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Biosurfactant-enhanced phytoremediation of soils contaminated by crude oil using maize (Zea mays. L)
Ecological Engineering 92 (2016) 10–17
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Biosurfactant-enhanced phytoremediation of soils contaminated by crude oil using maize (Zea mays. L) Changjun Liao a,b , Wending Xu a,c , Guining Lu a,d,e,∗ , Fucai Deng a , Xujun Liang a , Chuling Guo a,d , Zhi Dang a,d,e,∗ a
School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China Department of Environmental Engineering, Guangdong Vocational College of Environmental Protection Engineering, Foshan 528216, PR China c School of Municipal and Environmental Engineering, Henan University of Urban Construction, Pingdingshan 467036, PR China d The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, South China University of Technology, Guangzhou 510006, PR China e Guangdong Provincial Engineering and Technology Research Center for Environmental Risk Prevention and Emergency Disposal, South China University of Technology, Guangzhou 510006, PR China b
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 21 March 2016 Accepted 22 March 2016 Keywords: Biosurfactant Phytoremediation Crude oil Contaminated soil PAHs
a b s t r a c t Surfactant-enhanced phytoremediation is a green technology for the treatment of contaminated soil. In a pot experiment, two biosurfactants (rhamnolipid and soybean lecithin) and a synthetic surfactant (Tween 80) were used to facilitate phytoremediation of crude oil contaminated soil by maize (Zea mays. L). Results showed that these surfactants did not significantly affect the biomass production of maize, but they inhibited the chlorophyll fluorescence of the maize leaf. Rhamnolipid and soybean lecithin enhanced the soil microbial population, resulting in increased removal of total petroleum hydrocarbons from the soil. Saturated hydrocarbons were the main component of petroleum hydrocarbons decreased in the soil. In addition, the accumulation of polycyclic aromatic hydrocarbons was inhibited in the maize leaf by all selected surfactant treatments, but was facilitated in maize root by the treatments of rhamnolipid and Tween 80. This work indicates that biosurfactant amended phytoremediation may be a useful biotechnological approach for the remediation of petroleum hydrocarbon polluted soil. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Soil pollution by petroleum hydrocarbons is a serious environmental problem, which mainly arises from accidental spills and discharge of oil or oily waste and can pose a risk to human health (Urum et al., 2006; Lai et al., 2009). As a result of the persistence and toxicity, numerous studies have investigated innovative and environmentally compatible technologies to remove them from contaminated soil. Among these technologies, phytoremediation plays a particular role due to its solar-driven and cost-effective characteristics. In recent years, some higher plants, such as tall fescue, fire phoenix and maize have been used for remediation of petroleum-contaminated soil (Liu et al., 2014a,b; Zamani et al., 2014). However, the efficiency of phytoremediation is limited by the bioavailability of petroleum hydrocarbons due to their low
∗ Corresponding authors at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. E-mail addresses: [email protected] (G. Lu), [email protected] (Z. Dang). http://dx.doi.org/10.1016/j.ecoleng.2016.03.041 0925-8574/© 2016 Elsevier B.V. All rights reserved.
aqueous solubility and strong sorption to soil. A key step in enhancing the bioavailability of the oil contaminant is to transport the pollutant into the aqueous bulk phase (Mihelcic et al., 1993; Lai et al., 2009). Surfactants can improve the solubility of pollutants in aqueous phase, so they greatly enhance the bioavailability of hydrophobic organic compounds in soil (Calvo et al., 2009; Zhu and Aitken, 2010). In particular, some studies have shown that environmentally friendly biosurfactants have the ability to effectively solubilize and mobilize organic compounds adsorbed on soil particles (Mulligan, 2005; Whang et al., 2008). Others have reported on the remediation of petroleum hydrocarbon-contaminated soil by various surfactants (Lai et al., 2009; Zhu and Aitken, 2010; Von Lau et al., 2014). However, the effect of surfactant on the phytoremediation of petroleum hydrocarbon-contaminated soil is less fully investigated. Therefore, it is useful to investigate whether the surfactant can facilitate the phytoremediation process and to determine the extent of any toxicity to soil microorganisms and growing plants. Some polycyclic aromatic hydrocarbons (PAHs), one kind of target pollutants often used to represent petroleum hydrocarbons, are
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considered carcinogenic and mutagenic microorganisms, plants, animals and human beings (Fan et al., 2008; Zhang et al., 2010). Plant uptake of organic pollutants is a major route for the transfer of pollutants from soils to the food chain (Gao and Collins, 2009). In the presence of different surfactants, plants exhibit different behaviors as to the uptake of PAHs (Gao et al., 2008; Zhu and Zhang, 2008; Lu and Zhu, 2009). Organic pollutants, including PAHs, can enter vegetation through several routes, including from contaminated soils through plant roots and from the atmosphere through leaf stomata (Simonich and Hites, 1995; Lin et al., 2007; Tao et al., 2009). Therefore, the accumulation of PAHs in plant tissues during phytoremediation of contaminated soil amended with surfactants needs to be well understood. In this work, two biosurfactants (rhamnolipid and soybean lecithin) as well as a synthetic surfactant (Tween 80) were added into soil to facilitate phytoremediation of crude oil-contaminated soil. A comparison of the relative removal efficiency of total petroleum hydrocarbons (TPHs) in soil by different surfactants was examined for comparison. The effect of surfactants on the maize plants and soil microbial population was investigated. Also, the accumulation of PAHs in maize tissues was determined. The aim of this work is to assess the utility and possible risks of biosurfantantenhanced phytoremedation in treatment of oil-polluted soil. 2. Materials and methods 2.1. Chemicals and maize seeds Crude oil was obtained from Guangzhou Department, Sinopec Corporation, China. Tween 80 was purchased from Kelong Company, China. Rhamnolipid and soybean lecithin were obtained from Huangzhou Zijin Biotechnology Co., Ltd. All other reagents were of analytical grade. Seeds of maize CT 38 were purchased from Research Institution of Crop, Guangdong Academy of Agricultural Sciences, China. 2.2. Soil preparation The soil used in the experiment was collected from the upper layer (0–20 cm) of a farm in Guangzhou Higher Education Mega Center. The soil was air-dried, crushed and passed through a 4 mm sieve to remove stones and roots. The soil organic matter content and pH were 1.3% and 6.54, respectively. Nutrient levels were 24.5 g/kg ammoniac nitrogen, 4.32 g/kg total P and 0.40 g/kg total K. The soil (4 kg) was placed in a plastic crate, spiked with crude oil (5000 mg/kg, moderate TPH contamination in soil) and stirred for homogeneity with a wood spoon. Then the soil was transferred into a pot and equilibrated with water for two weeks in order to age the material. The equilibrated soil was stored at room temperature and prepared for the experiment. 2.3. Experimental design and sampling Seeds of maize were disinfected in 3% H2 O2 solution for 20 min, followed by washing with distilled water. Then the seeds were soaked in water for 24 h and planted (three to a pot) at a depth of about 4 cm in pots containing the soil from Section 2.2. After two weeks, two seedlings were removed from each of the pots, so that each pot contained one seeding of roughly the same height. The pots were placed on the top of the laboratory building in our school. Average temperature was 22.6 ◦ C during the experiment period. The date (Oct. 16, 2011) was the start time of the experiment. Every half a month, 200 mL of surfactant solution (10,000 mg/L) was added the pot holder, so that it could be absorbed into the soils. The concentration of surfactant solution used in this study was based on our previous work (Liao et al., 2015). In this pot experiment,
