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Role of Streptomyces pactum in phytoremediation of trace elements by Brassica juncea in mine polluted soils
Ecotoxicology and Environmental Safety 144 (2017) 387–395
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Role of Streptomyces pactum in phytoremediation of trace elements by Brassica juncea in mine polluted soils
MARK
Amjad Alia, Di Guoa, Amanullah Mahara,b, Zhen Wanga, Dost Muhammadc, Ronghua Lia, ⁎ Ping Wanga, Feng Shena, Quanhong Xuea, Zengqiang Zhanga, a b c
College of Natural Resources and Environment, Northwest A & F University, Yangling 712100, China Centre for Environmental Sciences, University of Sindh, Jamshoro 76080, Pakistan Department of Soil and Environmental Sciences, The University of Agriculture, Peshawar 25130, Pakistan
A R T I C L E I N F O
A B S T R A C T
Keywords: Brassica juncea Medical stone compost Mining Phytoremediation Streptomyces pactum Trace elements
The industrial expansion, smelting, mining and agricultural practices have increased the release of toxic trace elements (TEs) in the environment and threaten living organisms. The microbe-assisted phytoremediation is environmentally safe and provide an effective approach to remediate TEs contaminated soils. A pot experiment was conducted to test the potential of an Actinomycete, subspecies Streptomyces pactum (Act12) along with medical stone compost (MSC) by growing Brassica juncea in smelter and mines polluted soils of Feng County (FC) and Tongguan (TG, China), respectively. Results showed that Zn (7, 28%), Pb (54, 21%), Cd (16, 17%) and Cu (8, 10%) uptake in shoot and root of Brassica juncea was pronounced in FC soil. Meanwhile, the Zn (40, 14%) and Pb (82, 15%) uptake in the shoot and root were also increased in TG soil. Shoot Cd uptake remained below detection, while Cu decreased by 52% in TG soil. The Cd and Cu root uptake were increased by 17% and 33%, respectively. Results showed that TEs uptake in shoot increased with increasing Act12 dose. Shoot/root dry biomass, chlorophyll and carotenoid content in Brassica juncea were significantly influenced by the application of Act12 in FC and TG soil. The antioxidant enzymatic activities (POD, PAL, PPO and CAT) in Brassica juncea implicated enhancement in the plant defense mechanism against the TEs induced stress in contaminated soils. The extraction potential of Brasssica was further evaluated by TF (translocation factor) and MEA (metal extraction amount). Based on our findings, further investigation of Act12 assisted phytoremediation of TEs in the smelter and mines polluted soil and hyperaccumulator species are suggested for future studies.
1. Introduction Maximum food production and industrial activities are required in order to satisfy the basic needs of world growing population. The burgeoning population, disarrayed industrialization and technical innovations have led to the global increase in the release of trace elements (TEs) and widespread contamination of the environment. TEs pollution has pernicious effects on soil, air and human health due to soil-plant transfer to reach food-chain (Cheng et al., 2016). Sources of TEs include petrochemicals, textile, steel manufacturing, mining and smelting, as well as coal combustion (Alvarez et al., 2017). The impacts of TEs have been studied regarding soil biology, water/air quality as well as animals and human health. TEs reduce the soil enzymatic activities, degrade air and water quality in the close proximity of the source point, decrease the biosynthesis of chlorophyll, reduce respiration and limit antioxidant activities in plants (Ali et al., 2017; Xian et al., 2015). TEs reduce soil organic matter decomposition and ⁎
Corresponding author. E-mail address: [email protected] (Z. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2017.06.046 Received 28 January 2017; Received in revised form 13 June 2017; Accepted 16 June 2017 0147-6513/ © 2017 Published by Elsevier Inc.
nutrients cycling (Ma et al., 2015). Furthermore, TEs are carcinogenic and mutagenic and can cause fatal diseases in humans and animals, if exposed to higher levels (Cao et al., 2016). A variety of physical, chemical, and biological remediation techniques are being used for phytoextraction of TEs to treat the contaminated sites. These practices have produced secondary pollutants and account for contamination of the environment in the long-term adoption. The use of biotechnology in the remediation of TEs is getting the recognition due to the recent developments (Ali et al., 2017). The bacterial inoculation can potentially reduce phytotoxicity, increases TEs uptake and removal, and enhance plant biomass in TEs contaminated soil. Soil microbes affect the mobility and availability of TEs through chelation, acidification and siderophores formation (AbouShanab et al., 2008; Gopalakrishnan et al., 2013). Actinobacteria (Act) exhibit diverse physiological and metabolic properties, playing a vital role in phytoremediation by promoting the TEs uptake in plants and increase biomass production (Aparicio et al., 2015; Gopalakrishnan
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et al., 2015; Rajkumar et al., 2012). Members of the order Actinomycetales i.e. Streptomyces are known to promote the growth in Oryza sativa, Sorghum bicolor, Cicer arietinum and Amaranthus hypochondriacus (Cao et al., 2016; Gopalakrishnan et al., 2015). Streptomyces pactum (Act12) is reported to promote the plant growth, control pathogenic disease and promote the phytoremediation of Cd. However, its role in phytoremediation of TEs and antioxidant activities was not reported in smelter/mines contaminated soil (Zhao et al., 2012). Phytoremediation is a promising green technology by using plants to restore TEs contaminated soils (Cao et al., 2016). Phytoremediation can effectively be practiced to extract TEs and achieve sustainable rehabilitation of contaminated land (Babu et al., 2014). The microbeassisted phytoremediation technique depends on the interaction between the host plant and microorganism species. It is cost-effective and eco-friendly by inoculating hyperaccumulator plants with plant growthpromoting microorganisms (Alvarez et al., 2017; Ho et al., 2013; Ma et al., 2011). Remediation of mine sites is a complex process, as a variety of TEs and soil physicochemical conditions influence the process (Ma et al., 2015). Brassica juncea belongs to the Brassicaceae family and mainly grown for oil production and as a forage crop. Brassica is known as hyperaccumulator due to its potential to grow in the contaminated soil and accumulate huge amounts of the TEs in its shoot and root biomass (Mobin and Khan, 2007). Association of plant growth-promoting bacteria with brassica has been widely studied for the TEs translocation in the shoot and root biomass in contaminated soil (Kumar et al., 2009; Ma et al., 2009). The prime objective of the study was to assess the phytoremediation potential of Streptomyces pactum in smelter/mines polluted soils by growing Brassica juncea and evaluate its effects on enzymatic activities.
Northwest A & F University, Yangling, China (Zhao et al., 2012). Act12 carrier agent (2.6×1011 spores g−1) was applied in powder form to treated pots.
