Effect of different non-ionic surfactants on the biodegradation of PAHs by diverse aerobic bacteria

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Abstract

The aim of this work was to evaluate the effect of several non-ionic surfactants (Tween-80, Triton X-100 and Tergitol NP-10) on the ability of different bacteria (Enterobacter sp., Pseudomonas sp. and Stenotrophomonas sp.) to degrade polycyclic aromatic hydrocarbons (PAHs). Bacterial cultures were performed at 25 °C in an orbital shaker under dark conditions in BHB medium containing 1% of surfactant and 500 mg l−1 of each PAH. Experiments performed with Tween-80 showed the highest cell density values and maximum specific growth rate because this surfactant was used as a carbon source by all bacteria. High degree of PAHs degradation (>90%) was reached in 15 days in all experiments. Toxicity increased at early times using Tween-80 but decreased to low levels in a short time after the firsts 24 h. On the other hand, Triton X-100 and Tergitol NP-10 were not biodegraded and toxicity kept constant along time. However, PAHs-degradation rate was higher, especially by the action of Enterobacter sp. with Tween-80 or Triton X-100. Control experiments performed without surfactant showed a significant decrease in biomass growth rate with a subsequent loss of biodegradation activity likely due to a reduced solubility and bioavailability of PAHs in absence of surfactant.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are comprised of two or more fused aromatic rings. They are generally formed by incomplete combustion of fossil fuels and other organic material. They are known to be carcinogenic to humans and other organisms (Kim et al., 2001). PAHs should be undetectable in soil and groundwater under the drinking water standards and health advisory regulations of the US Environmental Protection Agency (USEPA), where 16 PAHs have been defined as priority pollutants and form a subset of “toxic pollutants” under the Clean Water Act including naphthalene, phenanthrene and anthracene (USEPA, 1972). Naphthalene is also considered a hazardous air pollutant (USEPA, 1994, USEPA, 2004). These administrative regulations have encouraged scientists and engineers to identify the best remediation technologies from a variety of physical, chemical and biological methods that can be used to remove these compounds from polluted sites. Most low molecular weight PAHs are biodegradable in presence of a suitable microbial population. However, the efficiency of PAHs biodegradation is limited because these compounds have a very low aqueous solubility and vapour pressure (Luning Prak and Pritchard, 2002).

Surfactants may be useful for the bioremediation of sites polluted with PAHs since they enhance the solubility of hydrophobic compounds (Boonchan et al., 1998). Many studies have been conducted to enhance the biodegradation of PAHs using surfactants to increase their solubility by decreasing the interfacial surface tension between PAHs and the soil/water interphase. When surfactant concentration is above the critical micelle concentration (CMC), micelle aggregates provide an additional hydrophobic area in the central region of micelles enhancing the aqueous solubility of PAHs (Li and Chen, 2009). The addition of non-ionic surfactants as additives has a positive effect on PAHs biodegradation (Grimberg et al., 1996, Volkering et al., 1995). However, other studies have reported negligible (Ghosh et al., 1995) or even negative effects (Laha and Luthy, 1991) of surfactants. Possible reasons for this include the competition for substrate utilization and the toxicity of the surfactants toward PAHs-degrading bacteria (Liu et al., 2001). Therefore, PAHs-degradation processes involving surfactant utilization need to be optimized for each of the factors influencing biodegradation, including surfactants type and concentration, PAH specificity and the microorganisms present in the process (Jin et al., 2007).

The purpose of the present study was to examine the effect of the presence of non-ionic surfactants (Tween-80, Triton X-100 and Tergitol NP-10) on the biodegradation of low molecular weight PAHs (naphthalene, phenanthrene and anthracene) in aqueous media inoculated with one of each of the aerobic bacterial strains identified as Pseudomonas sp., Enterobacter sp. and Stenotrophomonas sp. or with a mixed culture comprised of these three species.

Section snippets

Chemicals and media

Naphthalene, phenanthrene and anthracene (all >99% purity) used in the degradation experiments were purchased from Sigma–Aldrich (Steinheim, Germany) and Fluka (Steinheim, Germany). Reagent grade dichloromethane was supplied by Scharlau Chemie (Barcelona, Spain). Bushnell Haas Broth medium (BHB) was purchased from Panreac (Barcelona, Spain). All surfactants (Tween-80, Triton X-100 and Tergitol NP-10) were purchased from Sigma–Aldrich (Steinheim, Germany). Relevant properties of these

Effects of different surfactants on the bacterial growth

The growth curves of the three bacterial strains during the PAHs-biodegradation experiments (Fig. 1) show the expected latent, exponential and stationary phases. Depending on the surfactant used, the length of these phases was different. The end of the exponential phase was reached approximately after 48 h in experiments performed in presence of Tween-80 while 150 h were necessary for Triton X-100 and Tergitol NP-10. The stationary phase was usually completed after 15 days of incubation in all

Discussion

Data show that the addition of surfactants affect positively PAHs biodegradation, increasing the solubility of PAHs and allowing their progressive biodegradation up to concentration levels lower than the initial values. This effect was clearly observed considering the low solubility and biodegradation degree of PAHs when surfactant was not added. The abiotic depletion of naphthalene in presence of 1% w/w Tween-80 (kA = 2.2 × 10−3 ± 6.0 × 10−5 h−1), was not observed for both three-ring PAHs (kA

Acknowledgements

Authors gratefully acknowledge the financial support from the Spanish Ministry of Environment (Research Project 13/2006/2-1.1) and Fundación Alfonso Martín Escudero.

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