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Influence of saponins on the biodegradation of halogenated phenols
Ecotoxicology and Environmental Safety 131 (2016) 127–134
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Influence of saponins on the biodegradation of halogenated phenols Ewa Kaczorek a,n, Wojciech Smułek a, Agata Zdarta a, Agata Sawczuk a, Agnieszka Zgoła-Grześkowiak b a b
Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2016 Received in revised form 11 May 2016 Accepted 17 May 2016
Biotransformation of aromatic compounds is a challenge due to their low aqueous solubility and sorptive losses. The main obstacle in this process is binding of organic pollutants to the microbial cell surface. To overcome these, we applied saponins from plant extract to the microbial culture, to increase pollutants solubility and enhance diffusive massive transfer. This study investigated the efficiency of Quillaja saponaria and Sapindus mukorossi saponins-rich extracts on biodegradation of halogenated phenols by Raoultella planticola WS2 and Pseudomonas sp. OS2, as an effect of cell surface modification of tested strains. Both strains display changes in inner membrane permeability and cell surface hydrophobicity in the presence of saponins during the process of halogenated phenols biotransformation. This allows them to more efficient pollutants removal from the environment. However, only in case of the Pseudomonas sp. OS2 the addition of surfactants to the culture improved effectiveness of bromo-, chloro- and fluorophenols biodegradation. Also introduction of surfactant allowed higher biodegradability of halogenated phenols and can shorten the process. Therefore this suggests that usage of plant saponins can indicate more successful halogenated phenols biodegradation for selected strains. & 2016 Elsevier Inc. All rights reserved.
Keywords: Biodegradation Cell surface properties Halophenols HPLC-MS/MS Saponins
1. Introduction The chemical and petroleum industries generate a number of highly toxic organic pollutants that contribute to harmful effects on the environment. Waste water from chemical industry often contains aromatic organic compounds, which often are resistant to biological degradation by naturally existing microbes and therefore remains in the environment. It makes these compounds able to transport over long distances and bioaccumulate in the tissues of humans and animals (Monsalvo et al., 2009). Organic pollutants constitute a potential group of chemical compounds, which may pose a threat to human health and animals (Yong et al., 2009; Al-Khalid and El-Naas, 2012). Particularly dangerous to the environment are halogenated organic compounds, including halogenated phenols. Their sources are various industrial products, such as: herbicides, wood preservatives, paints, flame retardants or dyes (Sim et al., 2009). Furthermore, these compounds are difficult to remove from the environment and their biodegradation is relatively small (Juretic et al., 2014). These compounds are not only highly toxic but they also are characterized by mutagenicity and cancerogenicity properties n
Corresponding author. E-mail address: [email protected] (E. Kaczorek).
http://dx.doi.org/10.1016/j.ecoenv.2016.05.015 0147-6513/& 2016 Elsevier Inc. All rights reserved.
(Pera-Titus et al., 2004). Nonetheless, they can be removed from the environment via biological methods, which are more efficient than chemical methods (Olaniran and Igbinosa, 2011). Biodegradation of halogenated aromatic compounds can be performed under aerobic or anaerobic conditions, depending on the type of microorganisms and the structure of pollutant. Microorganisms, mainly bacteria and fungi capable of including these compounds in their metabolic pathways are isolated from samples of soil and water from locations retaining in prolonged contact with the impurities (Travkin et al., 2006; Bergauer et al., 2005; Demnerova et al., 2005). Biodegradation pathways of halogenated aromatic compounds are similar to those for aromatic compounds in general (Commandeur and Parsons, 1990). Many studies demonstrated that chlorophenol can be degraded by microorganisms present in water and sediments as well as in sludge (Crawford et al., 2007; Lallai and Mura, 2004; Liu et al., 2014; Karci, 2014; Moussavi et al., 2014). Furthermore, the biodegradation of halogenated phenols by bacteria strain may occur more efficiently, when is co-cultured with other bacteria or yeast (Ronen et al., 2005). According to some researchers, better results of biodegradation were observed when process was initiated by the less toxic compounds (for example phenol) as growth substrates – cometabolic degradation (Wang and Loh, 2000; Tobajas et al., 2012).
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The halogenated phenols can be degraded by different pathways, which are determined by the physical, chemical, and microbiological components of a particular environment. However, the pathways depend on number of halogen atoms in the molecule. In aerobic biodegradation of chlorophenol the ring is usually dihydroxylated by enzyme oxygenase in the first step. Two hydroxyl groups are positioned either ortho or para to one another on the ring (Pieper and Reineke, 2000). This is followed by cleavage of the ring. Bioremediation of organic compounds can be enhanced by the addition of surfactants to polluted soils. On the one hand surfactants increase solubility and/or dispersion of organic compounds leading to an increase in the contact area between hydrocarbons and microorganisms (Zeng et al., 2011). On the other hand the surface active agents due to sorption on cell surface of microorganisms cause the modification of cell surface hydrophobicity as well as an increase in the permeability of biological membrane (Shoji et al., 2012). Modification of these properties may determine the effectiveness of biodegradation process of different pollutants. In the bioremediation both synthetic and natural surfactants are used. The use of surfactants in bioremediation process should be preceded by their selection. Some of them may have toxic effects by causing membrane disruption leading to cellular lysis. They can also increase membrane permeability causing metabolite leakage. These changes may lead to disorders related to the proper functioning of membrane (Singh et al., 2007). Saponins are a surface active compounds, which are widely distributed in nature, most of all in the kingdom Plantae. They are known primarily with its pharmacological, hemolytic, emulsifying and foaming properties (Yücekutlu and Bildaci, 2008). They are glycosides – sugar derivatives. Furthermore, addition of saponins to polluted environment can cause increase of organic compounds biodegradation (Kobayashi et al., 2012; Choi et al., 2009; Tang et al., 2014; Huang et al., 2014). The aim of this work was to investigate the three halogenated aromatic compounds biodegradation by two environmental bacterial strains: Raoultella planticola WS2 and Pseudomonas sp. OS2. The study focused on the biodegradation of 4-bromophenol, 4-chlorophenol and 4-fluorophenol. An important novelty point of the research was to determine the influence of natural surfactants on effectiveness of halogenated phenols removal from the environment. Two saponins extracts were used as surfactants. The first one was a commercial product – the extract from the bark of the Quillaja saponaria Molina. The second one was obtained from fruits of Sapindus mukorossi tree directly before experiments. Moreover, cell surface hydrophobicity, zeta potential and membrane permeability investigations of tested strains were conducted to explain the interaction in the system surfactant-halophenol-cell surface. This subject has not been investigated so far. What is more, the biodegradation of halogenated phenols is mostly described in cometabolism with intermediants of these compounds.
