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International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil a
a
ab
a
ab
ab
Changjun Liao , Wending Xu , Guining Lu , Xujun Liang , Chuling Guo , Chen Yang Dang
& Zhi
ac
a
School of Environment and Energy, South China University of Technology, Guangzhou, PR China b
The Ministry of Education Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, South China University of Technology, Guangzhou, PR China c
The State Key Lab of Pulp and Paper Engineering, South China University of Technology, Guangzhou, PR China Published online: 15 May 2015.
Click for updates To cite this article: Changjun Liao, Wending Xu, Guining Lu, Xujun Liang, Chuling Guo, Chen Yang & Zhi Dang (2015) Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil, International Journal of Phytoremediation, 17:7, 693-700, DOI: 10.1080/15226514.2014.964840 To link to this article: http://dx.doi.org/10.1080/15226514.2014.964840
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
International Journal of Phytoremediation, 17: 693–700, 2015 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2014.964840
Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil CHANGJUN LIAO1, WENDING XU1, GUINING LU1,2, XUJUN LIANG1, CHULING GUO1,2, CHEN YANG1,2, and ZHI DANG1,3 1
School of Environment and Energy, South China University of Technology, Guangzhou, PR China The Ministry of Education Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, South China University of Technology, Guangzhou, PR China 3 The State Key Lab of Pulp and Paper Engineering, South China University of Technology, Guangzhou, PR China
Downloaded by [New York University] at 02:33 30 May 2015
2
This study has investigated the use of screened maize for remediation of soil contaminated with crude oil. Pots experiment was carried out for 60 days by transplanting maize seedlings into spiked soils. The results showed that certain amount of crude oil in soil (≤2 147 mg·kg−1) could enhance the production of shoot biomass of maize. Higher concentration (6 373 mg·kg−1) did not significantly inhibit the growth of plant maize (including shoot and root). Analysis of plant shoot by GC-MS showed that low molecular weight polycyclic aromatic hydrocarbons (PAHs) were detected in maize tissues, but PAHs concentration in the plant did not increase with higher concentration of crude oil in soil. The reduction of total petroleum hydrocarbon in planted soil was up to 52.21–72.84%, while that of the corresponding controls was only 25.85–34.22% in two months. In addition, data from physiological and biochemical indexes demonstrated a favorable adaptability of maize to crude oil pollution stress. This study suggested that the use of maize (Zea mays L.) was a good choice for remediation of soil contaminated with petroleum within a certain range of concentrations. Keywords: phytoremediation, PAHs; Maize, crude oil, soil contamination
Introduction The wide use of petroleum has caused serious environmental problem. For example, soil contamination by petroleum hydrocarbons has been attracting considerable attention over the past decades (Peng et al. 2009), which can occur from pipeline blow-outs, waste deposal of drilling operations, road accidents, leaking underground storage tanks, land-farming fields and uncontrolled landfills (Chaineau et al. 2003). As land resources become more scarce due to population growth and increased demands for food, it is urgent to restore and make full use of the polluted soils for food production. Current remediation methods for managing contaminated soils include physical, chemical and biological remediation technologies, such as soil washing, composting, and biopiles. However, traditional remediation methods are costly and prone to secondary contamination or damage to soil structure that renders it unsuitable for plant growth (Zand, Bidhendi and Mehrdadi 2010). Therefore, phytoremediation creates a new era, on which the public place hope as a promising green technology.
Address correspondence to Guining Lu, School of Environment and Energy, South China University of Technology, Guangzhou, 510006, PR China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp.