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each treatment had three replicates. The experiment was carried on for three months. After harvest, maize plants were washed with tap water and then with distilled water to remove dust, followed by drying in an oven at 50 ◦ C for 72 h. Plant tissues were separated, crushed and stored at −20 ◦ C prior to analysis. 2.4. Sample analysis 2.4.1. Photosynthetic efficiency and dry biomass of maize plants Chlorophyll fluorescence (ChFl) was measured at bell stage of maize growth to monitor the stress and determine the effect of the surfactant. ChFl is considered a good estimator of the quantum efficiency photosynthesis (Rosso et al., 2005). ChFl was measured with a Junior-PAM chlorophyll fluorometer (Walz, Germany). Two fluorescence parameters (variable fluorescence (Fv) and maximum fluorescence (Fm)) were recorded and their ratio was calculated and averaged per pot. Fv is the difference between minimum (in absence of light) and maximum (light-induced) fluorescence. Fv/Fm measures the proportion of maximum possible fluorescence used for photosynthesis, which tends to decrease with stress. Therefore, Fv/Fm was used as the stress indicator to investigate the surfactant amendment of the soil on maize photosynthesis efficiency. Three shoots per treatment were randomly chosen for these measurements. To determine the weight of maize plant biomass, shoot and root were separated and washed with tap water followed by distilled water, dried in an air oven at 50 ◦ C for 48 h and weighed. 2.4.2. Measurement of soil microbial activity To examine the toxic effect of surfactants on soil microbial activity, the population of living microorganisms in the soil was determined by the serial dilution method (Zhang et al., 2013). Briefly, 5 g soil and 45 mL sterile water were added to a conical flask and shaken at 150 rpm for 30 min. Soil suspension (1 mL) was mixed with 9 mL sterile water and serially diluted to obtain 10−2 to 10−7 dilutions. Then, 0.1 mL of three dilutions (10−5 , 10−6 and 10−7 ) were each spread on a nutrient agar plate and spread out evenly and incubated at 30 ◦ C for 48 h. The bacterial colonies in each plate were counted, averaged and expressed as the number of colony-forming units (CFU) per gram of dry soil. 2.4.3. Determination of total petroleum hydrocarbons in soil TPHs in soil and their fractions were determined by using the gravimetric method (Peng et al., 2009). About 5.00 g air-dried and sieved soil was transferred to a glass centrifuge tube, to which 15 mL dichloromethane was added. The tube was closed, and the sample was extracted by ultrasonic treatment for 15 min, followed by centrifugation at 3000 rpm for 10 min. This extraction was repeated twice more. The supernatants were combined in a beaker and allowed to evaporate at room temperature in a fume hood. The amount of residual TPHs was determined by the mass remaining in the air-dried beaker. Further fractionation of TPHs into saturated hydrocarbon, aromatic hydrocarbon, asphaltene and polar fraction was done by the silica gel column chromatography followed by the gravimetric analysis. A glass column was filled with 12 cm of activated silica gel (pre-baked at 300 ◦ C for 4 h) and 1 cm dry sodiumsulfate (pre-baked at 400 ◦ C for 6 h) on the upper layer. TPH extracts were dissolved in n-hexane and separated into soluble and insoluble fractions (asphaltene) by n-hexane precipitation. The soluble fractions were loaded on the silica gel column and eluted by solvents with different polarities. The saturated hydrocarbons were eluted by nhexane, followed by an n-hexane/dichloromethane (1:1) mixture to obtain aromatic hydrocarbons. Finally, methanol was employed to elute polar (resin) fraction.
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2.4.4. Analysis of n-alkanes and PAHs by GC–MS Measurement of n-alkanes in soil and PAHs in the maize seedling was conducted as in previous work (Tao et al., 2006). Briefly, 1.00 g dried sample was homogenized with about 1 g of anhydrous sodium sulfate in glass tube, followed by addition of 10 mL hexane/dichloromethane (1:1) solution and extracted with ultrasonic treatment for 30 min. Each sample was extracted three times. The extractions were collected in a beaker and purified by passage through a silica gel column. The eluate was subsequently vacuum-concentrated by rotary evaporation at 40 ◦ C. The sample was resuspended in hexane to a final volume of 1 mL and transferred to a vial with a syringe fitted with a 0.22 m filter for further analysis by GC–MS. The analysis was conducted using a GC–MS that coupled aThermo-Trace GC Ultra instrument to a-Thermo-DSQ II mass spectrometer (Thermo-Electron Corporation, Waltham, USA). Compounds were separated on a 30 m (id = 0.25 mm) capillary column coated with 0.25 m film. The GC temperature was programmed from an initial temperature of 80 ◦ C and increased at 10 ◦ C min−1 up to a final temperature of 290 ◦ C that was held for 10 min. Helium was used as the carrier gas. A 1.0 L aliquot of the extract was injected into an injector port at 280 ◦ C and operated in a splitless mode with a flow rate of 1.0 mL/min. Selective ion monitoring mode was used and the target ion was m/z 85 for n-alkanes and molecular weight for PAHs. The process blank was determined by going through the extraction and cleanup procedures using sand and vegetable samples. Recovery of individual PAHs ranged from 41.2% to 93.8% with a mean value of 72.6% for 7 PAHs. 2.5. Statistical analysis SPSS 17.0 was used for the statistical evaluation of the results, which were designed as completely randomized with three replicates for each parameter. Mean values followed by different letters were significantly different as determined by an analysis of variance (ANOVA). The differences were compared by a Duncan’s range at a significance level of p < 0.05. 3. Results and discussion 3.1. Effect of different surfactants on maize plants Plants are profoundly influenced by soil conditions. The dry biomass production and photosynthetic efficiency of maize plants grown in surfactants-treated soil are shown in Fig. 1, which shows no significant difference in dry biomass of maize plants grown with different surfactant treatments (Fig. 1A). Note especially that the biomass of maize grown in soil treated with rhamnolipid is 13% higher than the control. In addition, values of Fv/Fm of maize plants grown in surfactant treated soils were lower than that of the control (Fig. 1B), which indicated that ChFl in maize plants was sensitive to the stress associated with surfactant amendment of the soil. Fv/Fm in the plant grown in the soybean lecithin-treated soil is significantly less than that observed in the control. The addition of surfactants in soil has raised concern due to their potential phytotoxicity. In the present work, the addition of surfactants to the soil did not result in any inhibition on maize growth, which is an evidence that biosurfactant-enhanced phytoremediation of contaminated soil did not affect plant growth. In fact, there was a small positive effect on maize biomass production in the treatment with Rhamnolipid, a glycolipid biosurfactant produced by various bacterial species, including Pseudomonas sp. and Burkholderia sp. (Abdel-Mawgoud et al., 2010). The positive effects on the growth of maize grown in rhamnolipid-treated soil may be due to the degradation of rhamnolipid in soil, resulting in