2. Materials and methods 2.1. Chemicals The hydrocarbons and other fine chemicals employed in this study were of the highest purity grade (99%), produced by Merck (Germany). Haloaromatic compounds such as 4-bromophenol, 4-chlorophenol and 4-fluorophenol were obtained from SigmaAldrich. Halogenated phenol characteristic is shown in Table S1 (Supplementary materials). The surface active agents used in this study are saponins which are natural glycoside-based non-ionic surfactants obtained from two different plants. The first one is a commercial product, an
extract from the bark of the South American soap tree, Quillaja saponaria Molina (Sigma-Aldrich, USA, high grade purity). They are amphipathic glycosides belonging to non-ionic natural surfactants. Structurally they contain one or more hydrophilic glycoside moieties combined with a lipophilic triterpene or steroid derivative. The second one is obtained by special methanol extractions from fruits of Sapindus mukorossi tree (soap nut extract) and removal of both undesirable accompanying substances from the extracts and undesirable solvents (Smułek et al., 2016). Both surfactants contain triterpenoid saponins (Upadhyay and Singh, 2012; Guo and Kenne, 2000). These natural surfactants are characterized by lower toxicity and greater biodegradability compared to synthetic surfactants. Therefore, they can be used in bioremediation of pollutants as an alternative to them.
2.2. Microorganisms and growth conditions for the biodegradation test and measurement of cell surface changes There were two environmental bacterial strains used in the experiments. The Raoultella planticola WS2 was isolated from soil contaminated with crude oil. Contaminated samples were collected from Polish northern areas. The Pseudomonas sp. OS2 was isolated from the activated sludge. Bacterial strains were identified using ID 32 GN biochemical tests (bio-Merieux, France) and molecular techniques. The 16S rRNA gene sequence of the tested strain has been deposited in the GeneBank database of NCBI under accession number KP096507.1 (Raoultella planticola WS2) and KP0965011.1 (Pseudomonas sp. OS2). The mineral salts medium (MSM) used throughout these studies was described previously (Kaczorek et al., 2010). A liquid culture was started by adding a loopful of cells from an agar plate into a 250 mL Erlenmeyer flask containing 50 mL of medium. After approximately 24 h 3–5 mL of this liquid culture was used for the inoculation of the final culture to reach an OD600 0.1 (108 cells per mL).
2.3. Cell surface properties 2.3.1. Microbial adhesion to hydrocarbons (MATH) The cell surface hydrophobicity of the tested bacterial strains was assayed using the method of microbial adhesion to the hydrocarbon (Górna et al., 2011). It checked the influence of growth condition on cell surface modification of the two tested bacterial strains. Raoultella planticola WS2 and Pseudomonas sp. OS2 were grown on different carbon sources: 4-bromophenol, 4-chlorophenol and 4-fluorophenol at concentration 20 mg L 1. Moreover, the addition of natural surfactants to the solution with halophenols was also analysed to determine the effect of this arrangement on the surface of bacteria. The surfactants concentration was determined on the basis of their critical micelle concentration (CMC). For commercial product it was 1.0 g L 1, and for extract from fruits of Sapindus mukorossi tree 0.1 g L 1. The growth temperature was 30 °C, and cells in the exponential growth phase were centrifuged at 8000 g for 5 min and washed twice with the MSM in order to remove residual hydrocarbons and surfactant. The cells were then re-suspended in the MSM to fit an optical density of ca. 1.0. Optical density was measured at 600 nm (OD600) on a UV-Visible Spectrophotometer (Jasco, Germany). Next, 500 mL of hexadecane was added to 5 mL of microbial suspension and vortexed for 2 min. After 30 min the optical density of the aqueous phase was measured. Bacterial adhesion to hydrocarbon was calculated as:
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Cell surface hydrophobicity ( %) = (1–OD600 of aqueous phase after mixing withhexadecane/OD600
Table 1 Parameters of mass spectrometric detection characteristic to phenols, MRM 1 – analytical multiple reaction monitoring transition, MRM 2 – confirmatory multiple reaction monitoring transition. Analyte
of initial aqueous phase)⋅100 2.3.2. Zeta potential The zeta potential was calculated from the Smoluchowski equation (Sze et al., 2003) following measurements of electrophoresis mobility using the ZetaPlus instrument (Brookhaven Instruments Co., USA). The same bacterial systems, such as for the cell surface hydrophobicity, were prepared and analysed to determine the influence of growth condition on the zeta potential of bacterial cells. Bacterial cells from the exponential growth phase were centrifuged at 8000 g for 5 min and washed twice with the MSM to remove residual hydrocarbons. The bacteria were then suspended in the same buffer to a final concentration of ca 108 cfu mL 1. 2.3.3. Inner membrane permeability Bacterial cells from the exponential growth phase were centrifuged at 8000 g for 5 min and cell suspension in MSM was prepared. Next, to 5 mL of cell suspension (OD600 ¼0.8) was added 250 mL o-nitrophenyl-β-d-galactopyranoside (ONGP) in concentration 30 mmol L 1. It was incubated for 2 h at 28 °C. After this time suspension was centrifuged at 4000 g for 5 min and the supernatant was determined on a Jasco UV-Visible Spectrophotometer at 415 nm (Zhang et al., 2013).
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Phenol Fluorophenol Chlorophenol Bromophenol
MRM transitions (precursor ion [M-H] m/z-product ion m/z) MRM 1
Collision energy [V]
MRM 2
Collision energy [V]
93-93 111-91 127-91 171-79
5 25 24 29
93-65 111-63 127-35 173-81
30 31 36 30
to 100 ms. The detected mass transitions and specific parameters of each analyte are summarised in Table 1. Validation parameters of the LC-MS/MS method are given in Table S2 (Supplementary materials). 2.5. Statistical analysis Every experiment was repeated three times and the mean values and statistical error were calculated. Statistical significance between obtained results was marked by different small letters. All the results obtained were statistically analysed using SigmaPlot 11.0 software.
