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This study investigated the adsorption of two biosurfactants, non‐ionic saponin and anionic Reco-10 (a mixture of rhamnolipids). The experiments were performed with three different soils (sandy clay loam, clay loam, clay) and at two soil/biosurfactant ratios, m/V=1/10 and 1/40. Using a tensiometer, surface tension in aqueous biosurfactant solutions and their supernatants was measured and the critical micelle concentration © UNIVERSITY OF WARMIA AND MAZURY IN OLSZTYN INTRODUCTION Surfactants are amphiphilic compounds (containing hydrophobic and hydrophilic portions) that reduce the free energy of a system by replacing bulk molecules of higher energy at an interface (Mulligan 2005). Due to their ability to lower surface/interfacial tension, and to increase solubility, detergency power, wetting ability and foaming capacity, surfactants have a wide range of applications in many fields, such as the petroleum or pharmaceutical industries. In addition, surfactants monomers aggregate in micelles at a specific concentration, which not only reduces surface and interfacial tension, but also facilitates the desorption of pollutants and increases their bioavailability in soils or sediments. These properties mean that surfactants can be used in many surfactant-enhanced remediation systems like soil washing (Mao et al. 2015; Mulligan 2009), electrokinetic processes (Saichek and Reddy 2005), phytoremediation (Liu et al. 2013) and bioremediation (Pacwa-Plociniczak et al. 2011). Up to now, different ionic (anionic, cationic) and non-ionic surfactants have been tested for soil remediation. Anionic synthetic surfactants that have been tested include sodium dodecyl sulphate (SDS), bis(2-ethylhexyl) sulfosuccinate sodium (AOT) and linear sodium alkene sulfonates (Spolapon AOS). As a cationic surfactant, cetyltrialkyl ammonium bromide (CTAB) has been used. In contrast to ionic surfactants, nonionic surfactants have lower toxicity and greater capacity to solubilize contaminants, so they are more commonly used in remediation projects than ionic (Zheng et al. 2012). Although ionic surfactants are highly efficient at removing various pollutants 28 ENVIRONMENTAL BIOTECHNOLOGY 11 (2) 2015 such as PCBs, petroleum,NAPLs andBTEX, their toxicity can limit their usefulness (Mao et al. 2015). Currently, biosurfactants appear more attractive than synthetic surfactants for surfactant-based soil remediation. Biosurfactants are natural surface active agents produced by bacteria, fungi and yeast, or extracted from plants (Paria 2008). They have a larger molecular structure and more functional groups than synthetic surfactants, which enables them to remove both hydrophobic organics and heavy metals. The biosurfactants commonly used in soil remediation are anionic rhamnolipids secreted by Pseudomonas aeruginosa (Juwarkar et al. 2007; Muligan 2009) and non-ionic saponin of plant origin (Hong et al. 2002). Biosurfactants differ in their properties and can behave in soil in different ways. Although biosurfactants have a low environmental impact, and can be left in soil after treatment (Wouter et al. 2004), their adsorption can lower the efficiency of surfactant-based soil remediation. The degree of their adsorption depends primarily on soil properties, i.e. its organic carbon content and cation exchange capacity, and on the chemical nature of the surfactant. Anionic surfactants are generally adsorbed less than nonionic surfactants and much less than cationic surfactants (Lee et al. 2004). As a result of surfactants being adsorbed to soil, the hydrophobicity of the soil can be increased, and previously removed pollutants, especially organic ones, can be re-adsorbed on the soil surface (Paria 2008). In many remediation projects, the biosurfactant concentration is chosen based on the critical micelle concentration (CMC) (Zhang et al. 2011). If the degree of adsorption is great, surfactant concentrations could drop below the CMC and pollutants will not be solubilized (Chu 2003). Therefore, selection of the proper biosurfactant concentration for soil remediation should be preceded by determination of the CMC in the soil-surfactant solution system. To determine biosurfactant adsorption, there are some methods based on the measurement of selected surfactant properties, i.e. surface tension, absorbance or chemical oxygen demand (COD) (Liu et al. 1992). However, methods based on measurement of absorbance or COD can be problematic, because compounds released from the soil can affect the extract color and concentration of organics. As a result, surfactant adsorption may be overestimated. Zhou et al. (2013) confirmed that, after soil sorption experiments, it is difficult to accurately quantify by UV spectrometry the total concentration of Sapindus saponin in aqueous solution. Thus, methods using measurement of surface tension seem to be more adequate. Although the adsorption of various synthetic surfactants has been determined, little is known about biosurfactant adsorption on soil, especially plant-biosurfactants. Therefore, the aim of the present study was to determine the adsorption of two commercially available biosurfactants (saponin and rhamnolipids) at their CMC, using a surface tension technique. The experiments were performed with three soils with different properties and at two ratios of soil to biosurfactant solution. MATERIALS AND METHODS Biosurfactants Two different biosurfactants were used. Chemically-pure saponin (Product No. 16109), a non-ionic plant-derived biosurfactant, was purchased as a powder from RiedeldeHaën, Switzerland. Saponin is an acidic biosurfactant (pH 4.5–5.5) with a density of 1.015-1.020g·mL-1 at 20°C (5% in H2O). It is a mixture of triterpene-glycosides extracted from the bark of the tree Quillaja saponaria, and its hydrophilic part is composed of sugar chains with functional groups. Purum saponin contains 42.3% carbon (C), 6.2% hydrogen (H), 0.2% nitrogen (N), and 51.3% oxygen (O). Reco-10, a 10% mixture of two major rhamnolipids, RLL (R1, C26H48O9) and RRLL (R2, C32H58O13) was purchased from the Jeneil Biosurfactant Co LLC, USA. Chemically, rhamnolipids are glycosides of rhamnose (6-deoxymannose) and p-hydroxydecanoic acid. The rhamnolipids were produced from sterilized and centrifuged fermentation broth. The commercially available product is in the form of a dark brown solution. In contrast to that of saponin, the pH of rhamnolipids ranges from 6 to 7. The chemical structure of both biosurfactants is given in Figure 1. Soils Three soils were collected from different sites in Warmia and Mazury province, Poland: sandy clay loam, SCL-B (Baranowo), clay loam, CL-W (Wanguty) and clay, C-W (Wiktorowo). The soils were air-dried and ground to pass through a 1-mm sieve. The physico-chemical properties of the soils are given in Table 1. Determination of biosurfactant adsorption on soils To determine biosurfactant adsorption on soils, the surface tension of fresh biosurfactant solutions at concentrations from 1 to 10 000mg·L-1 was measured with a Krüss K100 tensiometer employing the Wilhelmy plate method. Then, each biosurfactant solution at a given concentration was shaken with soil (SCL-B, CLW, C-W) at soil/biosurfactant ratios of 1/10 and 1/40 (m/V) on a rotary shaker at 150 rpm for 24h. The supernatants were centrifuged at 8000 rpm for 1h, filtered, and then the surface tension was measured again. The surface tension values were plotted vs. the logarithm of the surfactant concentration. The point of intersection of the two regression lines made on the basis of the experimental data indicates the critical micelle concentration (CMC). The CMC is the lowest aqueous concentration of surfactant at which the surface tension of the solution shows the smallest tensional force (Urum and Pekdemir 2004). The amount of biosurfactant adsorbed on soil at the critical micelle concentration was calculated using the following formula (Zheng and Obbard 2002): Gusiatin Adsorption of biosurfactants to soil 29 Table 1. Physico‐chemical characteristics of the soils.

Surface tension technique as a strategy to evaluate the adsorption of biosurfactants used in soil remediation