2. Material and methods
Chlorophyll (a, b) and carotenoid contents in Brassica juncea were extracted by grinding leaves (0.2 g) in 80% acetone in the dark and calculated from the absorbance of extract at 663, 645 and 470 nm. The concentrations were expressed as mg g−1 FW (Wang et al., 2014). After 7 weeks, the shoot and root of Brassica juncea were harvested, thoroughly washed with running tap water followed by deionized water and dried up to a constant weight at 105 °C. The dried biomass was crushed into fine powder and stored. Shoot (0.50 g) and root (0.25 g) samples were digested with HNO3–HClO4 (3:1) and total TEs (Zn, Pb, Cd, and Cu) were determined (Hu et al., 2014). PPO and PAL activities were detected as per the standard method (Ahammed et al., 2013). Guaiacol peroxidase (POD) and catalase (CAT) activities were determined according to Cheng et al. (2016).
2.2. Experimental methods 2.2.1. Pot experiment The pot experiment was laid out following a completely randomized design under a mobile shelter house in an open environment. Soil treatments (T) were T1 (Control), T2 (0.5 g kg−1 Act12), T3 (1.0 g kg−1 Act12), and T4 (2.0 g kg−1 Act12). Each pot (15×12×10 cm3 dimension) was filled with thoroughly mixed one kg soil (2 mm) as growing media and 2.5% MSC as a nutritional supplement. Ten sterilized (3% H2O2) seeds of Brassica juncea (Shaan you 16) were sown per pot with three replicates per treatment. The shoot/root dry biomasses were recorded after 7 weeks on the harvesting of Brassica plants. 2.3. Soil and medical stone compost analysis Soil pH (1:2), electrical conductivity (EC) and organic matter were measured according to Li et al. (2012). Soil particle size distribution (Mastersizer 2000E laser diffractometer, UK) and cation exchange capacity (CEC) were measured according to Mahar et al. (2016). Total trace elements (Zn, Pb, Cd and Cu) in soil (FC, TG) and MSC were measured by ICP-AES (Hu et al., 2014). Similarly, DTPA (0.005 M) extractable TEs were tested according to Burges et al. (2016). Analytical grade chemicals and double deionized water was used for all chemical analysis in the laboratory. 2.4. Plant analysis
2.1. Samples collection Mines and smelter contaminated soil was collected from Feng County (106°24′~107°7′ N, 33°34′~34°18′ E) and Tongguan (34°27′~34°37′ N, 110°10′~110°23′ E) stations of Shaanxi province, China. Feng County (FC) and Tongguan (TG), are located in the southwest and east of Shaanxi Province, China. Feng County, surrounded by Qinling Mountains, is one of the largest production units in China, with Pb/Zn mines reserves around 4.5 million tons. The climate is dry, the temperature ranging −1.1 to 22.7 °C and average annual rainfall is 613 mm. Surface water and soil pollution are the main environmental hazards in FC, due to the discharge of mines wastewater, atmospheric deposition and mine tailings (Xu et al., 2012). Tongguan is famous for gold mining operations at domestic and industrial scale. The area is mainly polluted with mining, mineral processing and atmospheric deposition of TEs (Ali et al., 2017; Mahar et al., 2016). Climate is humid subtropical with an average temperature and rainfall of 13.4 °C and 581 mm, respectively (Feng et al., 2006). Samples were collected from contaminated surface soil (0–20 cm), stored in polyethylene bags and transferred to the laboratory. Soil samples were homogenized, air-dried, crushed manually and passed through 2 mm sieve. Pig manure was collected from a local pig farm and sawdust from a wood-processing factory in Yangling town, Shaanxi, China. The medical stone (MS) was purchased from Shijiazhuang Building Materials Co. Ltd., China. Medical stone (2.5% dw) added pig manure compost (MSC) was prepared by mixing pig manure and sawdust (2:1 dry weight) in 130 L PVC composter (Li et al., 2012; Wang et al., 2016). The PVC composters were turned over and the compost temperature was daily monitored during the composting process (45 days). Actinomycete was isolated from the Qinghai-Tibet Plateau of China and identified as Streptomyces pactum based on 16S rDNA sequence analysis. Streptomyces pactum carrier was obtained from Laboratory of Microbial Resources at the College of Natural Resources and Environment,
2.5. Phytoextraction indices The amount of TEs translocated from soil to shoot and root was calculated using the following equations (Cao et al., 2016; Shi et al., 2016).
Translocation Factor(TF) =
Metal concentration in shoot Metal concentration in roots
(1)
Metal Extraction Amount (MEA) = Metal concentration in aerial parts × Biomass (2) 2.6. Quality control and statistical analysis All the analytical samples were prepared in three replicates and average readings are presented with standard deviation. Reagent blanks were used to correct the analytical errors. Standard reference materials for wheat and soil (GBW10011 and GBW07405, respectively) were purchased from the National Research Center of Certified Reference Materials (Beijing, China). The recovery of the standard wheat sample 388
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and is of great concern with respect to the environment, plant uptake, and development (Hu et al., 2014). DTPA-extractable concentration of Zn, Pb, Cd and Cu were 584, 40.2, 36.4 and 1.77 mg kg−1 in FC soil, while reported as 15.59, 200, 0.549 and 11.65 mg kg−1 in TG soil. FC and TG are basically mining and smelting sites in Shaanxi province, which contributes specifically to the higher bioavailable content of Zn, Pb and Cd in surface soil (Xu et al., 2012). The extractable Zn, Pb, Cd, and Cu in MSC were 171.11, 5.50, 0.30 and 20.56 mg kg−1, respectively. The extractable TEs contents in MSC were in decreasing order of Zn > Cu > Pb > Cd.