3. Results and discussion 3.1. Cell surface properties of the tested strains
2.4. Biodegradation tests Biodegradation of halophenols in the presence of saponins and without them was tested. The concentration of halogenated aromatic compounds in all biodegradation experiments was 20 mg L 1. The influence of both saponins was also tested. The surfactants concentration was determined on the basis of their CMC. The experiments were performed in twisted 250 mL Duran Schotts glass bottles filled with 50 mL MSM. Experimental samples contained suitable halophenol, a culture medium and a few mL of bacteria stock cultures (to reach an OD of ca. 0.1). In experiments with saponins an appropriate amount of each surfactant was added to such samples. Samples were inoculated on a rotary shaker (120 rpm) at 30 °C during 25 days. Biodegradation was determined using LC-MS/MS chromatography after 6, 9, 12, 17, 22 and 25 days. The LC-MS/MS analysis was carried out using the UltiMate 3000 RSLC chromatographic system from Dionex (Sunnyvale, CA, USA) connected with API 4000 QTRAP triple quadrupole mass spectrometer from AB Sciex (Foster City, CA, USA). Five microliters samples were injected into phenyl XBridge column (50 mm 3 mm I.D; 2.5 mm) from Waters. The mobile phase used for the analysis consisted of 5 mmol L 1 ammonium acetate in water and methanol at a flow rate of 0.3 mL min 1. The following gradient was used: 0 min 50%; 2 min 100%; 4 min 100% of methanol. The LC column effluent was directed to the mass spectrometer through the electrospray ionization source (Turbo Ion Spray). The ionization source operated in negative ion mode. All phenols were detected using the following settings for the ion source and mass spectrometer: curtain gas 10 psi, nebulizer gas 45 psi, auxiliary gas 45 psi, temperature 450 °C, ion spray voltage 4500 V and collision gas set to medium. Declustering potential was set to 40 V and the dwell time for each mass transition detected in the MS/MS multiple reaction monitoring mode was set
3.1.1. Membrane permeability The influence of halogenated phenols in the absence and presence of two surfactants on permeate the inner membrane of two tested bacterial strains: R. planticola WS2 (Fig. 1a) and Pseudomonas sp. OS2 (Fig. 1b) was examined. This study was based on the release of cytoplasmic β-galactosidase, which concentration was determinated. Among tested halogenated phenols, 4-bromophenol caused significantly increase of the inner membrane permeability for both tested strains (0.0647 mM/min for R. planticola WS2 strain and 0.0902 mM/min for Pseudomonas sp. OS2 strain). Nevertheless, addition of surfactants to systems containing 4-bromophenol reduced β-galactosidase release. For R. planticola WS2 strain suppression reached level observed in the control sample (0.0144 mM/min). Presence of 4-chloro- and 4-fluorophenol in the system with and without surfactants has no influence on the inner membrane permeability for R. planticola WS2 strain. On the other hand, presence of 4-chloro- and 4-fluorophenol showed enhanced β-galactosidase release in comparison to the control sample (growth on glucose) for Pseudomonas sp. OS2 strain (0.0403 mM/min for 4-chlorophenol and 0.0443 mM/min for 4-fluorophenol). For system with 4-chlorophenol addition of surfactants reduced the inner membrane permeability to 0.0278 mM /min for commercial saponins and to 0.0255 mM/min for soap nut extract. However, level observed in the control sample was not reached. Surfactants at low concentration may change permeability of the membrane (Van Hamme et al., 2006). Moreover, Shoji et al. (2012) demonstrated, that increase of the number of surfactants molecule in the membrane may cause that it becomes weaker and shrunk. As it was presented in this study addition of surfactants can play an important role only in some systems. 3.1.2. Microbial adhesion to hydrocarbon A study was conducted to determine the impact of halogenated
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Fig. 1. Effects of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract) on the inner membrane permeability of Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) cells.
phenols and tested surfactants on the surface properties of the bacteria used in experiments. The presence of halogenated phenols in the system differently modifies surface of tested strains. Results obtained in this study showed that during cultivation of halogenated phenols R. planticola WS2 cells are characterized by hydrophilic properties (Fig. 2a). Among halogenated phenols, presence of 4-fluorophenol only caused the highest modification of bacteria cell surface (24%). The addition of surfactants, did not affect significantly the surface modification of the tested strain. What is more, it was observed that the addition of soap nuts extract to 4-fluorophenol system results in decrease of CSH (from 24% for 4-fluorophenol to 11% for 4-fluorophenol-soap nuts extract). Such dependency was not observed for commercial saponins. Among of the three halogenated phenols, 4-fluorophenol, modifies the cell surface of Pseudomonas sp. OS2 strain the most (Fig. 2b), as for R. planticola WS2. However, the Pseudomonas sp. OS2 strain was characterized by hydrophobic properties (57%) unlike R. planticola WS2. As was shown in Fig. 2b both surfactants promoted CSH in 4-bromophenol system. For 4-chlorophenol CSH was promoted for soap nut extract while addition of commercial surfactant had little influence. On the other hand, in the case of 4-fluorophenol system CSH increase was observed for commercial saponins only (70% for Pseudomonas sp. OS2 and 24% for R. planticola WS2). The maximum CSH appeared in the presence of surfactants. It was observed for commercial saponins added to
Fig. 2. Microbial adhesion of hydrocarbon to Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) cells. Cells were grown on halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract). Process was carried out at 30 °C and cells in the exponential growth phase were used in experiments. Each value is the average of triplicate determinations with the standard deviation in the range of 71.5%.
4-bromophenol system (78%) and 4-fluorophenol system (70%), as well as for soap nut extract added for 4-chlorophenol system (80%). The interaction between surface active agents and bacteria may play a crucial role to make use of various hydrocarbon pollution as a carbon and energy source by microorganisms. The result of this may be more effective biodegradation of organic compounds. Cell surface hydrophobicity (CSH) is an important factor governing the growth and adhesion of microorganisms to various surfaces (Chakrabortya et al., 2010; Zita and Hermansson, 1997). CSH between 0% and 30% means that cells with hydrophilic properties dominate in the culture (Kaczorek and Olszanowski, 2011). Moreover, it has been assumed that CSH above 50% indicates a preponderance of cells with hydrophobic properties. In the case of biodegradation of hydrophobic organic compounds, having by microorganisms hydrophobic properties seems to be important factor. Nevertheless, strains of microorganisms with hydrophilic characteristics are also able to dispose of organic pollutants from environment (Obuekwe et al., 2009). Moreover, Tribedi and Sil (2013) demonstrated that CSH plays a vital role in the bioremediation process of nonpolar polymers by promoting cellular attachment and formation of biofilm on polymer surface.
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The modification of cell surface properties may be the result of surfactants adsorption. These compounds are often used in the bioremediation process to increase the effectiveness of the removal of hydrocarbon contaminants from the environment. Moreover, Lanzon and Brown (2013) demonstrated that surfactant sorption onto the bacterial cell surface is mostly in the form of hemi-micelles. Furthermore, the presence of surfactants can lead to the removal of lipopolysaccharide (LPS) and changes in surface properties of cells (Al-Tahhan et al., 2000). The presence of surfactants may either increase or decrease the cell surface hydrophobicity and therefore influence the uptake and biodegradation of organic pollutants by microorganisms (Zhao et al., 2011). Surfactants can also interact with microbial membrane proteins and modify enzyme conformation (Kamiya et al., 2000). Results obtained in this study indicate that saponins modify the cell surface properties of tested bacterial strains in different way. In the presence of surfactants, the Pseudomonas sp. OS2 strain is characterized by more hydrophobic properties than R. planticola WS2 strain. 3.1.3. Zeta potential The surface charge on bacteria due to the presence of anionic surface groups at neutral pH is negative. It is determined by measuring the zeta potential, which besides CSH is important factor in bacterial adhesion. The changes in this parameter after the introduction of surfactants to halogenated phenols were studied (Fig. 3a and b). The zeta potential of halogenated-grown cells of Pseudomonas sp. OS2 was much higher than for R. planticola WS2 strain. In the case of Pseudomonas sp. OS2 the potential zeta was determinated from 9.1 mV (for 4-fluorophenol) to 11.9 mV (for bromiophenol). However, for R. planticola WS2 cells the zeta-potential was 18.7 mV (for 4-chlorophenol) to 24.4 mV (for 4-bromophenol). What is more, Pseudomonas sp. OS2 strain was more susceptible to changes in surface charge after the addition of saponins to the system (Fig. 3b). Results of the zeta potential were comparable with the results obtained for the R. planticola WS2 strain in the same systems halogenated phenols surfactants respectively. Liu et al. (2012) showed in their study that the pre-treatments by surfactants increased the zeta potential and CSH of Penicillium simplicissimum. The addition of surfactants changed the cell surface functional groups. According to Poortinga et al. (2002) an impact on bacterial adhesion to surface is related to the structure of bacterial cell wall. It is structurally and chemically more complex and heterogeneous than the surface of synthetic colloidal particles. Mohanty and Mukherji (2013) demonstrated that the zeta potential of Burkholderia multivorans, when grown on model hydrocarbon and surfactants depends not only on kind of surfactants but also on hydrocarbon composition. 3.2. Biodegradation of halogenated aromatic compounds Biodegradation of halogenated phenols depends on type of halogen atom, kind of bacteria species and presence of surfactants. For the tested halogenated phenols biodegraded by R. planticola WS2 and Pseudomonas sp. OS2 the highest primary biodegradation rate was noted for 4-fluorophenol and the lowest for 4-bromophenol. R. planticola WS2 bacteria degraded 4-fluorophenol in 12 days (Fig. 4). Addition of commercial saponins do not considerably affect biodegradation of 4-fluorophenol by this strain while addition of soap nut extract led to a few days faster biodegradation (total primary biodegradation after 9 days). More considerable influence of surfactant addition was observed for biodegradation of 4-fluorophenol with Pseudomonas sp. OS2 (Fig. 5). Both saponins and soap nut extract considerably improved biodegradation of 4-fluorophenol by Pseudomonas sp. OS2. The total primary biodegradation of 4-fluorophenol after soap nut extract addition
Fig. 3. Zeta potential of Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) grown on halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract). For each sample, three measurements were made, the accuracies of the measurements being 7 0.01 mV.