more plant growth-promoting microorganisms in the rhizosphere and better physical soil conditions for nutrition uptake by the plant (Sheng et al., 2008). According to previous work (Vatsa et al., 2010), rhamnolipid has been shown to be a powerful additive when used in bioremediation application at sites contaminated with organic pollutants and heavy metals. Therefore, this study indicates that coupling rhamnolipid with maize plants is a good choice for remediating contaminated soil. Inhibition of ChFl by surfactants indicates that the photosynthesis might be inhibited in maize plants, even though biomass production was not significantly affected by various surfactants. This observation implies that maize can readily adapt to some stress conditions to ensure a steady biomass production. The value of Fv/Fm is normally about 0.85, but it will decrease significantly in stress conditions (Björkman and Demmig, 1987). In this study, values of Fv/Fm in different treatments were less than 0.8, indicating maize plants grown in crude oil contaminated soil exhibited a reduced ChFl and then less photosynthesis. Therefore, ChFl was an important indicator for assessing the overall health of plants. 3.2. Effect of different surfactants on soil microbial number The effect of surfactants on soil microbes is also a concern. As shown in Fig. 2, microbial number significantly increased in the treatments with biosurfactant rhamnolipid or natural surfactant soybean lecithin, suggesting that degradable surfactants had a promoting effect on microbial numbers in soil. In addition, the microbial number in Tween 80 treated soil was similar to that of the control with maize, but it was significantly decreased in the treatment without maize planting. This result suggests that maize plants had a positive effect on microbial activity, which would enhance the removal efficiency of hydrocarbons from the contaminated soil. The increase of soil microbial number in surfactant-treated soil might be directly due to the surfactants, per se, or to the higher levels of dissolved organic matter released by the surfactants in soil pore water, serving as a new carbon source for additional microbial growth, which result in higher microbial number. This observation is in agreement with previous work (Mathurasa et al., 2012) and suggests that biosurfactants from microorganisms or plants should be explored more widely for possible use in the remediation of contaminated soil. As a dynamic group in soil ecosystems, microorganisms play a crucial role in material cycles. Therefore, the effects of the addition of surfactants to soil on the microbial activity should be further investigated. In particular, evaluating the dynamics of hydrocarbon-degrading bacteria in soils is critical to understand and optimize the remediation of contaminated soil. The plate counting method used in this study only revealed the number of cells from soil, but not the structure of the microbial community. Further investigation is needed on soil microbial community structure, exploring advanced biotechnologies, such as taxon-specific quantitative PCR assays and phospholipid fatty acid analysis. 3.3. Effect of surfactant on TPH removal efficiency from soil The removal efficiencies of TPHs from soils after three months remediation are shown in Fig. 3. The removal efficiencies of TPHs in the treatments with soybean lecithin, rhamnolipid and Tween 80 were 62%, 58% and 47%, respectively, while that of the control (without surfactant) was 52%, indicating that addition of natural surfactant soybean lecithin or biosurfactant rhamnolipid enhanced the removal of hydrocarbons from soil. Meanwhile, removal efficiency of TPHs in the control treatment without maize plants was only 13%, suggesting that maize plants significantly enhanced the removal of hydrocarbons from the soil.
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Fig. 1. Effect of surfactant on (A) maize plant biomass and (B) chlorophyll fluorescence. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Pollutants in soil are removed mainly by three pathways: (1) abiotic dissipation, such as leaching loss and volatilization; (2) degradation by soil microorganisms and (3) plant uptake and accumulation. Desorption capability of surfactants might contribute to the bioavailability of pollutants for microbial degradation, leading to enhanced reduction of TPHs. In our study, leaching loss was controlled by the addition of adequate volume of surfactant solution, and plant uptake was not significant. This result showed that maize plants, coupled with surfactant treatment, could be employed for remediation of petroleum hydrocarbon-contaminated soil. Special consideration should be given to natural surfactants, such as soybean lecithin and biosurfactants, such as rhamnolipid should be considered, as priority treatments for remediation of contaminated soil due to their greater environmental compatibility, lower toxicity and higher biodegradability. In previous studies, Fava et al. (2004) reported that the overall removal of PAHs in the presence of soya lecithin is faster and more extensive under slurry-phase conditions. Moreover, Xu et al. (2011) demonstrated that increasing concern over the supply, price and environmental impact of petrochemical surfactants has resulted in increased demand for surfactants derived from natural sources, especially from plants. Therefore, it would be useful to explore more surfactants from plants for use in the remediation of contaminated soil.
3.4. Change of TPH fractions in soil TPHs can be divided into saturated hydrocarbons, aromatic hydrocarbons, asphaltene and polar fractions (Zhang et al., 2010). The changes in TPH fractions in soils before and after remediation are shown in Fig. 5. Following remediation, the proportion of saturated hydrocarbons in TPHs decreased from 60% to 36%, while that of aromatic hydrocarbons increased from 22% to 38%, and that of asphaltene and polar fraction increased from 18% to 26%, indicating that the aromatic hydrocarbons, and asphaltene and polar fractions were more resistant to the treatment (Fig. 4). In contrast, the lower n-alkanes (≤C22) were notably decreased. Further, the reduction seen in the odd-numbered carbon compounds was greater than that seen in the even-numbered carbon compounds in this group, indicating they were more degradable. Also, branched paraffin (phytane) was less degradable than n-alkane (n-octadecane) in soil (Fig. 5). Saturated hydrocarbons were possibly more degradable than other fractions in soil, because they are less toxic to microbes in the rhizosphere and can serve as a carbon source involved in the microbial metabolisms (Zhang et al., 2010). In contrast, because the aromatic hydrocarbon and asphaltene and polar fractions were more resistant to microbial degradation in the soil, the relative amounts of these compounds increased as a result of the remediation.
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Fig. 2. Effect of surfactant on soil microbial activity. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Fig. 3. Effect of surfactant on reduction of total petroleum hydrocarbons (TPHs) in soil. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Since biodegradability of hydrocarbons is related to their molecular sizes (Schaefer and Juliane, 2007), lighter chain n-alkanes are degraded preferentially by microorganisms in soil. Moreover, oddnumbered n-alkanes are asymmetric in molecular structure and are unstable, so that they are more easily attached by microorganisms. Also, the bond energy in a branched paraffin is higher than that
in n-alkanes (Schaefer and Juliane, 2007), resulting in the greater persistence of branched paraffin in soil.
3.5. Effect of surfactant on accumulation of PAHs in maize tissues Considering the use of crops for remediation of soil contaminated with crude oil, accumulation of some toxic chemicals, such
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Fig. 4. Comparison of crude oil fraction in soils before and after phytoremediation. Mean and standard deviation of three replicates are shown.