Table 1 Basic characteristics of Feng County, Tongguan soil and medical stone compost. Soil characteristics
Feng County soil
Tongguan soil
Medical stone compost
Clay % 1.56* 0.50 – Silt % 48.43 22.10 – Sand % 50.01 77.33 – Soil texture Sandy loam Loamy sand – pH 7.72 8.06 6.57 −1 422 201.3 732.3 EC (µS cm ) CEC (cmol + /kg) 96.51 19.5 – Organic matter (g kg−1) 14.9 28.40 665 Total Nitrogen (g kg−1) 1.23 0.689 15.64 Total Phosphorus 0.848 0.861 22.50 −1 (g kg ) 5.51 2.9 – Total potassium (mg kg−1) 8.45 16.47 352.87 Total organic carbon (g kg−1) Total TEs in soil (Feng County and Tongguan) and medical stone compost (mg kg−1) Zn 6625 230.4 384.54 Pb 204.4 393.2 8.09 Cd 117.7 1.58 0.557 Cu 51.1 141.36 77.42 31,629 40,529 – Al (mg kg−1) As (mg kg−1) 0.08 9.075 – Ca (mg kg−1) 15,538 24,938 – Co (mg kg−1) 16.20 11.95 – −1 Cr (mg kg ) 61.75 52.548 – −1 Fe (mg kg ) 25,694 19,215 – Hg (mg kg−1) 0.30 0.783 – Mg (mg kg−1) 8205 8592 – Mn (mg kg−1) 729.7 545 – Mo (mg kg−1) 0.85 0.908 – Na (mg kg−1) 9200 10,574 – Extractable TEs in soil (Feng County and Tongguan) and medical stone compost (mg kg−1) Zn 584 15.59 171.11 Pb 40.2 200.0 5.50 Cd 36.4 0.549 0.30 Cu 1.77 11.65 20.56
3.2. Effect of Streptomyces pactum on TEs uptake by Brassica juncea TEs in Brassica shoot grown on FC and TG soils were determined after acid digestion. TEs uptake in the shoot was significantly increased after the application of Act12 to FC soil. However, the uptake was less at low doses and comparatively higher in the case of TG soil. Adsorption on the microbial cells and compost may be the possible reasons for the decreased uptake of Cd and Cu in shoot of TG soil (Babu et al., 2014). 3.2.1. Uptake of TEs by Brassica juncea shoot The accumulation of TEs (Zn, Pb, Cd, and Cu) in the shoot of Brassica grown in FC and TG soils is shown in the Fig. 1. The data showed that TEs uptake in shoot collected from FC soil was significantly (p < 0.05) increased as compared to control. Overall, there was a significant increase in Zn and Pb uptake in TG shoot, while a decrease in Cd and Cu in shoots samples at the application of 0.5–2 g kg−1 Act12. A maximum of 7% increase in Zn was reported at 2 g kg−1, as compared to control in FC soil. Zn content is very high in FC soil as compared to TG soil, as the samples were collected from the close proximity of the Zn/Pb smelter. The plant tissue is almost saturated with Zn and no further Zn accumulation occurred in Brassica. Zn shoot uptake in TG shoot raised from 30.45 to 39.41 mg kg−1, which is still below the concentration reported in the control (47.01 mg kg−1). Metal-resistant Streptomyces AR17 also enhanced Zn and Cd uptake by Salix caprea (Kuffner et al., 2008). Pb uptake in shoot increased significantly (p < 0.05) by 54% and 82% in FC and TG soil at 2 g kg−1 Act12, as compared to their respective control pots. Pb uptake in shoot collected from FC and TG soil showed a steady increase as observed in Zn with the higher dose of Act12. The concentration of Pb is lower than the Zn in soil and the uptake is enhanced in the shoot by the application of Act12. The Pb uptake was well facilitated by the addition of Act12 in the smelter polluted soil of FC. Burkholderia sp. J62 also increased Pb uptake in the shoot of Zea mays by siderophores production (Jiang et al., 2008). Coinoculation of Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 also increased the TEs translocation in the shoot by 69% (Pb) and 82% (Zn), while Cu remained constant (Babu et al., 2014). Cd uptake was enhanced up to 16% by application of 2 g kg−1 Act12 in shoots collected from FC soil. However, Cd was below the detection level in TG shoot samples. The uptake was significantly (p < 0.05) influenced by the inoculation of Act12. As shown in Table 1, DTPA extractable Cd in FC (36.4 mg kg−1) is 66 times greater than TG (0.549 mg kg−1). This resulted in higher shoot uptake in FC shoot samples and the concentration was even below the detection in TG samples. Similarly, Streptomyces sp. F4 also improved the uptake of Cd from culture medium and soil (Haferburg et al., 2008). Cao et al. (2016) also reported 86.5% increase in shoot Cd uptake by Amaranthus hypochondriacus after being inoculated with Act12. The lowering of Cd in TG shoots is attributed to adsorption on the MSC surfaces, added to soil as a nutritional supplement (Babu et al., 2014). Farm yard manure also lowered Cd concentration in wheat grains, but the response varied with types of the soil (Dahlin et al., 2016). Cu uptake in the shoot increased up to 8% (2 g kg−1 Act12) in FC, while decreased by 52% (0.5 g kg−1 Act12) in TG shoot as compared to their respective controls. Cu in the shoot of Brassica juncea increased
* Values indicate mean of one sample with three replications.
ranged from 97.5% to 102.2%, 96.2–101.5%, 96.4–101.5% and 98.1–102.2% for Cd, Cu, Pb and Zn, respectively. The recovery of the standard soil sample ranged from 93.2% to 101.5%, 97.8–105.5%, 96.4–104.8% and 94.5–102.3% for Cd, Cu, Pb, and Zn, respectively. The results obtained for the standard wheat and soil samples were within the acceptable ranges. Data were subjected to one-way ANOVA at p < 0.05 for independent variable analysis and significant differences among the mean values were calculated using SPSS 22.0 (IBM SPSS, Somers, NY, USA). All graphs were drawn using Origin-Pro (7.5 version).
3. Results and discussion 3.1. Characteristics of the studied soils and medical stone compost The main characteristics of soil (FC and TG) and MSC are illustrated in Table 1. The higher Zn, Pb and Cd content in FC and TG soil is attributed to the mining and smelting activities in these areas over a longtime period (Mahar et al., 2016; Xu et al., 2012). Total TEs concentration in FC and TG soil were in decreasing order of Zn > Pb > Cd > Cu and Pb > Zn > Cd > Cu, respectively. Trace elements content in FC and TG soil was higher than the National Environmental Quality Standards for Agricultural Soil (Environmental Quality Standards, China, GB15618-1995). Bioavailability of trace elements in soil is a dynamic process limited by chemical, biological, and environmental factors (Ali et al., 2017). The DTPA-extractable proportion of TEs is considered as bioavailable 389
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Fig. 1. Effect of Streptomyces pactum (Act12) on shoot uptake (mg kg−1 DW) of TEs in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
from 4.47 to 4.76 mg kg−1 in FC samples, while, it raised from 1.16 to 1.74 mg kg−1 in TG samples after the inoculation of Act12. Similarly, Achromobacter xylosoxidans also increased Cu uptake in Brassica juncea grown on the mines contaminated soil in São Domingos, Portugal (Ma et al., 2009). Furthermore, the addition of MSC is also reported to reduce the bioavailability of the TEs, especially Cu uptake by Brassica juncea due to adsorption and immobilization in the soil (Rizwan et al., 2016; Wang et al., 2016). Poor translocation in Brassica juncea shoots could be due to confinement of TEs in the vacuoles of root and Act12 cells to render them and reduce toxicity in the plant (Babu et al., 2013; Hanikenne and Nouet, 2011). Babu et al. (2013) and Rosselli et al. (2003) also reported that Alnus firma growing in polluted soil contained the low concentration of TEs in shoots.