Phytoremediation, the use of plants to detoxify polluted environments, is under active investigation as a low-cost and environmentally friendly technique for reclaiming contaminated soils and waters (Pilon-Smits 2005). In the past, a number of different plant species, such as ornamental plants (Peng et al. 2009), alfalfa plants (Muratova et al. 2008; Wei and Pan 2010), tall fescue (Chen, Banks and Schwab 2003; Liu et al. 2010), ryegrass (Cheema et al. 2010; Rezek et al. 2008), and maize (Kaimi, Mukaidani and Tamaki 2007; Zand et al. 2010) have been used by researchers to investigate the effectiveness of phytoremediation for hydrocarbons-contaminated environments. These efforts yield fruitful results for removal of pollutants. On the other hand, leguminous plants with nitrogen-fixing bacterial have also applied in soils contaminated with high levels of crude oil, which are often nitrogen (N) limited (Kirkpatrick et al. 2006). Also, using cultivation of multispecies (ryegrass, white clover, and celery) appears to be a more effective approach to remove PAHs from industrial soils as compared with monoculture cropping (Meng, Qiao and Arp 2011). However, due to the growing demands placed on agricultural lands for food in many nations, especially in China and other developing countries, an alternative approach is needed. Specifically, the overall efficiency of the process will be enhanced if the remediating plant cannot only remove pollutants from the soil but simultaneously produce a commercially viable food stock or energy biomass, as well. Therefore, it is
Downloaded by [New York University] at 02:33 30 May 2015
694 important to investigate possible crops which can perform the dual purpose of soil remediation and commercial production. Previous studies (Jansson et al. 2009) have investigated a variety of crops for biofuel production. We believe that maize is a strong candidate that should be considered for this purpose, since maize has greater adaptability to stressed conditions around the world and is already a key crop for biomassfor-energy production. For example, maize accounts for 27.3% of the total agricultural production acreage in the world (Yi et al. 2010) and has given indications of a promising future in renewable energy production and CO2 abatement (Sticklen 2008; Torney et al. 2007; Witters et al. 2012). In our previous work, a maize cultivars CT 38 was used to successfully remediate soils co-polluted with pyrene and cadmium (Zhang et al. 2009a; Zhang et al. 2009b). Based on the results of earlier work, the current study examines whether the crop can adapt to soil contaminated with crude oil. If so, then the extent of bioaccumulation of petroleum hydrocarbons from soil into the maize will be determined, since hydrocarbons can be beneficial to in bioenergy production, while PAHs in crude oil may latently threaten to food chain safety. Thus, we investigated the physiological and biochemical responses of maize to crude oil pollutant in soil and examined the accumulation of n-alkanes and PAHs in the plant’s tissue.
Materials and Methods Chemicals and Seed of Maize Crude oil without refining was obtained from Guangzhou Department, Sinopec Corporation, China. All other agents used in the study were analytical grade except hexane and dichloromethane, which were HPLC grade for chromatographic analysis. Seed of CT 38 was purchased at Research Institution of Crop, Guangdong Academy of Agricultural Sciences, China. Soil The soil used for experiment was collected from the upper layer of abandoned farmland in Guangzhou Higher Education Mega Center, Guangzhou, China. The soil was air-dried, smashed and passed through a 4 mm sieve to remove stones and roots. The organic matter content of the soil was 1.3%, the pH was 6.54. Nutrient levels were 24.5 g·kg−1 ammoniac nitrogen, 4.32 g·kg−1 total P and 397 mg·kg−1 total K. Experimental Design and Management The soil was placed in plastic crate, spiked with four different amounts of crude oil and stirred for homogeneity with a wood spoon. Crude oil concentration was selected as 0, 1500, 2500, 5000, and 10000 mg·kg−1 based on report by Peng et al. (2009). The soil was then put into plastic pots and placed outdoors for four months in order to adequately evaporate the volatile fractions of crude oil. The measured Total Petroleum