was observed after 9 days and for commercial saponins after 12 days. According to Kim et al. (2010) electron-withdrawing nature of fluorine has a significant impact on the catabolism of fluorinated organic compounds. These compounds due to the stability of carbon-fluorine bond are less biodegradable in comparison to their unsubstituted analogues (Key et al., 1997). However, there are known microorganisms capable to grow with these compounds as a sole source of carbon and energy, as Arthrobacter strain IF1 (Ferreira et al., 2008), Rhodococcus (Finkelstein et al., 2000) or Pseudonocardia benzenivorans (Kim et al., 2010). The results obtained in this study show that the two tested strains are also capable to grow on fluorinated compounds i.e. on 4-fluorophenol. Biodegradation of 4-chlorophenol by R. planticola WS2 was slower than it was noted for 4-fluorophenol but still practically complete primary degradation was achieved after 2 weeks. Positive influence of commercial saponins and soap nut extract was also noted. However, Pseudomonas sp. OS2 could not considerably degrade 4-chlorophenol. Less than 20% primary biodegradation was achieved for this strain during 25 days of process. Addition of surfactants improved biodegradation, but not more than to 40% after 22 days. The biodegradation of 4-chlorophenol usually occurs by oxidation to 4-chlorocatechol, followed by ortho cleavage of the aromatic ring (Park and Kim, 2003). Subsequently, the chlorine atom is removed. What is more, 4-chlorophenol may be also
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Fig. 4. The influence of surfactants (S – commercial saponins, SN – soap nut extract) on the primary biodegradation of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol by Raoultella planticola WS2 strain. Process was carried out at 30 °C during 25 days. Biodegradation was determined after 6, 9, 12, 17, 22 and 25 days.
Fig. 5. The influence of surfactants (S – commercial saponins, SN – soap nut extract) on the primary biodegradation of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol by Pseudomonas sp. OS2 strain. Process was carried out at 30 °C during 25 days. Biodegradation was determined after 6, 9, 12, 17, 22 and 25 days.
transformed into hydroquinone and next hydroxylated to yield hydroxyquinol (Nordin et al., 2005). Different bacterial genera are capable to degrade chlorophenolic compounds, and some of them possess plasmids with code for the catabolic genes (Olaniran and Igbinosa, 2011). Biodegradation of 4-bromophenol by two tested bacterial species was very low and it did not exceed 20% during 25 days of process. Only very small effect of surfactants was observed for R. planticola WS2 and there was no influence of surfactants when Pseudomonas sp. OS2 was used for biodegradation of 4-bromophenol. These compounds due to their wide usage in chemical industry can accumulate in the environment and may have a negative impact on living organisms (Howe et al., 2005). Their biodegradation is possible by selected microorganisms (Sahoo et al., 2014), although more efficiency is observed when degradation of 4-bromophenol is co-cultured with yeast and other bacteria (Ronen et al., 2005).
Of the two bacterial strains tested better efficiency of biodegradation of halogenated phenols was obtained for R. planticola WS2. The surfactants addition did not affect significantly the result of biodegradation. This strain was characterized by the hydrophilic properties of the cell surface and relatively low cell surface charge. Generally, the introduction of surfactants did not affect in a significant way to modify the surface properties of strain (CSH and zeta potential). Moreover, different type of surfactants in different way impacts the biodegradation of organic compounds. It may be modification of cell surface hydrophobicity, as well as disruption of bacterial membrane (Zhang et al., 2013). In the biodegradation of phenol and its derivatives particularly active are bacteria of the genus Pseudomonas. However, also Ralstonia eutropha (Tepe and Dursun, 2008) or Arthobacter (Ferreira et al., 2008) have ability to use a variety of aromatic compounds including halogenated compounds. In the case of the second test (strain Pseudomonas sp. OS2) the addition of surfactants to halogenated phenols system improve effectiveness of their biodegradation. Better results were obtained after application of the extract from the soap nuts than
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for commercial saponins. The introduction of surfactant allowed higher biodegradability of halogenated phenols and in the case of 4-fluorophenol the total primary biodegradation was achieved after 9 days of the experiment. However, the total primary biodegradation in the absence of surfactant was obtained after 25 days. The introduction of surfactants caused significantly changes in cell surface modification. An increase in CSH and surface charge was observed, which resulted in higher halogenated phenols biodegradation. The tested bacterial strains and the natural surfactants as well may be used in industrial wastewater treatment plants, similarly as it was described e.g. by Gonzales et al. (2001). In their study they used fluidized bed bioreactor to decrease concentration of the phenolic compounds in raw wastewaters from phenolic resins production. The saponins surfactants can also be used during treatment of highly toxic wastewaters in full-scale bioreactor like that presented by Dao et al. (2014).
4. Conclusion The efficiency of biodegradation of halogenated phenols is related to halogen atom, surfactants used in experiments, as well as cell surface properties of microorganisms. The addition of saponins may enhance biodegradation or reduce the time required to completely remove impurities from the environment. Saponins differently influence cell surface properties of tested strain. In the case of Pseudomonas sp. OS2, increase of biodegradation was correlated with higher cell surface hydrophobicity and lower zeta potential than in system with absence of surfactants. However, for Raoultella planticola WS2 significant cell surface modification after surfactant addition was not observed.
Acknowledgments This study was supported by The National Science Centre (Poland) awarded by decisions number DEC-2012/07/B/NZ9/00950.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.05. 015.