Cr and lindane uptake in Lactuca sativa (Aparicio et al., 2015). The Pb uptake was significantly (p < 0.05) increased as high as 21% and 15% (2 g kg−1 Act12) in shoot in FC and TG soil, respectively. Although, the addition of Act12 to FC soils showed 25% and 16% decrease (0.5 and 1 g kg−1 Act12) in Pb uptake in Brassica juncea root, while higher dose (2 g kg−1 Act12) increased the uptake. It is believed that Pb is immobile in plants and the translocation is low in the Brassica juncea root system. Diminution in Pb uptake was also reported in Phaseolus vulgaris and pine (Krupa and Kozdrój, 2007). In another study, Streptomyces sp. MC1 also immobilized Cr in the soil, while improved maize growth (Polti et al., 2011). Soil microbes reduce the TEs phytotoxicity by employing biosorption and bioaccumulation (Ma et al., 2011). It is believed that microbial adsorption in FC soil might have occurred, which reduced (25, 16%) the Pb uptake in Brassica roots (Babu et al., 2014). The concentration of Pb is higher in soil but the root translocation is lower than Cd ions. TEs translocation in roots depends on the selection of specific ions by the plants and soil physicochemical characteristics (Ansari and Malik, 2007). Cd uptake in root was significantly (p < 0.05) improved by 17% and 33% in the FC and TG (2 g kg−1 Act12) samples, respectively. The DPTA extractable Cd (36.4 mg kg−1) is higher in the FC soil, which was 95 times more translocated than in the TG root samples. The shoot/root growth and Cd uptake were amplified by Bacillus megaterium H3 in Pennisetum purpureum. IAA, organic acids, and siderophores production are believed to promote the growth, reduce stress symptoms and increase TEs tolerance as well as translocation in plants (Li et al., 2016). In another study, Amaranthus hypochondriacus grown in spiked soil, the root Cd uptake was enhanced by 23.6% after Act12 inoculation, which confirmed the results obtained in this study (Cao et al., 2016).
3.2.2. Uptake of TEs by Brassica juncea root The accumulation of TEs in Brassica roots collected from FC and TG soils is shown in Fig. 2. Root data revealed that TEs uptake was significantly (p < 0.05) influenced in both soils by Act12. Act12 enhanced the root and shoot development, which in turn promoted TEs uptake in Brassica juncea shoot (Gopalakrishnan et al., 2013). The Zn uptake was significantly (p < 0.05) increased by 28% and 14% (2 g kg−1 Act12) in FC and TG root samples compared to their controls. The higher Zn uptake in FC roots is attributed to the higher degree of Zn contamination in FC soil. The samples were collected near the main smelter installation, where the extractable Zn (584 mg kg−1) was above the permissible limit. Dary et al. (2010) and Sheng et al. (2008) also reported an increase in Zn, Pb and Cd uptake due to high biomass production after inoculation of seed/soil with plant growthpromoting microbes. Similarly, Streptomyces sp. M7 also increased the 390
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Fig. 2. Effect of Streptomyces pactum (Act12) on root uptake (mg kg−1 DW) of TEs in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
Cu uptake in the FC and TG roots was higher than their respective controls. The translocation was improved by 10% and 17% in FC and TG at 2 g kg−1 Act12, respectively in their respective soil samples. Combine inoculation of Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 improved the root TEs translocation as much as 39% (Cu), 50% (Pb) and 38% (Zn) (Babu et al., 2014). These findings confirmed the role of Act12 in phytoextraction of TEs in polluted soil. TEs uptake in TG shoot is reduced while the accumulation in roots is increased after the inoculation of Act12. The plant-associated microbes have the ability to immobilize the TEs in root and reduce their uptake through binding, accumulation or dilution in the host plant (Babu et al., 2013; Rajkumar et al., 2012). Our findings are in line with the previous scientific reports (Ali et al., 2017). Fig. 3. Effect of Streptomyces pactum (Act12) on the shoot and root dry biomass (g pot−1) of Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
3.3. Effect of Streptomyces pactum on growth attributes of Brassica juncea The effect of Act12 on the shoot and root dry biomass, chlorophyll (a and b) and carotenoid content in Brassica juncea grown on contaminated soils were assessed. Mining activities have adverse effects on the plant physiology, which can be minimized by biotechnological approaches (Alvarez et al., 2017).
mining site in Ronneburg, Germany (Schütze et al., 2014). Similarly, a Cd-resistant Streptomyces tendae F4 secreted siderophores and significantly promoted the growth of Helianthus annuus (Dimkpa et al., 2009). Another study, conducted in Cd contaminated soil inoculated with Act12 also reported 45% and 20% increase in the shoot and root dry weight, respectively (Cao et al., 2016). Act12 promoted the plant development in the TEs rich soil of FC and TG due to enough nutrition available to the Brassica plant. Root dry biomass of Brassica juncea was significantly (p < 0.05) increased by 79% and 33% at 1 g kg−1 Act12 (FC) and 2 g kg−1 Act12 (TG) as compared to their respective controls. Streptomyces develop the association with the plant roots and their impact was also pronounced in the root. Shoot and root development was almost high in the TG soil
3.3.1. Effect of Streptomyces pactum on shoot and root dry biomass The inoculation of contaminated soil with Act12 significantly (p < 0.05) increased the shoot and root dry biomass of Brassica juncea as compared to control in both soils (Fig. 3). The maximum increase in shoot dry weight was 65% and 36% in case of FC and TG at 2 g kg−1 Act12, respectively, as compared to their respective controls. Antioxidants and siderophores promoted the shoot development in the TEs stress condition. Streptomyces mirabilis is also reported to improve biomass of Sorghum bicolor grown on soil collected from a former uranium 391
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as compared to FC soil, showing the adversaries of TEs on plant growth and development in mines polluted sites (Babu et al., 2013). The dry biomass of rapeseed grown on Cd and Pb spiked soil inoculated with Fusarium sp. CBRF44 was 47% and 32% higher than control, respectively (Shi et al., 2016). Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 inoculation of sorghum grown in mine tailings produced 37–95% more biomass due to higher P availability (Babu et al., 2014). Similarly, Bacillus thuringiensis GDB-1 inoculation of mines contaminated soil also increased the shoot dry biomass (170%) and root length (141%) as compared to control. Plant growth is enhanced by the inhibition of ethylene production in the roots by Act12 by producing ACC deaminase in TEs stress. Plant dry biomass was positively correlated with Act12 application rates, might be due to the release of specific sugars and amino acids into the plant rhizosphere (Babu et al., 2014; Cao et al., 2016). In addition, 2.5% MSC also promoted root growth, increased the availability of major cations and provide a substrate for Act12 growth.