C. Liao et al. Hydrocarbon (TPH) levels (T0-4) were 0, 973, 2147, 3047 and 6373 mg·kg−1, respectively. The corresponding control without planting maize was C1-4. Each treatment was replicated three times. The maize seeds were germinated and grown in moist culture dishes with filter paper. After 15 days, seedlings with the same height were transplanted into contaminated soil in the plastic pots. The pots were placed outdoors at the laboratory building of our school. The date (September 15, 2011) at which the seedlings were transplanted to the pots was considered as the starting time of experiment, and the plants were harvested after growing for 60 days in pot. Mean air temperature was 23.2 ◦ C during the experiment. Analytical Methods Plant growth and biomass Before harvesting, the heights and stem diameters at 10 cm above the soil of seedlings were measured with a flexible ruler and a vernier caliper, respectively. To determine the weight of the biomass of the plants, shoots and roots were separated and washed with tap water followed by distilled water, and dried in an air oven at 50 ◦ C for 48 h. Malondialdehyde determination The malondialdehyde (MDA) content in the leaves of the maize seedlings was conducted as described by Liu and Li (2007). The fresh plant sample (0.5 g) was homogenized with 5 mL 5% trichloroacetic acid (TCA). The homogenates were centrifuged at 3 000 rpm for 10 min. Two mL supernatant and 2 mL 5% TBA were mixed and kept in the water bath (100 ◦ C) for 30 min, and centrifuged after cooling. The MDA content was calculated utilizing the extinction coefficient of 155 m·mol−1·cm−1 and expressed as nmol·g−1 fresh weight (FW). Antioxidant enzymes activity Leaf tissues (0.2 g) were homogenized in ice-cold phosphate-buffered solution (0.5 M, pH 7), containing 1% polyvinylpyrrolidone (PVP), centrifuged at 5 000 rpm and 4 ◦ C for 15 min. The supernatant was used immediately to determine the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and content of protein. The SOD activity was assayed at 560 nm by the nitro-blue-tetrazolium (NBT) photoreduction method (Spychalla and Desborough 1990). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT and the SOD activity was expressed as U·mg−1·protein·min−1. The POD activity was determined at 470 nm using the guaiacol method. The measurement was initiated by adding H2 O2 and lasted for 180 seconds. The CAT activity was determined from the decrease in absorbance at 240 nm for 180 seconds, fol¨ lowing H2 O2 consumption. (Turkan et al. 2005). The protein content was determined using the Coomassie blue staining method (Liu and Li 2007). The ascorbate peroxidase (APX) activity was measured by following the decrease in absorbance at 290 nm on a spectrometer (UV 2550, Shimadzu Corporation). Leaf tissues
Accumulation of Hydrocarbons by Maize
Downloaded by [New York University] at 02:33 30 May 2015
(0.5 g) were homogenized in ice-cold 5 mL enzyme exact (pH 7.80) containing 0.5 M PBS buffer, 15 mM ethylenediamine tetraacetic acid (EDTA), 12 mM ascorbate (AsA), followed by centrifugation at 8 000 rpm and 4 ◦ C for 15 min. The supernatant was used immediately to determine the activity of APX. The assay mixture (1 mL) contained 0.5 M PBS (pH 7.00), 0.1 mM EDTA, 0.5 mM AsA, enzyme extract, and 0.8 mM H2 O2 . The measurement was initiated by adding H2 O2 and lasted for 60 seconds (Liu et al. 2009). Root activity Root activity was measured at the end the experiment using the reduction of triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF) (Liu and Li 2007). Briefly, 0.5 g root sample was incubated for 24 h at 37 ◦ C in 5 ml of TTC solution (5 g·L−1 in 0.2 mol·L−1 Tris-HCl buffer, pH 7.4). The sample was then blended with 5 mL of toluene to extract TPF and absorbance in the extract was measured at 492 nm. Root activity was expressed as μg·TPF·g−1 fresh mass (FM) of root 24 h−1. Determination of total petroleum hydrocarbon in soil TPH in soil was determined by using the gravimetric method (Peng et al. 2009). About 5.00 g air-dried and sieved (through a 100 mesh sieve) soil was transferred to a glass centrifuge tube, with 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 3 000 rpm for 10 min. This extraction was repeated twice more. The supernatant were combined in a beaker and allowed to evaporate at room temperature in a fuming cupboard. The amount of residual TPH was determined by the remaining in the air-dried beaker. Analysis of n-alkanes and PAHs in plant shoots Measurement of n-alkanes and PAHs in the maize seedling was conducted according to Tao et al. (2006). Briefly, 1.00 g dried shoot sample was homogenized with about 1 g of anhydrous sodium sulfate in glass tube, followed by adding 10 mL hexane/dichloromethane (1:1) into the tube 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 and 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 μm filter for further analysis by GC-MS. The analysis was conducted using a GC-MS that coupled a-Thermo-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 μm 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 μL aliquot of the extract was injected into an injector port held at 280 ◦ C and operated in a splitless mode with a flow rate of 1.0 mL·min−1. Selective ion monitoring model was used and the target ion was m/z 57 for n-alkanes and molecular weight for PAHs. The process blank was determined by going
695 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. Statistical Analysis SPSS 17.0 was used for the statistical evaluation of the results. The results were designed as completely randomized with three replicates of each parameter. Mean values followed by the same letter were not 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.