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Influence of saponins on the biodegradation of halogenated phenols Ewa Kaczorek a,n, Wojciech Smułek a, Agata Zdarta a, Agata Sawczuk a, Agnieszka Zgoła-Grześkowiak b a b
Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, Berdychowo 4, 60-965 Poznan, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 February 2016 Received in revised form 11 May 2016 Accepted 17 May 2016
Biotransformation of aromatic compounds is a challenge due to their low aqueous solubility and sorptive losses. The main obstacle in this process is binding of organic pollutants to the microbial cell surface. To overcome these, we applied saponins from plant extract to the microbial culture, to increase pollutants solubility and enhance diffusive massive transfer. This study investigated the efficiency of Quillaja saponaria and Sapindus mukorossi saponins-rich extracts on biodegradation of halogenated phenols by Raoultella planticola WS2 and Pseudomonas sp. OS2, as an effect of cell surface modification of tested strains. Both strains display changes in inner membrane permeability and cell surface hydrophobicity in the presence of saponins during the process of halogenated phenols biotransformation. This allows them to more efficient pollutants removal from the environment. However, only in case of the Pseudomonas sp. OS2 the addition of surfactants to the culture improved effectiveness of bromo-, chloro- and fluorophenols biodegradation. Also introduction of surfactant allowed higher biodegradability of halogenated phenols and can shorten the process. Therefore this suggests that usage of plant saponins can indicate more successful halogenated phenols biodegradation for selected strains. & 2016 Elsevier Inc. All rights reserved.
Keywords: Biodegradation Cell surface properties Halophenols HPLC-MS/MS Saponins
1. Introduction The chemical and petroleum industries generate a number of highly toxic organic pollutants that contribute to harmful effects on the environment. Waste water from chemical industry often contains aromatic organic compounds, which often are resistant to biological degradation by naturally existing microbes and therefore remains in the environment. It makes these compounds able to transport over long distances and bioaccumulate in the tissues of humans and animals (Monsalvo et al., 2009). Organic pollutants constitute a potential group of chemical compounds, which may pose a threat to human health and animals (Yong et al., 2009; Al-Khalid and El-Naas, 2012). Particularly dangerous to the environment are halogenated organic compounds, including halogenated phenols. Their sources are various industrial products, such as: herbicides, wood preservatives, paints, flame retardants or dyes (Sim et al., 2009). Furthermore, these compounds are difficult to remove from the environment and their biodegradation is relatively small (Juretic et al., 2014). These compounds are not only highly toxic but they also are characterized by mutagenicity and cancerogenicity properties n
Corresponding author. E-mail address: [email protected] (E. Kaczorek).
http://dx.doi.org/10.1016/j.ecoenv.2016.05.015 0147-6513/& 2016 Elsevier Inc. All rights reserved.
(Pera-Titus et al., 2004). Nonetheless, they can be removed from the environment via biological methods, which are more efficient than chemical methods (Olaniran and Igbinosa, 2011). Biodegradation of halogenated aromatic compounds can be performed under aerobic or anaerobic conditions, depending on the type of microorganisms and the structure of pollutant. Microorganisms, mainly bacteria and fungi capable of including these compounds in their metabolic pathways are isolated from samples of soil and water from locations retaining in prolonged contact with the impurities (Travkin et al., 2006; Bergauer et al., 2005; Demnerova et al., 2005). Biodegradation pathways of halogenated aromatic compounds are similar to those for aromatic compounds in general (Commandeur and Parsons, 1990). Many studies demonstrated that chlorophenol can be degraded by microorganisms present in water and sediments as well as in sludge (Crawford et al., 2007; Lallai and Mura, 2004; Liu et al., 2014; Karci, 2014; Moussavi et al., 2014). Furthermore, the biodegradation of halogenated phenols by bacteria strain may occur more efficiently, when is co-cultured with other bacteria or yeast (Ronen et al., 2005). According to some researchers, better results of biodegradation were observed when process was initiated by the less toxic compounds (for example phenol) as growth substrates – cometabolic degradation (Wang and Loh, 2000; Tobajas et al., 2012).
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The halogenated phenols can be degraded by different pathways, which are determined by the physical, chemical, and microbiological components of a particular environment. However, the pathways depend on number of halogen atoms in the molecule. In aerobic biodegradation of chlorophenol the ring is usually dihydroxylated by enzyme oxygenase in the first step. Two hydroxyl groups are positioned either ortho or para to one another on the ring (Pieper and Reineke, 2000). This is followed by cleavage of the ring. Bioremediation of organic compounds can be enhanced by the addition of surfactants to polluted soils. On the one hand surfactants increase solubility and/or dispersion of organic compounds leading to an increase in the contact area between hydrocarbons and microorganisms (Zeng et al., 2011). On the other hand the surface active agents due to sorption on cell surface of microorganisms cause the modification of cell surface hydrophobicity as well as an increase in the permeability of biological membrane (Shoji et al., 2012). Modification of these properties may determine the effectiveness of biodegradation process of different pollutants. In the bioremediation both synthetic and natural surfactants are used. The use of surfactants in bioremediation process should be preceded by their selection. Some of them may have toxic effects by causing membrane disruption leading to cellular lysis. They can also increase membrane permeability causing metabolite leakage. These changes may lead to disorders related to the proper functioning of membrane (Singh et al., 2007). Saponins are a surface active compounds, which are widely distributed in nature, most of all in the kingdom Plantae. They are known primarily with its pharmacological, hemolytic, emulsifying and foaming properties (Yücekutlu and Bildaci, 2008). They are glycosides – sugar derivatives. Furthermore, addition of saponins to polluted environment can cause increase of organic compounds biodegradation (Kobayashi et al., 2012; Choi et al., 2009; Tang et al., 2014; Huang et al., 2014). The aim of this work was to investigate the three halogenated aromatic compounds biodegradation by two environmental bacterial strains: Raoultella planticola WS2 and Pseudomonas sp. OS2. The study focused on the biodegradation of 4-bromophenol, 4-chlorophenol and 4-fluorophenol. An important novelty point of the research was to determine the influence of natural surfactants on effectiveness of halogenated phenols removal from the environment. Two saponins extracts were used as surfactants. The first one was a commercial product – the extract from the bark of the Quillaja saponaria Molina. The second one was obtained from fruits of Sapindus mukorossi tree directly before experiments. Moreover, cell surface hydrophobicity, zeta potential and membrane permeability investigations of tested strains were conducted to explain the interaction in the system surfactant-halophenol-cell surface. This subject has not been investigated so far. What is more, the biodegradation of halogenated phenols is mostly described in cometabolism with intermediants of these compounds.