3.3.2. Effect of Streptomyces pactum on chlorophyll and carotenoid Act12 application significantly (p < 0.05) increased the chlorophyll (a and b) and carotenoid content in Brassica juncea leaves collected from FC soil, while remained non-significant in TG soil as shown in Fig. 4. TEs adversely affect the chlorophyll and carotene content in plants. TEs mediated ROS production can damage the biosynthesis of chlorophyll and carotene production in plant cells and the severe condition can lead to chlorosis in the plant (Xian et al., 2015). Chlorophyll a, b and carotenoid content were significantly increased in treatments which received Act12 inoculum as compared to control. Chlorophyll a, b and carotenoid content were enhanced by 68.2%, 94.5% and 88.3% in FC (2 g kg−1 Act12) and 9%, 7% and 11% in TG (1 g kg−1 Act12) leaves samples. Meanwhile, there was a maximum of 18% reduction at 0.5 g kg−1 Act12 in chlorophyll in TG samples. Published reports have confirmed the harmful effects of TEs on carotene production in Ulva prolifera (Jiang et al., 2013) and Spirogyra setiformis (Çelekli et al., 2015). Previously, siderophores and ACC producing bacteria have been reported to enhance the chlorophyll by inhibiting ethylene production (Dimkpa et al., 2009; Ma et al., 2011). Pseudomonas aeruginosa KUCd1 and Enterobacter aerogenes NBRI also enhanced the chlorophyll, shoot and root dry biomass in Cucurbita pepo and Brassica juncea (Kumar et al., 2009). Pseudomonas inoculation improved chlorophyll (a, b), carotenoids as high as 69% and 65% in Festuca rubra plant grown on abandoned Pb/Zn mines in Biscay, Spain (Burges et al., 2016). Our results confirmed the role of Act12 to enhance chlorophyll and carotene content in Brassica juncea grown on mines contaminated soil.
Fig. 5. (a, b). Effect of Streptomyces pactum (Act12) on leaf antioxidant enzymatic activities in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
3.4. Effect of Streptomyces pactum on leaf antioxidant activities Biotic and abiotic stresses lead to the formation and accumulation of ROS (O2, H2O2 and OH·), generated by different metabolic pathways. Excessive production of ROS may lead to oxidative stress, DNA and protein damage, membrane permeability, lipid peroxidation, loss of cell function and even cell death (Ding et al., 2007). To scavenge ROS and avoid oxidative stress posed by TEs, plants develop antioxidant defense enzymes such as POD, PPO, PAL, and CAT (Cheng et al., 2016; Wang et al., 2014). Application of Act12 has significantly affected antioxidant biomarkers in FC and TG plants as shown in Fig. 5. Enhancement of antioxidants improves the detoxification of TEs by plants (Cao et al., 2016). POD is involved in multiple catalytic activities and its activity changes in response to TEs stress (Fig. 5a). It is involved in photosynthesis, protein metabolism and reduces TEs toxicity (Chen et al., 2015; Muratova et al., 2015). POD activity is influenced by high Zn concentration in FC and TG soil (Weisany et al., 2012). POD activity significantly (p < 0.05) declined by 44% and 20% in FC and TG leaf samples after inoculation with 0.5 g kg−1 Act12. Induced POD might be due to the protective measurements adopted by Brassica juncea against oxidative damage pronounced by ROS accumulation in TE stress. The higher TEs content stimulated the POD activation in both soils. Our findings are in line with Siddiqui and Meon (2009), who reported 30% POD reduction in Capsicum annuum with Pseudomonas aeruginosa inoculation. PPO play a vital role in oxidation of phenols to chinone and used as biomarkers for metal-induced oxidative stress in plants (Weisany et al., 2012). PPO activity in Brassica juncea leaves also showed significant (p < 0.05) diminution with the application of Act12 (Fig. 5a). This diminution was more (34% and 37%) in the treatments receiving 0.5 g kg−1 Act12 as compared to control. Hence, it can be concluded that Act12 showed more potential to enhance the defense mechanism in Brassica juncea to resist against the TEs stress. Similarly, phenanthrene and Cd contamination induced the PPO activities in tomato (Ahammed
Fig. 4. Effect of Streptomyces pactum (Act12) on leaf chlorophyll (a & b) and carotenoid (mg g−1 FW) in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
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extraction (451, 43.3 µg plant−1) was far high in FC and TG plant tissues (2 g kg−1 Act12) as compared to the other TEs (Pb, Cd, and Cu). MEA of Cd was also high in FC (79.8 µg plant−1), while it was below detection level in the TG plant tissues. Similarly, MEA values for Pb was high in TG (21.1 µg plant−1 at 2 g kg−1 Act12) as compared to FC plant tissues. The MEA value for the Cu was very low compared to the other elements in this study. MEA results confirmed that Act12 promoted the uptake of TEs in Brassica juncea shoot collected from FC and TG soil. Meanwhile, an unusual decreasing trend in the Cu MEA confirmed the adsorption of the TEs on the surface of microbial cells, the effect of soil physicochemical characteristics and the plant's selection for specific ions under certain conditions (Ansari and Malik, 2007).
et al., 2013). Plant PAL activity is increased after Act12 application in all treatments except at a dose of 0.5 g kg−1 Act12 (FC soil). PAL activity was significantly (p < 0.05) amplified by 8% and 27% in FC and TG, due to reduced formation of ROS in Brassica juncea as implicated from POD data (Fig. 5b). Act12 proved to alleviate the TEs stress by increasing the PAL production in their respective treatments. Soil microbes boast plant survival in TEs contaminated soil by promoting antioxidant activities (Rajkumar et al., 2012). Higher antioxidant enzymatic activities were also reported in Ricinus communis and Helianthus annuus collected from polluted soil (Ma et al., 2010). CAT is an ubiquitous enzyme, which is involved in the removal of toxic peroxides (Cheng et al., 2016). CAT quenches H2O2 to H2O and molecular oxygen. CAT activity was also significantly (p < 0.05) increased in treatments receiving Act12 (Fig. 5b). The higher Act12 rates have a positive effect on the CAT levels in leaf as compared to lower doses. CAT activity was increased up to 16% and 12% after Act12 application at 1 g kg−1 (FC) and 2 g kg−1 (TG), respectively. The increased CAT level can be assumed an adaptive mechanism developed by Brassica juncea in the mines and smelter polluted soil (Reddy et al., 2005). Reduction in CAT activity at 0.5 g kg−1 Act12 (FC) might be attributed to inactivation of the enzyme by TEs stimulated ROS, decrease in enzyme synthesis as well as changes in the assembly of its subunits (Verma and Dubey, 2003). Similarly, Cd spiked soil was inoculated with Act12 and reported 11.9% and 29.8% increase in CAT and SOD activities in Amaranthus hypochondriacus, respectively (Cao et al., 2016). Our findings suggest that Act12 is more effective in the single metal polluted soil than the multi-metals polluted mines soil. Further experiments are needed to investigate the detailed effects of Act12 in TEs polluted soil.