Results and Discussion Plant Growth and Biomass Plant height and stem diameter of maize seedlings was measured at the end of experiment to explore the adaptability of maize to crude oil. As shown in Table 1, the plant height and stem diameter increased and then decreased with increasing of crude oil contents. Among the five treatments, plant growth (expressed as height and stem diameter) in treatment 2 (T2) was significantly increased, while that in treatment 4 (T4), the lowest one, was not significantly different compared to the control (T0). The results indicated crude oil in soil within some limits might enhance the growth of maize plant. It was considered that soil spiked with certain amount of crude oil could have an influence on the maize bacterial community possessing many plant promoting growth bacteria species, which produce the growth regulators stimulating plant growth, protecting from soil disease, supplying with nitrogen (Dilfuza 2007). Compared with other treatments, T2 possibly contained appropriate content of crude oil, which was suitable for activating soil microorganism to enhance maize growth. Similar results could be seen from the dry biomass of shoot in Table 1, exhibited the highest biomass production, which was 1.2 times to T0. Meanwhile, biomass production was lower in T3 and T4 but there was no significant difference compared to the control. These results were different from other studies that reported decreased growth and production of plant biomass in soils contaminated with petroleum hydrocarbons (Barrutia et al. 2011; Sharonova and Breus 2012). From the above results, it seems that maize cultivar CT38 could benefit from crude oil in soils at lower concentration (
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil a
a
ab
a
ab
ab
Changjun Liao , Wending Xu , Guining Lu , Xujun Liang , Chuling Guo , Chen Yang Dang
& Zhi
ac
a
School of Environment and Energy, South China University of Technology, Guangzhou, PR China b
The Ministry of Education Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, South China University of Technology, Guangzhou, PR China c
The State Key Lab of Pulp and Paper Engineering, South China University of Technology, Guangzhou, PR China Published online: 15 May 2015.
Click for updates To cite this article: Changjun Liao, Wending Xu, Guining Lu, Xujun Liang, Chuling Guo, Chen Yang & Zhi Dang (2015) Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil, International Journal of Phytoremediation, 17:7, 693-700, DOI: 10.1080/15226514.2014.964840 To link to this article: http://dx.doi.org/10.1080/15226514.2014.964840
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
International Journal of Phytoremediation, 17: 693–700, 2015 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2014.964840
Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil CHANGJUN LIAO1, WENDING XU1, GUINING LU1,2, XUJUN LIANG1, CHULING GUO1,2, CHEN YANG1,2, and ZHI DANG1,3 1
School of Environment and Energy, South China University of Technology, Guangzhou, PR China The Ministry of Education Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, South China University of Technology, Guangzhou, PR China 3 The State Key Lab of Pulp and Paper Engineering, South China University of Technology, Guangzhou, PR China
Downloaded by [New York University] at 02:33 30 May 2015
2
This study has investigated the use of screened maize for remediation of soil contaminated with crude oil. Pots experiment was carried out for 60 days by transplanting maize seedlings into spiked soils. The results showed that certain amount of crude oil in soil (≤2 147 mg·kg−1) could enhance the production of shoot biomass of maize. Higher concentration (6 373 mg·kg−1) did not significantly inhibit the growth of plant maize (including shoot and root). Analysis of plant shoot by GC-MS showed that low molecular weight polycyclic aromatic hydrocarbons (PAHs) were detected in maize tissues, but PAHs concentration in the plant did not increase with higher concentration of crude oil in soil. The reduction of total petroleum hydrocarbon in planted soil was up to 52.21–72.84%, while that of the corresponding controls was only 25.85–34.22% in two months. In addition, data from physiological and biochemical indexes demonstrated a favorable adaptability of maize to crude oil pollution stress. This study suggested that the use of maize (Zea mays L.) was a good choice for remediation of soil contaminated with petroleum within a certain range of concentrations. Keywords: phytoremediation, PAHs; Maize, crude oil, soil contamination