2. Materials and methods 2.1. Chemicals The hydrocarbons and other fine chemicals employed in this study were of the highest purity grade (99%), produced by Merck (Germany). Haloaromatic compounds such as 4-bromophenol, 4-chlorophenol and 4-fluorophenol were obtained from SigmaAldrich. Halogenated phenol characteristic is shown in Table S1 (Supplementary materials). The surface active agents used in this study are saponins which are natural glycoside-based non-ionic surfactants obtained from two different plants. The first one is a commercial product, an
extract from the bark of the South American soap tree, Quillaja saponaria Molina (Sigma-Aldrich, USA, high grade purity). They are amphipathic glycosides belonging to non-ionic natural surfactants. Structurally they contain one or more hydrophilic glycoside moieties combined with a lipophilic triterpene or steroid derivative. The second one is obtained by special methanol extractions from fruits of Sapindus mukorossi tree (soap nut extract) and removal of both undesirable accompanying substances from the extracts and undesirable solvents (Smułek et al., 2016). Both surfactants contain triterpenoid saponins (Upadhyay and Singh, 2012; Guo and Kenne, 2000). These natural surfactants are characterized by lower toxicity and greater biodegradability compared to synthetic surfactants. Therefore, they can be used in bioremediation of pollutants as an alternative to them.
2.2. Microorganisms and growth conditions for the biodegradation test and measurement of cell surface changes There were two environmental bacterial strains used in the experiments. The Raoultella planticola WS2 was isolated from soil contaminated with crude oil. Contaminated samples were collected from Polish northern areas. The Pseudomonas sp. OS2 was isolated from the activated sludge. Bacterial strains were identified using ID 32 GN biochemical tests (bio-Merieux, France) and molecular techniques. The 16S rRNA gene sequence of the tested strain has been deposited in the GeneBank database of NCBI under accession number KP096507.1 (Raoultella planticola WS2) and KP0965011.1 (Pseudomonas sp. OS2). The mineral salts medium (MSM) used throughout these studies was described previously (Kaczorek et al., 2010). A liquid culture was started by adding a loopful of cells from an agar plate into a 250 mL Erlenmeyer flask containing 50 mL of medium. After approximately 24 h 3–5 mL of this liquid culture was used for the inoculation of the final culture to reach an OD600 0.1 (108 cells per mL).
2.3. Cell surface properties 2.3.1. Microbial adhesion to hydrocarbons (MATH) The cell surface hydrophobicity of the tested bacterial strains was assayed using the method of microbial adhesion to the hydrocarbon (Górna et al., 2011). It checked the influence of growth condition on cell surface modification of the two tested bacterial strains. Raoultella planticola WS2 and Pseudomonas sp. OS2 were grown on different carbon sources: 4-bromophenol, 4-chlorophenol and 4-fluorophenol at concentration 20 mg L 1. Moreover, the addition of natural surfactants to the solution with halophenols was also analysed to determine the effect of this arrangement on the surface of bacteria. The surfactants concentration was determined on the basis of their critical micelle concentration (CMC). For commercial product it was 1.0 g L 1, and for extract from fruits of Sapindus mukorossi tree 0.1 g L 1. The growth temperature was 30 °C, and cells in the exponential growth phase were centrifuged at 8000 g for 5 min and washed twice with the MSM in order to remove residual hydrocarbons and surfactant. The cells were then re-suspended in the MSM to fit an optical density of ca. 1.0. Optical density was measured at 600 nm (OD600) on a UV-Visible Spectrophotometer (Jasco, Germany). Next, 500 mL of hexadecane was added to 5 mL of microbial suspension and vortexed for 2 min. After 30 min the optical density of the aqueous phase was measured. Bacterial adhesion to hydrocarbon was calculated as:
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Cell surface hydrophobicity ( %) = (1–OD600 of aqueous phase after mixing withhexadecane/OD600
Table 1 Parameters of mass spectrometric detection characteristic to phenols, MRM 1 – analytical multiple reaction monitoring transition, MRM 2 – confirmatory multiple reaction monitoring transition. Analyte
of initial aqueous phase)⋅100 2.3.2. Zeta potential The zeta potential was calculated from the Smoluchowski equation (Sze et al., 2003) following measurements of electrophoresis mobility using the ZetaPlus instrument (Brookhaven Instruments Co., USA). The same bacterial systems, such as for the cell surface hydrophobicity, were prepared and analysed to determine the influence of growth condition on the zeta potential of bacterial cells. Bacterial cells from the exponential growth phase were centrifuged at 8000 g for 5 min and washed twice with the MSM to remove residual hydrocarbons. The bacteria were then suspended in the same buffer to a final concentration of ca 108 cfu mL 1. 2.3.3. Inner membrane permeability Bacterial cells from the exponential growth phase were centrifuged at 8000 g for 5 min and cell suspension in MSM was prepared. Next, to 5 mL of cell suspension (OD600 ¼0.8) was added 250 mL o-nitrophenyl-β-d-galactopyranoside (ONGP) in concentration 30 mmol L 1. It was incubated for 2 h at 28 °C. After this time suspension was centrifuged at 4000 g for 5 min and the supernatant was determined on a Jasco UV-Visible Spectrophotometer at 415 nm (Zhang et al., 2013).
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Phenol Fluorophenol Chlorophenol Bromophenol
MRM transitions (precursor ion [M-H] m/z-product ion m/z) MRM 1
Collision energy [V]
MRM 2
Collision energy [V]
93-93 111-91 127-91 171-79
5 25 24 29
93-65 111-63 127-35 173-81
30 31 36 30
to 100 ms. The detected mass transitions and specific parameters of each analyte are summarised in Table 1. Validation parameters of the LC-MS/MS method are given in Table S2 (Supplementary materials). 2.5. Statistical analysis Every experiment was repeated three times and the mean values and statistical error were calculated. Statistical significance between obtained results was marked by different small letters. All the results obtained were statistically analysed using SigmaPlot 11.0 software.