Fig. 5. Comparison of n-alkane in soils (A) before and (B) after phytoremediation.
as PAHs in plant tissues, has raised considerable concern among the public. Therefore, concentration of some representative priority PAH pollutants including naphthalene (Nap), acenaphthylene (Any), fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), and pyrene (Pyr) were studied because they are prominent PAHs in crude oil. As shown in Fig. 6, the concentration of PAH in maize leaf in the control treatment group was higher than that in the surfactant
treatment groups. Concentrations of PAHs in the roots of maize grown in Tween 80- and rhamnolipid-treated soils were higher than that in the other two treatment groups. There were no significant difference in the PAH concentrations in the stems of maize grown in different treatment groupsGenerally, uptake of PAHs by plants may occur through air-to-plant and soil-to-plant pathways, principally through atmospheric deposition in gaseous or particulate forms and root uptake (Tao et al., 2009). In the air-to-plant
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Fig. 6. Effect of surfactant on PAH accumulation in maize tissues. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05), no letter on the bar means no significant difference (p < 0.05).
pathway, PAHs can enter into plant inner tissues through leaf stomata (Desalme et al., 2013), which indicates that the changes of stomata will influence the accumulation of PAHs in the plant leaf. In the present work, leaf PAH concentration in the control was higher than the other treatments, which may ascribe to a decrease of number and size of stomata in maize leaf when surfactant is present in the soil. In the soil-to-plant pathway, organic pollutants can desorb from soil particles into soil solution, followed by plant root uptake (Collins et al., 2006), which can be influenced by the existence of surfactants. In previous work, Gao et al. (2008) reported that Tween 80 enhanced the plant uptake of Phe and Pyr at the concentrations lower than 13.2 mg/L, while inhibiting the uptake of both PAH compounds from an aqueous solution by the red clover (Trifolium pretense L.). Also, rhamnolipids have been shown to enhance the
uptake of PAHs by ryegrass roots in a hydroponic experiment (Zhu and Zhang, 2008). In the present work, the accumulation of PAHs by maize root in the treatments of Tween 80 and rhamnolipids amendments were higher than with the other two treatments, which might be ascribed to different abilities of the surfactants to desorb PAHs from soil.
4. Conclusions This work has investigated the effect of surfactants on the phytoremediation of crude oil-contaminated soil. The addition of surfactants did not significantly affect the biomass production of maize plant, but it inhibited the ChFl in the maize leaf. The soil microbial population increased in the treatments with soybean
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lecithin and rhamnolipid, which improved the removal efficiency of petroleum hydrocarbons from soil. At the end of soil remediation, the proportion of saturated hydrocarbons in TPHs decreased, but those of the aromatic hydrocarbon, and asphaltene/polar fraction increased. In addition, under the treatments of Tween 80 and rhamnolipid the accumulation of PAHs was inhibited in maize leaf, but enhanced in maize root. In summary, treatment with biosurfactants and growth of maize is a good alternative technology for the remediation of petroleum-contaminated soil. Acknowledgements The research was financially supported by the National Natural Science Foundation of China (No. 41573091), the Natural Science Foundation of Guangdong Province (No. 2015A030306005), and the Program for Pearl River Young Talents of Science and Technology in Guangzhou (No. 2013J2200007). The authors would like to thank Dr. Donald Barnes for language editing on the manuscript. References Abdel-Mawgoud, A.M., Lépine, F., Déziel, E., 2010. Rhamnolipids: diversity of structures, microbial origins and roles. Appl. Microbiol. Biotechnol. 86, 1323–1336. Björkman, O., Demmig, B., 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489–504. Calvo, C., Manzanera, M., Silva-Castro, G.A., Uad, I., González-López, J., 2009. Application of bioemulsifiers in soil oil bioremediation processes. Future prospects. Sci. Total Environ. 407, 3634–3640. Collins, C., Fryer, M., Grosso, A., 2006. Plant uptake of non-ionic organic chemicals. Environ. Sci. Technol. 40, 45–52. Desalme, D., Binet, P., Chiapusio, G., 2013. Challenges in tracing the fate and effects of atmospheric polycyclic aromatic hydrocarbon deposition in vascular plants. Environ. Sci. Technol. 47, 3967–3981. Fan, S., Li, P., Gong, Z., Ren, W., He, N., 2008. Promotion of pyrene degradation in rhizosphere of alfalfa (Medicago sativa L.). Chemosphere 71, 1593–1598. Fava, F., Berselli, S., Conte, P., Piccolo, A., Marchetti, L., 2004. Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs). Biotechnol. Bioeng. 88, 214–223. Gao, Y., Collins, C.D., 2009. Uptake pathways of polycyclic aromatic hydrocarbons in white clover. Environ. Sci. Technol. 43, 6190–6195. Gao, Y., Shen, Q., Ling, W., Ren, L., 2008. Uptake of polycyclic aromatic hydrocarbons by Trifolium pretense L. from water in the presence of a nonionic surfactant. Chemosphere 72, 636–643. Lai, C., Huang, Y., Wei, Y., Chang, J., 2009. Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil. J. Hazard. Mater. 167, 609–614. Liao, C., Liang, X., Lu, G., Thai, T., Xu, W., Dang, Z., 2015. Effect of surfactant amendment to PAHs-contaminated soil for phytoremediation by maize (Zea mays L.). Ecotoxicol. Environ. Saf. 112, 1–6. Lin, H., Tao, S., Zuo, Q., Coveney, R.M., 2007. Uptake of polycyclic aromatic hydrocarbons by maize plants. Environ. Pollut. 148, 614–619. Liu, R., Xiao, N., Wei, S., Zhao, L., An, J., 2014a. Rhizosphere effects of PAH-contaminated soil phytoremediation using a special plant named Fire Phoenix. Sci. Total Environ. 473, 350–358.
