteristics and Concentration of PAH in Contaminated Soil PAH in the soil PAH
water solubilitya (mg/L)
log octanol-water coeffa (log Kow)
concn (mg/kg)
standard deviationb (%)
naphthalene (NAP) acenaphthylene (ACY) acenaphthene (ACE) fluorene (FLU) phenanthrene (PHE) anthracene (ANT) fluoranthene (FLA) pyrene (PYR) benz[a]anthracene (BaA) chrysene (CHR) benzo[b]fluoranthene (BbF) benzo[k]fluoranthene (BkF) benzo[a]pyrene (BaP)
30.0 3.47 3.93 1.98 1.29 0.07 0.26 0.14 0.014 0.002 0.0012 0.0006 0.0038
3.37 4.33 4.07 4.18 4.46 4.45 5.33 5.32 5.61 5.61 6.57 6.84 6.04
871 294 97 308 964 284 681 448 254 173 125 158 169
10.3 14.7 16.7 13.3 9.5 9.6 9.0 8.2 8.5 6.4 7.7 5.1 9.8
a
According to ref 44.
b
Number of samples: n ) 15.
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TABLE 2. Calculation of PAH Biodegraded after Treatment without Surfactant and with Surfactant Additiona PAH remaining after treatment soil
aqueous phase
PAH taken out by sampling
PAH biodegraded
treatment
(mg)
(%)
(mg)
(%)
(mg)
(%)
(mg)
(%)
without surfactant, 53 d with 5 mM Arkopal N-300, 19 d with 2 × 2.5 mM Sapogenat T-300, 62 d
8443 6188 3640
64.8 47.5 27.9
3 934 76b