3. Results and discussion 3.1. Cell surface properties of the tested strains
2.4. Biodegradation tests Biodegradation of halophenols in the presence of saponins and without them was tested. The concentration of halogenated aromatic compounds in all biodegradation experiments was 20 mg L 1. The influence of both saponins was also tested. The surfactants concentration was determined on the basis of their CMC. The experiments were performed in twisted 250 mL Duran Schotts glass bottles filled with 50 mL MSM. Experimental samples contained suitable halophenol, a culture medium and a few mL of bacteria stock cultures (to reach an OD of ca. 0.1). In experiments with saponins an appropriate amount of each surfactant was added to such samples. Samples were inoculated on a rotary shaker (120 rpm) at 30 °C during 25 days. Biodegradation was determined using LC-MS/MS chromatography after 6, 9, 12, 17, 22 and 25 days. The LC-MS/MS analysis was carried out using the UltiMate 3000 RSLC chromatographic system from Dionex (Sunnyvale, CA, USA) connected with API 4000 QTRAP triple quadrupole mass spectrometer from AB Sciex (Foster City, CA, USA). Five microliters samples were injected into phenyl XBridge column (50 mm 3 mm I.D; 2.5 mm) from Waters. The mobile phase used for the analysis consisted of 5 mmol L 1 ammonium acetate in water and methanol at a flow rate of 0.3 mL min 1. The following gradient was used: 0 min 50%; 2 min 100%; 4 min 100% of methanol. The LC column effluent was directed to the mass spectrometer through the electrospray ionization source (Turbo Ion Spray). The ionization source operated in negative ion mode. All phenols were detected using the following settings for the ion source and mass spectrometer: curtain gas 10 psi, nebulizer gas 45 psi, auxiliary gas 45 psi, temperature 450 °C, ion spray voltage 4500 V and collision gas set to medium. Declustering potential was set to 40 V and the dwell time for each mass transition detected in the MS/MS multiple reaction monitoring mode was set
3.1.1. Membrane permeability The influence of halogenated phenols in the absence and presence of two surfactants on permeate the inner membrane of two tested bacterial strains: R. planticola WS2 (Fig. 1a) and Pseudomonas sp. OS2 (Fig. 1b) was examined. This study was based on the release of cytoplasmic β-galactosidase, which concentration was determinated. Among tested halogenated phenols, 4-bromophenol caused significantly increase of the inner membrane permeability for both tested strains (0.0647 mM/min for R. planticola WS2 strain and 0.0902 mM/min for Pseudomonas sp. OS2 strain). Nevertheless, addition of surfactants to systems containing 4-bromophenol reduced β-galactosidase release. For R. planticola WS2 strain suppression reached level observed in the control sample (0.0144 mM/min). Presence of 4-chloro- and 4-fluorophenol in the system with and without surfactants has no influence on the inner membrane permeability for R. planticola WS2 strain. On the other hand, presence of 4-chloro- and 4-fluorophenol showed enhanced β-galactosidase release in comparison to the control sample (growth on glucose) for Pseudomonas sp. OS2 strain (0.0403 mM/min for 4-chlorophenol and 0.0443 mM/min for 4-fluorophenol). For system with 4-chlorophenol addition of surfactants reduced the inner membrane permeability to 0.0278 mM /min for commercial saponins and to 0.0255 mM/min for soap nut extract. However, level observed in the control sample was not reached. Surfactants at low concentration may change permeability of the membrane (Van Hamme et al., 2006). Moreover, Shoji et al. (2012) demonstrated, that increase of the number of surfactants molecule in the membrane may cause that it becomes weaker and shrunk. As it was presented in this study addition of surfactants can play an important role only in some systems. 3.1.2. Microbial adhesion to hydrocarbon A study was conducted to determine the impact of halogenated
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Fig. 1. Effects of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract) on the inner membrane permeability of Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) cells.
phenols and tested surfactants on the surface properties of the bacteria used in experiments. The presence of halogenated phenols in the system differently modifies surface of tested strains. Results obtained in this study showed that during cultivation of halogenated phenols R. planticola WS2 cells are characterized by hydrophilic properties (Fig. 2a). Among halogenated phenols, presence of 4-fluorophenol only caused the highest modification of bacteria cell surface (24%). The addition of surfactants, did not affect significantly the surface modification of the tested strain. What is more, it was observed that the addition of soap nuts extract to 4-fluorophenol system results in decrease of CSH (from 24% for 4-fluorophenol to 11% for 4-fluorophenol-soap nuts extract). Such dependency was not observed for commercial saponins. Among of the three halogenated phenols, 4-fluorophenol, modifies the cell surface of Pseudomonas sp. OS2 strain the most (Fig. 2b), as for R. planticola WS2. However, the Pseudomonas sp. OS2 strain was characterized by hydrophobic properties (57%) unlike R. planticola WS2. As was shown in Fig. 2b both surfactants promoted CSH in 4-bromophenol system. For 4-chlorophenol CSH was promoted for soap nut extract while addition of commercial surfactant had little influence. On the other hand, in the case of 4-fluorophenol system CSH increase was observed for commercial saponins only (70% for Pseudomonas sp. OS2 and 24% for R. planticola WS2). The maximum CSH appeared in the presence of surfactants. It was observed for commercial saponins added to
Fig. 2. Microbial adhesion of hydrocarbon to Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) cells. Cells were grown on halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract). Process was carried out at 30 °C and cells in the exponential growth phase were used in experiments. Each value is the average of triplicate determinations with the standard deviation in the range of 71.5%.
4-bromophenol system (78%) and 4-fluorophenol system (70%), as well as for soap nut extract added for 4-chlorophenol system (80%). The interaction between surface active agents and bacteria may play a crucial role to make use of various hydrocarbon pollution as a carbon and energy source by microorganisms. The result of this may be more effective biodegradation of organic compounds. Cell surface hydrophobicity (CSH) is an important factor governing the growth and adhesion of microorganisms to various surfaces (Chakrabortya et al., 2010; Zita and Hermansson, 1997). CSH between 0% and 30% means that cells with hydrophilic properties dominate in the culture (Kaczorek and Olszanowski, 2011). Moreover, it has been assumed that CSH above 50% indicates a preponderance of cells with hydrophobic properties. In the case of biodegradation of hydrophobic organic compounds, having by microorganisms hydrophobic properties seems to be important factor. Nevertheless, strains of microorganisms with hydrophilic characteristics are also able to dispose of organic pollutants from environment (Obuekwe et al., 2009). Moreover, Tribedi and Sil (2013) demonstrated that CSH plays a vital role in the bioremediation process of nonpolar polymers by promoting cellular attachment and formation of biofilm on polymer surface.
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The modification of cell surface properties may be the result of surfactants adsorption. These compounds are often used in the bioremediation process to increase the effectiveness of the removal of hydrocarbon contaminants from the environment. Moreover, Lanzon and Brown (2013) demonstrated that surfactant sorption onto the bacterial cell surface is mostly in the form of hemi-micelles. Furthermore, the presence of surfactants can lead to the removal of lipopolysaccharide (LPS) and changes in surface properties of cells (Al-Tahhan et al., 2000). The presence of surfactants may either increase or decrease the cell surface hydrophobicity and therefore influence the uptake and biodegradation of organic pollutants by microorganisms (Zhao et al., 2011). Surfactants can also interact with microbial membrane proteins and modify enzyme conformation (Kamiya et al., 2000). Results obtained in this study indicate that saponins modify the cell surface properties of tested bacterial strains in different way. In the presence of surfactants, the Pseudomonas sp. OS2 strain is characterized by more hydrophobic properties than R. planticola WS2 strain. 3.1.3. Zeta potential The surface charge on bacteria due to the presence of anionic surface groups at neutral pH is negative. It is determined by measuring the zeta potential, which besides CSH is important factor in bacterial adhesion. The changes in this parameter after the introduction of surfactants to halogenated phenols were studied (Fig. 3a and b). The zeta potential of halogenated-grown cells of Pseudomonas sp. OS2 was much higher than for R. planticola WS2 strain. In the case of Pseudomonas sp. OS2 the potential zeta was determinated from 9.1 mV (for 4-fluorophenol) to 11.9 mV (for bromiophenol). However, for R. planticola WS2 cells the zeta-potential was 18.7 mV (for 4-chlorophenol) to 24.4 mV (for 4-bromophenol). What is more, Pseudomonas sp. OS2 strain was more susceptible to changes in surface charge after the addition of saponins to the system (Fig. 3b). Results of the zeta potential were comparable with the results obtained for the R. planticola WS2 strain in the same systems halogenated phenols surfactants respectively. Liu et al. (2012) showed in their study that the pre-treatments by surfactants increased the zeta potential and CSH of Penicillium simplicissimum. The addition of surfactants changed the cell surface functional groups. According to Poortinga et al. (2002) an impact on bacterial adhesion to surface is related to the structure of bacterial cell wall. It is structurally and chemically more complex and heterogeneous than the surface of synthetic colloidal particles. Mohanty and Mukherji (2013) demonstrated that the zeta potential of Burkholderia multivorans, when grown on model hydrocarbon and surfactants depends not only on kind of surfactants but also on hydrocarbon composition. 3.2. Biodegradation of halogenated aromatic compounds Biodegradation of halogenated phenols depends on type of halogen atom, kind of bacteria species and presence of surfactants. For the tested halogenated phenols biodegraded by R. planticola WS2 and Pseudomonas sp. OS2 the highest primary biodegradation rate was noted for 4-fluorophenol and the lowest for 4-bromophenol. R. planticola WS2 bacteria degraded 4-fluorophenol in 12 days (Fig. 4). Addition of commercial saponins do not considerably affect biodegradation of 4-fluorophenol by this strain while addition of soap nut extract led to a few days faster biodegradation (total primary biodegradation after 9 days). More considerable influence of surfactant addition was observed for biodegradation of 4-fluorophenol with Pseudomonas sp. OS2 (Fig. 5). Both saponins and soap nut extract considerably improved biodegradation of 4-fluorophenol by Pseudomonas sp. OS2. The total primary biodegradation of 4-fluorophenol after soap nut extract addition
Fig. 3. Zeta potential of Raoultella planticola WS2 (a) and Pseudomonas sp. OS2 (b) grown on halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol in the absence and presence of surfactants (S – commercial saponins, SN – soap nut extract). For each sample, three measurements were made, the accuracies of the measurements being 7 0.01 mV.