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Liu, W., Hou, J., Wang, Q., Ding, L., Luo, Y., 2014b. Isolation and characterization of plant growth-promoting rhizobacteria and their effects on phytoremediation of petroleum-contaminated saline-alkali soil. Chemosphere 117, 303–308. Lu, L., Zhu, L., 2009. Reducing plant uptake of PAHs by cationic surfactant-enhanced soil retention. Environ. Pollut. 157, 1794–1799. Mathurasa, L., Tongcumpou, C., Sabatini, D.A., Luepromchai, E., 2012. Anionic surfactant enhanced bacterial degradation of tributyltin in soil. Int. Biodeterior. Biodegrad. 75, 7–14. Mihelcic, J.R., Lueking, D.R., Mitzell, R.J., Stapleton, J.M., 1993. Bioavailability of sorbed-and separate-phase chemicals. Biodegradation 4, 141–153. Mulligan, C.N., 2005. Environmental applications for biosurfactants. Environ. Pollut. 133, 183–198. Peng, S., Zhou, Q., Cai, Z., Zhang, Z., 2009. Phytoremediation of petroleum contaminated soils by Mirabilis jalapa L. in a greenhouse plot experiment. J. Hazard. Mater. 168, 1490–1496. Rosso, P.H., Pushnik, J.C., Lay, M., Ustin, S.L., 2005. Reflectance properties and physiological responses of Salicornia virginica to heavy metal and petroleum contamination. Environ. Pollut. 137, 241–252. Schaefer, M., Juliane, F., 2007. The influence of earthworms and organic additives on the biodegradation of oil contaminated soil. Appl. Soil Ecol. 36, 53–62. Sheng, X., He, L., Wang, Q., Ye, H., Jiang, C., 2008. Effects of inoculation of biosurfactant-producing Bacillus sp. J119 on plant growth and cadmium uptake in a cadmium-amended soil. J. Hazard. Mater. 155, 17–22. Simonich, S.L., Hites, R.A., 1995. Organic pollutant accumulation in vegetation. Environ. Sci. Technol. 29, 2905–2914. Tao, S., Jiao, X.C., Chen, S.H., Liu, W.X., Coveney Jr, R.M., Zhu, L.Z., Luo, Y.M., 2006. Accumulation and distribution of polycyclic aromatic hydrocarbons in rice (Oryza sativa). Environ. Pollut. 140, 406–415. Tao, Y., Zhang, S., Zhu, Y., Christie, P., 2009. Uptake and acropetal translocation of polycyclic aromatic hydrocarbons by wheat (Triticum aestivum L.) grown in field-contaminated soil. Environ. Sci. Technol. 43, 3556–3560. Urum, K., Grigson, S., Pekdemir, T., McMenamy, S., 2006. A comparison of the efficiency of different surfactants for removal of crude oil from contaminated soils. Chemosphere 62, 1403–1410. Vatsa, P., Sanchez, L., Clement, C., Baillieul, F., Dorey, S., 2010. Rhamnolipid biosurfactants as new players in animal and plant defense against microbes. Int. J. Mol. Sci. 11, 5095–5108. Von Lau, E., Gan, S., Ng, H.K., Poh, P.E., 2014. Extraction agents for the removal of polycyclic aromatic hydrocarbons (PAHs) from soil in soil washing technologies. Environ. Pollut. 184, 640–649. Whang, L., Liu, P.G., Ma, C., Cheng, S., 2008. Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil. J. Hazard. Mater. 151, 155–163. Xu, Q., Nakajima, M., Liu, Z., Shiina, T., 2011. Biosurfactants for microbubble preparation and application. Int. J. Mol. Sci. 12, 462–475. Zamani, J., Hajabbasi, M.A., Alaie, E., 2014. Effect of Piriformospora indica inoculation on root development and distribution of maize (Zea mays L.) in the presence of petroleum contaminated soil. EGU General Assembly Conference Abstracts, p17027. Zhang, Z., Zhou, Q., Peng, S., Cai, Z., 2010. Remediation of petroleum contaminated soils by joint action of Pharbitis L. and its microbial community. Sci. Total Environ. 408, 5600–5605. Zhang, X., Wang, Z., Liu, X., Hu, X., Liang, X., Hu, Y., 2013. Degradation of diesel pollutants in Huangpu-Yangtze River estuary wetland using plant-microbe systems. Int. Biodeterior. Biodegrad. 76, 71–75. Zhu, H., Aitken, M.D., 2010. Surfactant-enhanced desorption and biodegradation of polycyclic aromatic hydrocarbons in contaminated soil. Environ. Sci. Technol. 44, 7260–7265. Zhu, L., Zhang, M., 2008. Effect of rhamnolipids on the uptake of PAHs by ryegrass. Environ. Pollut. 156, 46–52.
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Biosurfactant-enhanced phytoremediation of soils contaminated by crude oil using maize (Zea mays. L) Changjun Liao a,b , Wending Xu a,c , Guining Lu a,d,e,∗ , Fucai Deng a , Xujun Liang a , Chuling Guo a,d , Zhi Dang a,d,e,∗ a
School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China Department of Environmental Engineering, Guangdong Vocational College of Environmental Protection Engineering, Foshan 528216, PR China c School of Municipal and Environmental Engineering, Henan University of Urban Construction, Pingdingshan 467036, PR China d The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, South China University of Technology, Guangzhou 510006, PR China e Guangdong Provincial Engineering and Technology Research Center for Environmental Risk Prevention and Emergency Disposal, South China University of Technology, Guangzhou 510006, PR China b
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 21 March 2016 Accepted 22 March 2016 Keywords: Biosurfactant Phytoremediation Crude oil Contaminated soil PAHs
a b s t r a c t Surfactant-enhanced phytoremediation is a green technology for the treatment of contaminated soil. In a pot experiment, two biosurfactants (rhamnolipid and soybean lecithin) and a synthetic surfactant (Tween 80) were used to facilitate phytoremediation of crude oil contaminated soil by maize (Zea mays. L). Results showed that these surfactants did not significantly affect the biomass production of maize, but they inhibited the chlorophyll fluorescence of the maize leaf. Rhamnolipid and soybean lecithin enhanced the soil microbial population, resulting in increased removal of total petroleum hydrocarbons from the soil. Saturated hydrocarbons were the main component of petroleum hydrocarbons decreased in the soil. In addition, the accumulation of polycyclic aromatic hydrocarbons was inhibited in the maize leaf by all selected surfactant treatments, but was facilitated in maize root by the treatments of rhamnolipid and Tween 80. This work indicates that biosurfactant amended phytoremediation may be a useful biotechnological approach for the remediation of petroleum hydrocarbon polluted soil. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Soil pollution by petroleum hydrocarbons is a serious environmental problem, which mainly arises from accidental spills and discharge of oil or oily waste and can pose a risk to human health (Urum et al., 2006; Lai et al., 2009). As a result of the persistence and toxicity, numerous studies have investigated innovative and environmentally compatible technologies to remove them from contaminated soil. Among these technologies, phytoremediation plays a particular role due to its solar-driven and cost-effective characteristics. In recent years, some higher plants, such as tall fescue, fire phoenix and maize have been used for remediation of petroleum-contaminated soil (Liu et al., 2014a,b; Zamani et al., 2014). However, the efficiency of phytoremediation is limited by the bioavailability of petroleum hydrocarbons due to their low
∗ Corresponding authors at: School of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. E-mail addresses: [email protected] (G. Lu), [email protected] (Z. Dang). http://dx.doi.org/10.1016/j.ecoleng.2016.03.041 0925-8574/© 2016 Elsevier B.V. All rights reserved.