4. Conclusion In this study, phytoremediation was practiced on TEs rich mines polluted soils, inoculated with Streptomyces pactum. Results showed that the TEs (Zn, Pb, Cd, and Cu) uptake in shoot and root of Brassica juncea was pronounced in FC. The shoot uptake of TEs in TG samples was decreased (Cd and Cu) as compared to the control, but increased with increasing Act12 dose in each treatment. Root uptake of TEs was reportedly increased compared to the untreated pots. Plant physiological characters like shoot and root dry weight were improved in FC and TG samples. Chlorophyll (a and b) and carotenoid contents were significantly improved after the inoculation of Act12 in the mines contaminated soil. The antioxidant enzymatic activities of Brassica juncea were enhanced, showing the improvement of the plant defense mechanism against the TEs induced ROS. Overall, the goal of phytoremediation of the TEs in the mines polluted soil was assessed. Further investigation of phytoremediation mechanism of TEs and the proper dose of Act12 as well as suitable hyperaccumulator plant species are suggested for future studies.
3.5. Phytoextraction indices of trace elements The efficiency Act12 assisted phytoremediation by Brassica juncea in mines and smelter polluted soil was evaluated by TF and MEA as shown in Table 2. TF assesses the ability of a phytoextractor to translocate the absorbed TEs from the root to harvestable biomass (Shi et al., 2016). The mines and smelter contaminated soil from the FC and TG sites were inoculated with Act12. TF value of TEs (Zn, Pb, Cu, and Cd) in Brassica juncea was lower than the critical value for an ideal hyperaccumulator ( > 1.0) in FC and TG plant samples. However, TF for Zn was high in both cases. Previous results have also revealed that Act12 can better improve the phytoremediation efficiency of Cd spiked soil (Cao et al., 2016). But, our results showed that the efficiency can reduce in aged and multiple trace elements contaminated soil of mines and smelters. MEA represents the amount of the TEs extracted within the plant biomass, grown on the polluted substrate. MEA results showed that Zn
Authors contribution The present study is part of Amjad Ali's Ph.D. thesis. Dr. Zengqiang Zhang and Dr. Ronghua Li played an important role to design this experiment. Di Guo, Ping Wang, and Amanullah Mahar helped in soil and plant analysis, while Feng Shen and Zhen Wang helped in soil sampling and assisted to draw the figures. Dr. Quanhong Xue provided the inoculum for this experiment. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests.
Table 2 Translocation Factor (TF) and Metal Extraction Amount (MEA) of Brassica juncea after Act12 inoculation in mine polluted soils. MEA FC (µg plant−1 shoot)
TF (FC) Treatments Control 0.5 g kg−1 Act12 1.0 g kg−1 Act12 2.0 g kg−1 Act12
Zn
Pb *
0.54 ± 0.01 0.51 + 0.02 0.52 + 0.02 0.45 + 0.01
a a a b
0.22 0.31 0.33 0.28
Cd + + + +
0.07 0.01 0.02 0.06
b ab a ab
0.26 0.27 0.24 0.26
Cu + + + +
0.02 0.01 0.00 0.01
ab a b ab
0.09 0.09 0.08 0.08
Zn + + + +
0.00 0.01 0.00 0.01
a a a a
257 ± 14 389 ± 28 405 ± 22 451 ± 34
Pb b a a a
6.03 ± 3.4 9.29 ± 0.9 11.8 ± 1.3 15.3 ± 2.4
c bc ab a
Cd
Cu
41.8 ± 12 b 65.5 ± 3.2 a 64.0 ± 6.0 a 79.8 ± 7.7 a
0.95 ± 0.4 b 1.42 ± 0.10 a 1.53 ± 0.16 a 1.70 ± 0.22 a
Cd
Cu
0 0 0 0
1.16 ± 0.31 0.62 ± 0.14 0.78 ± 0.42 1.14 ± 0.20
MEA TG (µg plant−1 shoot)
TF (TG) Treatments
Zn
Control 0.5 g kg−1 Act12 1.0 kg−1 Act12 2.0 g kg−1 Act12
0.39 0.40 0.43 0.49
Pb + + + +
0.05 0.02 0.02 0.02
b b ab a
0.12 0.14 0.17 0.19
+ + + +
0.00 0.02 0.01 0.00
c b a a
Cd
Cu
0 0 0 0
0.04 0.02 0.02 0.03
Zn + + + +
0.01 0.00 0.00 0.00
a b b ab
22.7 ± 5.6 25.9 ± 3.0 34.3 ± 3.7 43.3 ± 1.7
Pb c bc ab a
8.54 ± 1.3 11.1 ± 1.3 16.9 ± 2.2 21.1 ± 1.1
d c b a
* Values indicate mean of one sample with three replications. Different letters within the same column represent significant differences at p < 0.05.
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Role of Streptomyces pactum in phytoremediation of trace elements by Brassica juncea in mine polluted soils
MARK
Amjad Alia, Di Guoa, Amanullah Mahara,b, Zhen Wanga, Dost Muhammadc, Ronghua Lia, ⁎ Ping Wanga, Feng Shena, Quanhong Xuea, Zengqiang Zhanga, a b c
College of Natural Resources and Environment, Northwest A & F University, Yangling 712100, China Centre for Environmental Sciences, University of Sindh, Jamshoro 76080, Pakistan Department of Soil and Environmental Sciences, The University of Agriculture, Peshawar 25130, Pakistan
A R T I C L E I N F O
A B S T R A C T
Keywords: Brassica juncea Medical stone compost Mining Phytoremediation Streptomyces pactum Trace elements
The industrial expansion, smelting, mining and agricultural practices have increased the release of toxic trace elements (TEs) in the environment and threaten living organisms. The microbe-assisted phytoremediation is environmentally safe and provide an effective approach to remediate TEs contaminated soils. A pot experiment was conducted to test the potential of an Actinomycete, subspecies Streptomyces pactum (Act12) along with medical stone compost (MSC) by growing Brassica juncea in smelter and mines polluted soils of Feng County (FC) and Tongguan (TG, China), respectively. Results showed that Zn (7, 28%), Pb (54, 21%), Cd (16, 17%) and Cu (8, 10%) uptake in shoot and root of Brassica juncea was pronounced in FC soil. Meanwhile, the Zn (40, 14%) and Pb (82, 15%) uptake in the shoot and root were also increased in TG soil. Shoot Cd uptake remained below detection, while Cu decreased by 52% in TG soil. The Cd and Cu root uptake were increased by 17% and 33%, respectively. Results showed that TEs uptake in shoot increased with increasing Act12 dose. Shoot/root dry biomass, chlorophyll and carotenoid content in Brassica juncea were significantly influenced by the application of Act12 in FC and TG soil. The antioxidant enzymatic activities (POD, PAL, PPO and CAT) in Brassica juncea implicated enhancement in the plant defense mechanism against the TEs induced stress in contaminated soils. The extraction potential of Brasssica was further evaluated by TF (translocation factor) and MEA (metal extraction amount). Based on our findings, further investigation of Act12 assisted phytoremediation of TEs in the smelter and mines polluted soil and hyperaccumulator species are suggested for future studies.