Introduction The wide use of petroleum has caused serious environmental problem. For example, soil contamination by petroleum hydrocarbons has been attracting considerable attention over the past decades (Peng et al. 2009), which can occur from pipeline blow-outs, waste deposal of drilling operations, road accidents, leaking underground storage tanks, land-farming fields and uncontrolled landfills (Chaineau et al. 2003). As land resources become more scarce due to population growth and increased demands for food, it is urgent to restore and make full use of the polluted soils for food production. Current remediation methods for managing contaminated soils include physical, chemical and biological remediation technologies, such as soil washing, composting, and biopiles. However, traditional remediation methods are costly and prone to secondary contamination or damage to soil structure that renders it unsuitable for plant growth (Zand, Bidhendi and Mehrdadi 2010). Therefore, phytoremediation creates a new era, on which the public place hope as a promising green technology.
Address correspondence to Guining Lu, School of Environment and Energy, South China University of Technology, Guangzhou, 510006, PR China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp.
Phytoremediation, the use of plants to detoxify polluted environments, is under active investigation as a low-cost and environmentally friendly technique for reclaiming contaminated soils and waters (Pilon-Smits 2005). In the past, a number of different plant species, such as ornamental plants (Peng et al. 2009), alfalfa plants (Muratova et al. 2008; Wei and Pan 2010), tall fescue (Chen, Banks and Schwab 2003; Liu et al. 2010), ryegrass (Cheema et al. 2010; Rezek et al. 2008), and maize (Kaimi, Mukaidani and Tamaki 2007; Zand et al. 2010) have been used by researchers to investigate the effectiveness of phytoremediation for hydrocarbons-contaminated environments. These efforts yield fruitful results for removal of pollutants. On the other hand, leguminous plants with nitrogen-fixing bacterial have also applied in soils contaminated with high levels of crude oil, which are often nitrogen (N) limited (Kirkpatrick et al. 2006). Also, using cultivation of multispecies (ryegrass, white clover, and celery) appears to be a more effective approach to remove PAHs from industrial soils as compared with monoculture cropping (Meng, Qiao and Arp 2011). However, due to the growing demands placed on agricultural lands for food in many nations, especially in China and other developing countries, an alternative approach is needed. Specifically, the overall efficiency of the process will be enhanced if the remediating plant cannot only remove pollutants from the soil but simultaneously produce a commercially viable food stock or energy biomass, as well. Therefore, it is
Downloaded by [New York University] at 02:33 30 May 2015
694 important to investigate possible crops which can perform the dual purpose of soil remediation and commercial production. Previous studies (Jansson et al. 2009) have investigated a variety of crops for biofuel production. We believe that maize is a strong candidate that should be considered for this purpose, since maize has greater adaptability to stressed conditions around the world and is already a key crop for biomassfor-energy production. For example, maize accounts for 27.3% of the total agricultural production acreage in the world (Yi et al. 2010) and has given indications of a promising future in renewable energy production and CO2 abatement (Sticklen 2008; Torney et al. 2007; Witters et al. 2012). In our previous work, a maize cultivars CT 38 was used to successfully remediate soils co-polluted with pyrene and cadmium (Zhang et al. 2009a; Zhang et al. 2009b). Based on the results of earlier work, the current study examines whether the crop can adapt to soil contaminated with crude oil. If so, then the extent of bioaccumulation of petroleum hydrocarbons from soil into the maize will be determined, since hydrocarbons can be beneficial to in bioenergy production, while PAHs in crude oil may latently threaten to food chain safety. Thus, we investigated the physiological and biochemical responses of maize to crude oil pollutant in soil and examined the accumulation of n-alkanes and PAHs in the plant’s tissue.