aqueous solubility and strong sorption to soil. A key step in enhancing the bioavailability of the oil contaminant is to transport the pollutant into the aqueous bulk phase (Mihelcic et al., 1993; Lai et al., 2009). Surfactants can improve the solubility of pollutants in aqueous phase, so they greatly enhance the bioavailability of hydrophobic organic compounds in soil (Calvo et al., 2009; Zhu and Aitken, 2010). In particular, some studies have shown that environmentally friendly biosurfactants have the ability to effectively solubilize and mobilize organic compounds adsorbed on soil particles (Mulligan, 2005; Whang et al., 2008). Others have reported on the remediation of petroleum hydrocarbon-contaminated soil by various surfactants (Lai et al., 2009; Zhu and Aitken, 2010; Von Lau et al., 2014). However, the effect of surfactant on the phytoremediation of petroleum hydrocarbon-contaminated soil is less fully investigated. Therefore, it is useful to investigate whether the surfactant can facilitate the phytoremediation process and to determine the extent of any toxicity to soil microorganisms and growing plants. Some polycyclic aromatic hydrocarbons (PAHs), one kind of target pollutants often used to represent petroleum hydrocarbons, are
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considered carcinogenic and mutagenic microorganisms, plants, animals and human beings (Fan et al., 2008; Zhang et al., 2010). Plant uptake of organic pollutants is a major route for the transfer of pollutants from soils to the food chain (Gao and Collins, 2009). In the presence of different surfactants, plants exhibit different behaviors as to the uptake of PAHs (Gao et al., 2008; Zhu and Zhang, 2008; Lu and Zhu, 2009). Organic pollutants, including PAHs, can enter vegetation through several routes, including from contaminated soils through plant roots and from the atmosphere through leaf stomata (Simonich and Hites, 1995; Lin et al., 2007; Tao et al., 2009). Therefore, the accumulation of PAHs in plant tissues during phytoremediation of contaminated soil amended with surfactants needs to be well understood. In this work, two biosurfactants (rhamnolipid and soybean lecithin) as well as a synthetic surfactant (Tween 80) were added into soil to facilitate phytoremediation of crude oil-contaminated soil. A comparison of the relative removal efficiency of total petroleum hydrocarbons (TPHs) in soil by different surfactants was examined for comparison. The effect of surfactants on the maize plants and soil microbial population was investigated. Also, the accumulation of PAHs in maize tissues was determined. The aim of this work is to assess the utility and possible risks of biosurfantantenhanced phytoremedation in treatment of oil-polluted soil. 2. Materials and methods 2.1. Chemicals and maize seeds Crude oil was obtained from Guangzhou Department, Sinopec Corporation, China. Tween 80 was purchased from Kelong Company, China. Rhamnolipid and soybean lecithin were obtained from Huangzhou Zijin Biotechnology Co., Ltd. All other reagents were of analytical grade. Seeds of maize CT 38 were purchased from Research Institution of Crop, Guangdong Academy of Agricultural Sciences, China. 2.2. Soil preparation The soil used in the experiment was collected from the upper layer (0–20 cm) of a farm in Guangzhou Higher Education Mega Center. The soil was air-dried, crushed and passed through a 4 mm sieve to remove stones and roots. The soil organic matter content and pH were 1.3% and 6.54, respectively. Nutrient levels were 24.5 g/kg ammoniac nitrogen, 4.32 g/kg total P and 0.40 g/kg total K. The soil (4 kg) was placed in a plastic crate, spiked with crude oil (5000 mg/kg, moderate TPH contamination in soil) and stirred for homogeneity with a wood spoon. Then the soil was transferred into a pot and equilibrated with water for two weeks in order to age the material. The equilibrated soil was stored at room temperature and prepared for the experiment. 2.3. Experimental design and sampling Seeds of maize were disinfected in 3% H2 O2 solution for 20 min, followed by washing with distilled water. Then the seeds were soaked in water for 24 h and planted (three to a pot) at a depth of about 4 cm in pots containing the soil from Section 2.2. After two weeks, two seedlings were removed from each of the pots, so that each pot contained one seeding of roughly the same height. The pots were placed on the top of the laboratory building in our school. Average temperature was 22.6 ◦ C during the experiment period. The date (Oct. 16, 2011) was the start time of the experiment. Every half a month, 200 mL of surfactant solution (10,000 mg/L) was added the pot holder, so that it could be absorbed into the soils. The concentration of surfactant solution used in this study was based on our previous work (Liao et al., 2015). In this pot experiment,
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each treatment had three replicates. The experiment was carried on for three months. After harvest, maize plants were washed with tap water and then with distilled water to remove dust, followed by drying in an oven at 50 ◦ C for 72 h. Plant tissues were separated, crushed and stored at −20 ◦ C prior to analysis. 2.4. Sample analysis 2.4.1. Photosynthetic efficiency and dry biomass of maize plants Chlorophyll fluorescence (ChFl) was measured at bell stage of maize growth to monitor the stress and determine the effect of the surfactant. ChFl is considered a good estimator of the quantum efficiency photosynthesis (Rosso et al., 2005). ChFl was measured with a Junior-PAM chlorophyll fluorometer (Walz, Germany). Two fluorescence parameters (variable fluorescence (Fv) and maximum fluorescence (Fm)) were recorded and their ratio was calculated and averaged per pot. Fv is the difference between minimum (in absence of light) and maximum (light-induced) fluorescence. Fv/Fm measures the proportion of maximum possible fluorescence used for photosynthesis, which tends to decrease with stress. Therefore, Fv/Fm was used as the stress indicator to investigate the surfactant amendment of the soil on maize photosynthesis efficiency. Three shoots per treatment were randomly chosen for these measurements. To determine the weight of maize plant biomass, shoot and root were separated and washed with tap water followed by distilled water, dried in an air oven at 50 ◦ C for 48 h and weighed. 2.4.2. Measurement of soil microbial activity To examine the toxic effect of surfactants on soil microbial activity, the population of living microorganisms in the soil was determined by the serial dilution method (Zhang et al., 2013). Briefly, 5 g soil and 45 mL sterile water were added to a conical flask and shaken at 150 rpm for 30 min. Soil suspension (1 mL) was mixed with 9 mL sterile water and serially diluted to obtain 10−2 to 10−7 dilutions. Then, 0.1 mL of three dilutions (10−5 , 10−6 and 10−7 ) were each spread on a nutrient agar plate and spread out evenly and incubated at 30 ◦ C for 48 h. The bacterial colonies in each plate were counted, averaged and expressed as the number of colony-forming units (CFU) per gram of dry soil. 2.4.3. Determination of total petroleum hydrocarbons in soil TPHs in soil and their fractions were determined by using the gravimetric method (Peng et al., 2009). About 5.00 g air-dried and sieved soil was transferred to a glass centrifuge tube, to which 15 mL dichloromethane was added. The tube was closed, and the sample was extracted by ultrasonic treatment for 15 min, followed by centrifugation at 3000 rpm for 10 min. This extraction was repeated twice more. The supernatants were combined in a beaker and allowed to evaporate at room temperature in a fume hood. The amount of residual TPHs was determined by the mass remaining in the air-dried beaker. Further fractionation of TPHs into saturated hydrocarbon, aromatic hydrocarbon, asphaltene and polar fraction was done by the silica gel column chromatography followed by the gravimetric analysis. A glass column was filled with 12 cm of activated silica gel (pre-baked at 300 ◦ C for 4 h) and 1 cm dry sodiumsulfate (pre-baked at 400 ◦ C for 6 h) on the upper layer. TPH extracts were dissolved in n-hexane and separated into soluble and insoluble fractions (asphaltene) by n-hexane precipitation. The soluble fractions were loaded on the silica gel column and eluted by solvents with different polarities. The saturated hydrocarbons were eluted by nhexane, followed by an n-hexane/dichloromethane (1:1) mixture to obtain aromatic hydrocarbons. Finally, methanol was employed to elute polar (resin) fraction.