1. Introduction Maximum food production and industrial activities are required in order to satisfy the basic needs of world growing population. The burgeoning population, disarrayed industrialization and technical innovations have led to the global increase in the release of trace elements (TEs) and widespread contamination of the environment. TEs pollution has pernicious effects on soil, air and human health due to soil-plant transfer to reach food-chain (Cheng et al., 2016). Sources of TEs include petrochemicals, textile, steel manufacturing, mining and smelting, as well as coal combustion (Alvarez et al., 2017). The impacts of TEs have been studied regarding soil biology, water/air quality as well as animals and human health. TEs reduce the soil enzymatic activities, degrade air and water quality in the close proximity of the source point, decrease the biosynthesis of chlorophyll, reduce respiration and limit antioxidant activities in plants (Ali et al., 2017; Xian et al., 2015). TEs reduce soil organic matter decomposition and ⁎
Corresponding author. E-mail address: [email protected] (Z. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2017.06.046 Received 28 January 2017; Received in revised form 13 June 2017; Accepted 16 June 2017 0147-6513/ © 2017 Published by Elsevier Inc.
nutrients cycling (Ma et al., 2015). Furthermore, TEs are carcinogenic and mutagenic and can cause fatal diseases in humans and animals, if exposed to higher levels (Cao et al., 2016). A variety of physical, chemical, and biological remediation techniques are being used for phytoextraction of TEs to treat the contaminated sites. These practices have produced secondary pollutants and account for contamination of the environment in the long-term adoption. The use of biotechnology in the remediation of TEs is getting the recognition due to the recent developments (Ali et al., 2017). The bacterial inoculation can potentially reduce phytotoxicity, increases TEs uptake and removal, and enhance plant biomass in TEs contaminated soil. Soil microbes affect the mobility and availability of TEs through chelation, acidification and siderophores formation (AbouShanab et al., 2008; Gopalakrishnan et al., 2013). Actinobacteria (Act) exhibit diverse physiological and metabolic properties, playing a vital role in phytoremediation by promoting the TEs uptake in plants and increase biomass production (Aparicio et al., 2015; Gopalakrishnan
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et al., 2015; Rajkumar et al., 2012). Members of the order Actinomycetales i.e. Streptomyces are known to promote the growth in Oryza sativa, Sorghum bicolor, Cicer arietinum and Amaranthus hypochondriacus (Cao et al., 2016; Gopalakrishnan et al., 2015). Streptomyces pactum (Act12) is reported to promote the plant growth, control pathogenic disease and promote the phytoremediation of Cd. However, its role in phytoremediation of TEs and antioxidant activities was not reported in smelter/mines contaminated soil (Zhao et al., 2012). Phytoremediation is a promising green technology by using plants to restore TEs contaminated soils (Cao et al., 2016). Phytoremediation can effectively be practiced to extract TEs and achieve sustainable rehabilitation of contaminated land (Babu et al., 2014). The microbeassisted phytoremediation technique depends on the interaction between the host plant and microorganism species. It is cost-effective and eco-friendly by inoculating hyperaccumulator plants with plant growthpromoting microorganisms (Alvarez et al., 2017; Ho et al., 2013; Ma et al., 2011). Remediation of mine sites is a complex process, as a variety of TEs and soil physicochemical conditions influence the process (Ma et al., 2015). Brassica juncea belongs to the Brassicaceae family and mainly grown for oil production and as a forage crop. Brassica is known as hyperaccumulator due to its potential to grow in the contaminated soil and accumulate huge amounts of the TEs in its shoot and root biomass (Mobin and Khan, 2007). Association of plant growth-promoting bacteria with brassica has been widely studied for the TEs translocation in the shoot and root biomass in contaminated soil (Kumar et al., 2009; Ma et al., 2009). The prime objective of the study was to assess the phytoremediation potential of Streptomyces pactum in smelter/mines polluted soils by growing Brassica juncea and evaluate its effects on enzymatic activities.
Northwest A & F University, Yangling, China (Zhao et al., 2012). Act12 carrier agent (2.6×1011 spores g−1) was applied in powder form to treated pots.
2. Material and methods
Chlorophyll (a, b) and carotenoid contents in Brassica juncea were extracted by grinding leaves (0.2 g) in 80% acetone in the dark and calculated from the absorbance of extract at 663, 645 and 470 nm. The concentrations were expressed as mg g−1 FW (Wang et al., 2014). After 7 weeks, the shoot and root of Brassica juncea were harvested, thoroughly washed with running tap water followed by deionized water and dried up to a constant weight at 105 °C. The dried biomass was crushed into fine powder and stored. Shoot (0.50 g) and root (0.25 g) samples were digested with HNO3–HClO4 (3:1) and total TEs (Zn, Pb, Cd, and Cu) were determined (Hu et al., 2014). PPO and PAL activities were detected as per the standard method (Ahammed et al., 2013). Guaiacol peroxidase (POD) and catalase (CAT) activities were determined according to Cheng et al. (2016).
2.2. Experimental methods 2.2.1. Pot experiment The pot experiment was laid out following a completely randomized design under a mobile shelter house in an open environment. Soil treatments (T) were T1 (Control), T2 (0.5 g kg−1 Act12), T3 (1.0 g kg−1 Act12), and T4 (2.0 g kg−1 Act12). Each pot (15×12×10 cm3 dimension) was filled with thoroughly mixed one kg soil (2 mm) as growing media and 2.5% MSC as a nutritional supplement. Ten sterilized (3% H2O2) seeds of Brassica juncea (Shaan you 16) were sown per pot with three replicates per treatment. The shoot/root dry biomasses were recorded after 7 weeks on the harvesting of Brassica plants. 2.3. Soil and medical stone compost analysis Soil pH (1:2), electrical conductivity (EC) and organic matter were measured according to Li et al. (2012). Soil particle size distribution (Mastersizer 2000E laser diffractometer, UK) and cation exchange capacity (CEC) were measured according to Mahar et al. (2016). Total trace elements (Zn, Pb, Cd and Cu) in soil (FC, TG) and MSC were measured by ICP-AES (Hu et al., 2014). Similarly, DTPA (0.005 M) extractable TEs were tested according to Burges et al. (2016). Analytical grade chemicals and double deionized water was used for all chemical analysis in the laboratory. 2.4. Plant analysis
2.1. Samples collection Mines and smelter contaminated soil was collected from Feng County (106°24′~107°7′ N, 33°34′~34°18′ E) and Tongguan (34°27′~34°37′ N, 110°10′~110°23′ E) stations of Shaanxi province, China. Feng County (FC) and Tongguan (TG), are located in the southwest and east of Shaanxi Province, China. Feng County, surrounded by Qinling Mountains, is one of the largest production units in China, with Pb/Zn mines reserves around 4.5 million tons. The climate is dry, the temperature ranging −1.1 to 22.7 °C and average annual rainfall is 613 mm. Surface water and soil pollution are the main environmental hazards in FC, due to the discharge of mines wastewater, atmospheric deposition and mine tailings (Xu et al., 2012). Tongguan is famous for gold mining operations at domestic and industrial scale. The area is mainly polluted with mining, mineral processing and atmospheric deposition of TEs (Ali et al., 2017; Mahar et al., 2016). Climate is humid subtropical with an average temperature and rainfall of 13.4 °C and 581 mm, respectively (Feng et al., 2006). Samples were collected from contaminated surface soil (0–20 cm), stored in polyethylene bags and transferred to the laboratory. Soil samples were homogenized, air-dried, crushed manually and passed through 2 mm sieve. Pig manure was collected from a local pig farm and sawdust from a wood-processing factory in Yangling town, Shaanxi, China. The medical stone (MS) was purchased from Shijiazhuang Building Materials Co. Ltd., China. Medical stone (2.5% dw) added pig manure compost (MSC) was prepared by mixing pig manure and sawdust (2:1 dry weight) in 130 L PVC composter (Li et al., 2012; Wang et al., 2016). The PVC composters were turned over and the compost temperature was daily monitored during the composting process (45 days). Actinomycete was isolated from the Qinghai-Tibet Plateau of China and identified as Streptomyces pactum based on 16S rDNA sequence analysis. Streptomyces pactum carrier was obtained from Laboratory of Microbial Resources at the College of Natural Resources and Environment,
2.5. Phytoextraction indices The amount of TEs translocated from soil to shoot and root was calculated using the following equations (Cao et al., 2016; Shi et al., 2016).