Materials and Methods Chemicals and Seed of Maize Crude oil without refining was obtained from Guangzhou Department, Sinopec Corporation, China. All other agents used in the study were analytical grade except hexane and dichloromethane, which were HPLC grade for chromatographic analysis. Seed of CT 38 was purchased at Research Institution of Crop, Guangdong Academy of Agricultural Sciences, China. Soil The soil used for experiment was collected from the upper layer of abandoned farmland in Guangzhou Higher Education Mega Center, Guangzhou, China. The soil was air-dried, smashed and passed through a 4 mm sieve to remove stones and roots. The organic matter content of the soil was 1.3%, the pH was 6.54. Nutrient levels were 24.5 g·kg−1 ammoniac nitrogen, 4.32 g·kg−1 total P and 397 mg·kg−1 total K. Experimental Design and Management The soil was placed in plastic crate, spiked with four different amounts of crude oil and stirred for homogeneity with a wood spoon. Crude oil concentration was selected as 0, 1500, 2500, 5000, and 10000 mg·kg−1 based on report by Peng et al. (2009). The soil was then put into plastic pots and placed outdoors for four months in order to adequately evaporate the volatile fractions of crude oil. The measured Total Petroleum
C. Liao et al. Hydrocarbon (TPH) levels (T0-4) were 0, 973, 2147, 3047 and 6373 mg·kg−1, respectively. The corresponding control without planting maize was C1-4. Each treatment was replicated three times. The maize seeds were germinated and grown in moist culture dishes with filter paper. After 15 days, seedlings with the same height were transplanted into contaminated soil in the plastic pots. The pots were placed outdoors at the laboratory building of our school. The date (September 15, 2011) at which the seedlings were transplanted to the pots was considered as the starting time of experiment, and the plants were harvested after growing for 60 days in pot. Mean air temperature was 23.2 ◦ C during the experiment. Analytical Methods Plant growth and biomass Before harvesting, the heights and stem diameters at 10 cm above the soil of seedlings were measured with a flexible ruler and a vernier caliper, respectively. To determine the weight of the biomass of the plants, shoots and roots were separated and washed with tap water followed by distilled water, and dried in an air oven at 50 ◦ C for 48 h. Malondialdehyde determination The malondialdehyde (MDA) content in the leaves of the maize seedlings was conducted as described by Liu and Li (2007). The fresh plant sample (0.5 g) was homogenized with 5 mL 5% trichloroacetic acid (TCA). The homogenates were centrifuged at 3 000 rpm for 10 min. Two mL supernatant and 2 mL 5% TBA were mixed and kept in the water bath (100 ◦ C) for 30 min, and centrifuged after cooling. The MDA content was calculated utilizing the extinction coefficient of 155 m·mol−1·cm−1 and expressed as nmol·g−1 fresh weight (FW). Antioxidant enzymes activity Leaf tissues (0.2 g) were homogenized in ice-cold phosphate-buffered solution (0.5 M, pH 7), containing 1% polyvinylpyrrolidone (PVP), centrifuged at 5 000 rpm and 4 ◦ C for 15 min. The supernatant was used immediately to determine the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and content of protein. The SOD activity was assayed at 560 nm by the nitro-blue-tetrazolium (NBT) photoreduction method (Spychalla and Desborough 1990). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT and the SOD activity was expressed as U·mg−1·protein·min−1. The POD activity was determined at 470 nm using the guaiacol method. The measurement was initiated by adding H2 O2 and lasted for 180 seconds. The CAT activity was determined from the decrease in absorbance at 240 nm for 180 seconds, fol¨ lowing H2 O2 consumption. (Turkan et al. 2005). The protein content was determined using the Coomassie blue staining method (Liu and Li 2007). The ascorbate peroxidase (APX) activity was measured by following the decrease in absorbance at 290 nm on a spectrometer (UV 2550, Shimadzu Corporation). Leaf tissues
Accumulation of Hydrocarbons by Maize