was observed after 9 days and for commercial saponins after 12 days. According to Kim et al. (2010) electron-withdrawing nature of fluorine has a significant impact on the catabolism of fluorinated organic compounds. These compounds due to the stability of carbon-fluorine bond are less biodegradable in comparison to their unsubstituted analogues (Key et al., 1997). However, there are known microorganisms capable to grow with these compounds as a sole source of carbon and energy, as Arthrobacter strain IF1 (Ferreira et al., 2008), Rhodococcus (Finkelstein et al., 2000) or Pseudonocardia benzenivorans (Kim et al., 2010). The results obtained in this study show that the two tested strains are also capable to grow on fluorinated compounds i.e. on 4-fluorophenol. Biodegradation of 4-chlorophenol by R. planticola WS2 was slower than it was noted for 4-fluorophenol but still practically complete primary degradation was achieved after 2 weeks. Positive influence of commercial saponins and soap nut extract was also noted. However, Pseudomonas sp. OS2 could not considerably degrade 4-chlorophenol. Less than 20% primary biodegradation was achieved for this strain during 25 days of process. Addition of surfactants improved biodegradation, but not more than to 40% after 22 days. The biodegradation of 4-chlorophenol usually occurs by oxidation to 4-chlorocatechol, followed by ortho cleavage of the aromatic ring (Park and Kim, 2003). Subsequently, the chlorine atom is removed. What is more, 4-chlorophenol may be also
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Fig. 4. The influence of surfactants (S – commercial saponins, SN – soap nut extract) on the primary biodegradation of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol by Raoultella planticola WS2 strain. Process was carried out at 30 °C during 25 days. Biodegradation was determined after 6, 9, 12, 17, 22 and 25 days.
Fig. 5. The influence of surfactants (S – commercial saponins, SN – soap nut extract) on the primary biodegradation of halogenated phenols – H: 4-fluorophenol, 4-chlorophenol and 4-bromophenol by Pseudomonas sp. OS2 strain. Process was carried out at 30 °C during 25 days. Biodegradation was determined after 6, 9, 12, 17, 22 and 25 days.
transformed into hydroquinone and next hydroxylated to yield hydroxyquinol (Nordin et al., 2005). Different bacterial genera are capable to degrade chlorophenolic compounds, and some of them possess plasmids with code for the catabolic genes (Olaniran and Igbinosa, 2011). Biodegradation of 4-bromophenol by two tested bacterial species was very low and it did not exceed 20% during 25 days of process. Only very small effect of surfactants was observed for R. planticola WS2 and there was no influence of surfactants when Pseudomonas sp. OS2 was used for biodegradation of 4-bromophenol. These compounds due to their wide usage in chemical industry can accumulate in the environment and may have a negative impact on living organisms (Howe et al., 2005). Their biodegradation is possible by selected microorganisms (Sahoo et al., 2014), although more efficiency is observed when degradation of 4-bromophenol is co-cultured with yeast and other bacteria (Ronen et al., 2005).
Of the two bacterial strains tested better efficiency of biodegradation of halogenated phenols was obtained for R. planticola WS2. The surfactants addition did not affect significantly the result of biodegradation. This strain was characterized by the hydrophilic properties of the cell surface and relatively low cell surface charge. Generally, the introduction of surfactants did not affect in a significant way to modify the surface properties of strain (CSH and zeta potential). Moreover, different type of surfactants in different way impacts the biodegradation of organic compounds. It may be modification of cell surface hydrophobicity, as well as disruption of bacterial membrane (Zhang et al., 2013). In the biodegradation of phenol and its derivatives particularly active are bacteria of the genus Pseudomonas. However, also Ralstonia eutropha (Tepe and Dursun, 2008) or Arthobacter (Ferreira et al., 2008) have ability to use a variety of aromatic compounds including halogenated compounds. In the case of the second test (strain Pseudomonas sp. OS2) the addition of surfactants to halogenated phenols system improve effectiveness of their biodegradation. Better results were obtained after application of the extract from the soap nuts than
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for commercial saponins. The introduction of surfactant allowed higher biodegradability of halogenated phenols and in the case of 4-fluorophenol the total primary biodegradation was achieved after 9 days of the experiment. However, the total primary biodegradation in the absence of surfactant was obtained after 25 days. The introduction of surfactants caused significantly changes in cell surface modification. An increase in CSH and surface charge was observed, which resulted in higher halogenated phenols biodegradation. The tested bacterial strains and the natural surfactants as well may be used in industrial wastewater treatment plants, similarly as it was described e.g. by Gonzales et al. (2001). In their study they used fluidized bed bioreactor to decrease concentration of the phenolic compounds in raw wastewaters from phenolic resins production. The saponins surfactants can also be used during treatment of highly toxic wastewaters in full-scale bioreactor like that presented by Dao et al. (2014).
4. Conclusion The efficiency of biodegradation of halogenated phenols is related to halogen atom, surfactants used in experiments, as well as cell surface properties of microorganisms. The addition of saponins may enhance biodegradation or reduce the time required to completely remove impurities from the environment. Saponins differently influence cell surface properties of tested strain. In the case of Pseudomonas sp. OS2, increase of biodegradation was correlated with higher cell surface hydrophobicity and lower zeta potential than in system with absence of surfactants. However, for Raoultella planticola WS2 significant cell surface modification after surfactant addition was not observed.
Acknowledgments This study was supported by The National Science Centre (Poland) awarded by decisions number DEC-2012/07/B/NZ9/00950.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.05. 015.
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