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2.4.4. Analysis of n-alkanes and PAHs by GC–MS Measurement of n-alkanes in soil and PAHs in the maize seedling was conducted as in previous work (Tao et al., 2006). Briefly, 1.00 g dried sample was homogenized with about 1 g of anhydrous sodium sulfate in glass tube, followed by addition of 10 mL hexane/dichloromethane (1:1) solution and extracted with ultrasonic treatment for 30 min. Each sample was extracted three times. The extractions were collected in a beaker and purified by passage through a silica gel column. The eluate was subsequently vacuum-concentrated by rotary evaporation at 40 ◦ C. The sample was resuspended in hexane to a final volume of 1 mL and transferred to a vial with a syringe fitted with a 0.22 m filter for further analysis by GC–MS. The analysis was conducted using a GC–MS that coupled aThermo-Trace GC Ultra instrument to a-Thermo-DSQ II mass spectrometer (Thermo-Electron Corporation, Waltham, USA). Compounds were separated on a 30 m (id = 0.25 mm) capillary column coated with 0.25 m film. The GC temperature was programmed from an initial temperature of 80 ◦ C and increased at 10 ◦ C min−1 up to a final temperature of 290 ◦ C that was held for 10 min. Helium was used as the carrier gas. A 1.0 L aliquot of the extract was injected into an injector port at 280 ◦ C and operated in a splitless mode with a flow rate of 1.0 mL/min. Selective ion monitoring mode was used and the target ion was m/z 85 for n-alkanes and molecular weight for PAHs. The process blank was determined by going through the extraction and cleanup procedures using sand and vegetable samples. Recovery of individual PAHs ranged from 41.2% to 93.8% with a mean value of 72.6% for 7 PAHs. 2.5. Statistical analysis SPSS 17.0 was used for the statistical evaluation of the results, which were designed as completely randomized with three replicates for each parameter. Mean values followed by different letters were significantly different as determined by an analysis of variance (ANOVA). The differences were compared by a Duncan’s range at a significance level of p < 0.05. 3. Results and discussion 3.1. Effect of different surfactants on maize plants Plants are profoundly influenced by soil conditions. The dry biomass production and photosynthetic efficiency of maize plants grown in surfactants-treated soil are shown in Fig. 1, which shows no significant difference in dry biomass of maize plants grown with different surfactant treatments (Fig. 1A). Note especially that the biomass of maize grown in soil treated with rhamnolipid is 13% higher than the control. In addition, values of Fv/Fm of maize plants grown in surfactant treated soils were lower than that of the control (Fig. 1B), which indicated that ChFl in maize plants was sensitive to the stress associated with surfactant amendment of the soil. Fv/Fm in the plant grown in the soybean lecithin-treated soil is significantly less than that observed in the control. The addition of surfactants in soil has raised concern due to their potential phytotoxicity. In the present work, the addition of surfactants to the soil did not result in any inhibition on maize growth, which is an evidence that biosurfactant-enhanced phytoremediation of contaminated soil did not affect plant growth. In fact, there was a small positive effect on maize biomass production in the treatment with Rhamnolipid, a glycolipid biosurfactant produced by various bacterial species, including Pseudomonas sp. and Burkholderia sp. (Abdel-Mawgoud et al., 2010). The positive effects on the growth of maize grown in rhamnolipid-treated soil may be due to the degradation of rhamnolipid in soil, resulting in
more plant growth-promoting microorganisms in the rhizosphere and better physical soil conditions for nutrition uptake by the plant (Sheng et al., 2008). According to previous work (Vatsa et al., 2010), rhamnolipid has been shown to be a powerful additive when used in bioremediation application at sites contaminated with organic pollutants and heavy metals. Therefore, this study indicates that coupling rhamnolipid with maize plants is a good choice for remediating contaminated soil. Inhibition of ChFl by surfactants indicates that the photosynthesis might be inhibited in maize plants, even though biomass production was not significantly affected by various surfactants. This observation implies that maize can readily adapt to some stress conditions to ensure a steady biomass production. The value of Fv/Fm is normally about 0.85, but it will decrease significantly in stress conditions (Björkman and Demmig, 1987). In this study, values of Fv/Fm in different treatments were less than 0.8, indicating maize plants grown in crude oil contaminated soil exhibited a reduced ChFl and then less photosynthesis. Therefore, ChFl was an important indicator for assessing the overall health of plants. 3.2. Effect of different surfactants on soil microbial number The effect of surfactants on soil microbes is also a concern. As shown in Fig. 2, microbial number significantly increased in the treatments with biosurfactant rhamnolipid or natural surfactant soybean lecithin, suggesting that degradable surfactants had a promoting effect on microbial numbers in soil. In addition, the microbial number in Tween 80 treated soil was similar to that of the control with maize, but it was significantly decreased in the treatment without maize planting. This result suggests that maize plants had a positive effect on microbial activity, which would enhance the removal efficiency of hydrocarbons from the contaminated soil. The increase of soil microbial number in surfactant-treated soil might be directly due to the surfactants, per se, or to the higher levels of dissolved organic matter released by the surfactants in soil pore water, serving as a new carbon source for additional microbial growth, which result in higher microbial number. This observation is in agreement with previous work (Mathurasa et al., 2012) and suggests that biosurfactants from microorganisms or plants should be explored more widely for possible use in the remediation of contaminated soil. As a dynamic group in soil ecosystems, microorganisms play a crucial role in material cycles. Therefore, the effects of the addition of surfactants to soil on the microbial activity should be further investigated. In particular, evaluating the dynamics of hydrocarbon-degrading bacteria in soils is critical to understand and optimize the remediation of contaminated soil. The plate counting method used in this study only revealed the number of cells from soil, but not the structure of the microbial community. Further investigation is needed on soil microbial community structure, exploring advanced biotechnologies, such as taxon-specific quantitative PCR assays and phospholipid fatty acid analysis. 3.3. Effect of surfactant on TPH removal efficiency from soil The removal efficiencies of TPHs from soils after three months remediation are shown in Fig. 3. The removal efficiencies of TPHs in the treatments with soybean lecithin, rhamnolipid and Tween 80 were 62%, 58% and 47%, respectively, while that of the control (without surfactant) was 52%, indicating that addition of natural surfactant soybean lecithin or biosurfactant rhamnolipid enhanced the removal of hydrocarbons from soil. Meanwhile, removal efficiency of TPHs in the control treatment without maize plants was only 13%, suggesting that maize plants significantly enhanced the removal of hydrocarbons from the soil.
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Fig. 1. Effect of surfactant on (A) maize plant biomass and (B) chlorophyll fluorescence. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Pollutants in soil are removed mainly by three pathways: (1) abiotic dissipation, such as leaching loss and volatilization; (2) degradation by soil microorganisms and (3) plant uptake and accumulation. Desorption capability of surfactants might contribute to the bioavailability of pollutants for microbial degradation, leading to enhanced reduction of TPHs. In our study, leaching loss was controlled by the addition of adequate volume of surfactant solution, and plant uptake was not significant. This result showed that maize plants, coupled with surfactant treatment, could be employed for remediation of petroleum hydrocarbon-contaminated soil. Special consideration should be given to natural surfactants, such as soybean lecithin and biosurfactants, such as rhamnolipid should be considered, as priority treatments for remediation of contaminated soil due to their greater environmental compatibility, lower toxicity and higher biodegradability. In previous studies, Fava et al. (2004) reported that the overall removal of PAHs in the presence of soya lecithin is faster and more extensive under slurry-phase conditions. Moreover, Xu et al. (2011) demonstrated that increasing concern over the supply, price and environmental impact of petrochemical surfactants has resulted in increased demand for surfactants derived from natural sources, especially from plants. Therefore, it would be useful to explore more surfactants from plants for use in the remediation of contaminated soil.
3.4. Change of TPH fractions in soil TPHs can be divided into saturated hydrocarbons, aromatic hydrocarbons, asphaltene and polar fractions (Zhang et al., 2010). The changes in TPH fractions in soils before and after remediation are shown in Fig. 5. Following remediation, the proportion of saturated hydrocarbons in TPHs decreased from 60% to 36%, while that of aromatic hydrocarbons increased from 22% to 38%, and that of asphaltene and polar fraction increased from 18% to 26%, indicating that the aromatic hydrocarbons, and asphaltene and polar fractions were more resistant to the treatment (Fig. 4). In contrast, the lower n-alkanes (≤C22) were notably decreased. Further, the reduction seen in the odd-numbered carbon compounds was greater than that seen in the even-numbered carbon compounds in this group, indicating they were more degradable. Also, branched paraffin (phytane) was less degradable than n-alkane (n-octadecane) in soil (Fig. 5). Saturated hydrocarbons were possibly more degradable than other fractions in soil, because they are less toxic to microbes in the rhizosphere and can serve as a carbon source involved in the microbial metabolisms (Zhang et al., 2010). In contrast, because the aromatic hydrocarbon and asphaltene and polar fractions were more resistant to microbial degradation in the soil, the relative amounts of these compounds increased as a result of the remediation.