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(0.5 g) were homogenized in ice-cold 5 mL enzyme exact (pH 7.80) containing 0.5 M PBS buffer, 15 mM ethylenediamine tetraacetic acid (EDTA), 12 mM ascorbate (AsA), followed by centrifugation at 8 000 rpm and 4 ◦ C for 15 min. The supernatant was used immediately to determine the activity of APX. The assay mixture (1 mL) contained 0.5 M PBS (pH 7.00), 0.1 mM EDTA, 0.5 mM AsA, enzyme extract, and 0.8 mM H2 O2 . The measurement was initiated by adding H2 O2 and lasted for 60 seconds (Liu et al. 2009). Root activity Root activity was measured at the end the experiment using the reduction of triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF) (Liu and Li 2007). Briefly, 0.5 g root sample was incubated for 24 h at 37 ◦ C in 5 ml of TTC solution (5 g·L−1 in 0.2 mol·L−1 Tris-HCl buffer, pH 7.4). The sample was then blended with 5 mL of toluene to extract TPF and absorbance in the extract was measured at 492 nm. Root activity was expressed as μg·TPF·g−1 fresh mass (FM) of root 24 h−1. Determination of total petroleum hydrocarbon in soil TPH in soil was determined by using the gravimetric method (Peng et al. 2009). About 5.00 g air-dried and sieved (through a 100 mesh sieve) soil was transferred to a glass centrifuge tube, with 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 3 000 rpm for 10 min. This extraction was repeated twice more. The supernatant were combined in a beaker and allowed to evaporate at room temperature in a fuming cupboard. The amount of residual TPH was determined by the remaining in the air-dried beaker. Analysis of n-alkanes and PAHs in plant shoots Measurement of n-alkanes and PAHs in the maize seedling was conducted according to Tao et al. (2006). Briefly, 1.00 g dried shoot sample was homogenized with about 1 g of anhydrous sodium sulfate in glass tube, followed by adding 10 mL hexane/dichloromethane (1:1) into the tube 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 and 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 μm filter for further analysis by GC-MS. The analysis was conducted using a GC-MS that coupled a-Thermo-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 μm 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 μL aliquot of the extract was injected into an injector port held at 280 ◦ C and operated in a splitless mode with a flow rate of 1.0 mL·min−1. Selective ion monitoring model was used and the target ion was m/z 57 for n-alkanes and molecular weight for PAHs. The process blank was determined by going
695 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. Statistical Analysis SPSS 17.0 was used for the statistical evaluation of the results. The results were designed as completely randomized with three replicates of each parameter. Mean values followed by the same letter were not 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.
Results and Discussion Plant Growth and Biomass Plant height and stem diameter of maize seedlings was measured at the end of experiment to explore the adaptability of maize to crude oil. As shown in Table 1, the plant height and stem diameter increased and then decreased with increasing of crude oil contents. Among the five treatments, plant growth (expressed as height and stem diameter) in treatment 2 (T2) was significantly increased, while that in treatment 4 (T4), the lowest one, was not significantly different compared to the control (T0). The results indicated crude oil in soil within some limits might enhance the growth of maize plant. It was considered that soil spiked with certain amount of crude oil could have an influence on the maize bacterial community possessing many plant promoting growth bacteria species, which produce the growth regulators stimulating plant growth, protecting from soil disease, supplying with nitrogen (Dilfuza 2007). Compared with other treatments, T2 possibly contained appropriate content of crude oil, which was suitable for activating soil microorganism to enhance maize growth. Similar results could be seen from the dry biomass of shoot in Table 1, exhibited the highest biomass production, which was 1.2 times to T0. Meanwhile, biomass production was lower in T3 and T4 but there was no significant difference compared to the control. These results were different from other studies that reported decreased growth and production of plant biomass in soils contaminated with petroleum hydrocarbons (Barrutia et al. 2011; Sharonova and Breus 2012). From the above results, it seems that maize cultivar CT38 could benefit from crude oil in soils at lower concentration (