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Fig. 2. Effect of surfactant on soil microbial activity. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Fig. 3. Effect of surfactant on reduction of total petroleum hydrocarbons (TPHs) in soil. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05).
Since biodegradability of hydrocarbons is related to their molecular sizes (Schaefer and Juliane, 2007), lighter chain n-alkanes are degraded preferentially by microorganisms in soil. Moreover, oddnumbered n-alkanes are asymmetric in molecular structure and are unstable, so that they are more easily attached by microorganisms. Also, the bond energy in a branched paraffin is higher than that
in n-alkanes (Schaefer and Juliane, 2007), resulting in the greater persistence of branched paraffin in soil.
3.5. Effect of surfactant on accumulation of PAHs in maize tissues Considering the use of crops for remediation of soil contaminated with crude oil, accumulation of some toxic chemicals, such
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Fig. 4. Comparison of crude oil fraction in soils before and after phytoremediation. Mean and standard deviation of three replicates are shown.
Fig. 5. Comparison of n-alkane in soils (A) before and (B) after phytoremediation.
as PAHs in plant tissues, has raised considerable concern among the public. Therefore, concentration of some representative priority PAH pollutants including naphthalene (Nap), acenaphthylene (Any), fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), and pyrene (Pyr) were studied because they are prominent PAHs in crude oil. As shown in Fig. 6, the concentration of PAH in maize leaf in the control treatment group was higher than that in the surfactant
treatment groups. Concentrations of PAHs in the roots of maize grown in Tween 80- and rhamnolipid-treated soils were higher than that in the other two treatment groups. There were no significant difference in the PAH concentrations in the stems of maize grown in different treatment groupsGenerally, uptake of PAHs by plants may occur through air-to-plant and soil-to-plant pathways, principally through atmospheric deposition in gaseous or particulate forms and root uptake (Tao et al., 2009). In the air-to-plant
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Fig. 6. Effect of surfactant on PAH accumulation in maize tissues. Mean and standard deviation of three replicates are shown. Different letters on top of the bar indicate significant differences (p < 0.05), no letter on the bar means no significant difference (p < 0.05).
pathway, PAHs can enter into plant inner tissues through leaf stomata (Desalme et al., 2013), which indicates that the changes of stomata will influence the accumulation of PAHs in the plant leaf. In the present work, leaf PAH concentration in the control was higher than the other treatments, which may ascribe to a decrease of number and size of stomata in maize leaf when surfactant is present in the soil. In the soil-to-plant pathway, organic pollutants can desorb from soil particles into soil solution, followed by plant root uptake (Collins et al., 2006), which can be influenced by the existence of surfactants. In previous work, Gao et al. (2008) reported that Tween 80 enhanced the plant uptake of Phe and Pyr at the concentrations lower than 13.2 mg/L, while inhibiting the uptake of both PAH compounds from an aqueous solution by the red clover (Trifolium pretense L.). Also, rhamnolipids have been shown to enhance the
uptake of PAHs by ryegrass roots in a hydroponic experiment (Zhu and Zhang, 2008). In the present work, the accumulation of PAHs by maize root in the treatments of Tween 80 and rhamnolipids amendments were higher than with the other two treatments, which might be ascribed to different abilities of the surfactants to desorb PAHs from soil.
4. Conclusions This work has investigated the effect of surfactants on the phytoremediation of crude oil-contaminated soil. The addition of surfactants did not significantly affect the biomass production of maize plant, but it inhibited the ChFl in the maize leaf. The soil microbial population increased in the treatments with soybean
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lecithin and rhamnolipid, which improved the removal efficiency of petroleum hydrocarbons from soil. At the end of soil remediation, the proportion of saturated hydrocarbons in TPHs decreased, but those of the aromatic hydrocarbon, and asphaltene/polar fraction increased. In addition, under the treatments of Tween 80 and rhamnolipid the accumulation of PAHs was inhibited in maize leaf, but enhanced in maize root. In summary, treatment with biosurfactants and growth of maize is a good alternative technology for the remediation of petroleum-contaminated soil. Acknowledgements The research was financially supported by the National Natural Science Foundation of China (No. 41573091), the Natural Science Foundation of Guangdong Province (No. 2015A030306005), and the Program for Pearl River Young Talents of Science and Technology in Guangzhou (No. 2013J2200007). The authors would like to thank Dr. Donald Barnes for language editing on the manuscript. References Abdel-Mawgoud, A.M., Lépine, F., Déziel, E., 2010. Rhamnolipids: diversity of structures, microbial origins and roles. Appl. Microbiol. Biotechnol. 86, 1323–1336. Björkman, O., Demmig, B., 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489–504. Calvo, C., Manzanera, M., Silva-Castro, G.A., Uad, I., González-López, J., 2009. Application of bioemulsifiers in soil oil bioremediation processes. Future prospects. Sci. Total Environ. 407, 3634–3640. Collins, C., Fryer, M., Grosso, A., 2006. Plant uptake of non-ionic organic chemicals. Environ. Sci. Technol. 40, 45–52. Desalme, D., Binet, P., Chiapusio, G., 2013. Challenges in tracing the fate and effects of atmospheric polycyclic aromatic hydrocarbon deposition in vascular plants. Environ. Sci. Technol. 47, 3967–3981. Fan, S., Li, P., Gong, Z., Ren, W., He, N., 2008. Promotion of pyrene degradation in rhizosphere of alfalfa (Medicago sativa L.). Chemosphere 71, 1593–1598. Fava, F., Berselli, S., Conte, P., Piccolo, A., Marchetti, L., 2004. Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs). Biotechnol. Bioeng. 88, 214–223. Gao, Y., Collins, C.D., 2009. Uptake pathways of polycyclic aromatic hydrocarbons in white clover. Environ. Sci. Technol. 43, 6190–6195. Gao, Y., Shen, Q., Ling, W., Ren, L., 2008. Uptake of polycyclic aromatic hydrocarbons by Trifolium pretense L. from water in the presence of a nonionic surfactant. Chemosphere 72, 636–643. Lai, C., Huang, Y., Wei, Y., Chang, J., 2009. Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil. J. Hazard. Mater. 167, 609–614. Liao, C., Liang, X., Lu, G., Thai, T., Xu, W., Dang, Z., 2015. Effect of surfactant amendment to PAHs-contaminated soil for phytoremediation by maize (Zea mays L.). Ecotoxicol. Environ. Saf. 112, 1–6. Lin, H., Tao, S., Zuo, Q., Coveney, R.M., 2007. Uptake of polycyclic aromatic hydrocarbons by maize plants. Environ. Pollut. 148, 614–619. Liu, R., Xiao, N., Wei, S., Zhao, L., An, J., 2014a. Rhizosphere effects of PAH-contaminated soil phytoremediation using a special plant named Fire Phoenix. Sci. Total Environ. 473, 350–358.
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