Translocation Factor(TF) =
Metal concentration in shoot Metal concentration in roots
(1)
Metal Extraction Amount (MEA) = Metal concentration in aerial parts × Biomass (2) 2.6. Quality control and statistical analysis All the analytical samples were prepared in three replicates and average readings are presented with standard deviation. Reagent blanks were used to correct the analytical errors. Standard reference materials for wheat and soil (GBW10011 and GBW07405, respectively) were purchased from the National Research Center of Certified Reference Materials (Beijing, China). The recovery of the standard wheat sample 388
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and is of great concern with respect to the environment, plant uptake, and development (Hu et al., 2014). DTPA-extractable concentration of Zn, Pb, Cd and Cu were 584, 40.2, 36.4 and 1.77 mg kg−1 in FC soil, while reported as 15.59, 200, 0.549 and 11.65 mg kg−1 in TG soil. FC and TG are basically mining and smelting sites in Shaanxi province, which contributes specifically to the higher bioavailable content of Zn, Pb and Cd in surface soil (Xu et al., 2012). The extractable Zn, Pb, Cd, and Cu in MSC were 171.11, 5.50, 0.30 and 20.56 mg kg−1, respectively. The extractable TEs contents in MSC were in decreasing order of Zn > Cu > Pb > Cd.
Table 1 Basic characteristics of Feng County, Tongguan soil and medical stone compost. Soil characteristics
Feng County soil
Tongguan soil
Medical stone compost
Clay % 1.56* 0.50 – Silt % 48.43 22.10 – Sand % 50.01 77.33 – Soil texture Sandy loam Loamy sand – pH 7.72 8.06 6.57 −1 422 201.3 732.3 EC (µS cm ) CEC (cmol + /kg) 96.51 19.5 – Organic matter (g kg−1) 14.9 28.40 665 Total Nitrogen (g kg−1) 1.23 0.689 15.64 Total Phosphorus 0.848 0.861 22.50 −1 (g kg ) 5.51 2.9 – Total potassium (mg kg−1) 8.45 16.47 352.87 Total organic carbon (g kg−1) Total TEs in soil (Feng County and Tongguan) and medical stone compost (mg kg−1) Zn 6625 230.4 384.54 Pb 204.4 393.2 8.09 Cd 117.7 1.58 0.557 Cu 51.1 141.36 77.42 31,629 40,529 – Al (mg kg−1) As (mg kg−1) 0.08 9.075 – Ca (mg kg−1) 15,538 24,938 – Co (mg kg−1) 16.20 11.95 – −1 Cr (mg kg ) 61.75 52.548 – −1 Fe (mg kg ) 25,694 19,215 – Hg (mg kg−1) 0.30 0.783 – Mg (mg kg−1) 8205 8592 – Mn (mg kg−1) 729.7 545 – Mo (mg kg−1) 0.85 0.908 – Na (mg kg−1) 9200 10,574 – Extractable TEs in soil (Feng County and Tongguan) and medical stone compost (mg kg−1) Zn 584 15.59 171.11 Pb 40.2 200.0 5.50 Cd 36.4 0.549 0.30 Cu 1.77 11.65 20.56
3.2. Effect of Streptomyces pactum on TEs uptake by Brassica juncea TEs in Brassica shoot grown on FC and TG soils were determined after acid digestion. TEs uptake in the shoot was significantly increased after the application of Act12 to FC soil. However, the uptake was less at low doses and comparatively higher in the case of TG soil. Adsorption on the microbial cells and compost may be the possible reasons for the decreased uptake of Cd and Cu in shoot of TG soil (Babu et al., 2014). 3.2.1. Uptake of TEs by Brassica juncea shoot The accumulation of TEs (Zn, Pb, Cd, and Cu) in the shoot of Brassica grown in FC and TG soils is shown in the Fig. 1. The data showed that TEs uptake in shoot collected from FC soil was significantly (p < 0.05) increased as compared to control. Overall, there was a significant increase in Zn and Pb uptake in TG shoot, while a decrease in Cd and Cu in shoots samples at the application of 0.5–2 g kg−1 Act12. A maximum of 7% increase in Zn was reported at 2 g kg−1, as compared to control in FC soil. Zn content is very high in FC soil as compared to TG soil, as the samples were collected from the close proximity of the Zn/Pb smelter. The plant tissue is almost saturated with Zn and no further Zn accumulation occurred in Brassica. Zn shoot uptake in TG shoot raised from 30.45 to 39.41 mg kg−1, which is still below the concentration reported in the control (47.01 mg kg−1). Metal-resistant Streptomyces AR17 also enhanced Zn and Cd uptake by Salix caprea (Kuffner et al., 2008). Pb uptake in shoot increased significantly (p < 0.05) by 54% and 82% in FC and TG soil at 2 g kg−1 Act12, as compared to their respective control pots. Pb uptake in shoot collected from FC and TG soil showed a steady increase as observed in Zn with the higher dose of Act12. The concentration of Pb is lower than the Zn in soil and the uptake is enhanced in the shoot by the application of Act12. The Pb uptake was well facilitated by the addition of Act12 in the smelter polluted soil of FC. Burkholderia sp. J62 also increased Pb uptake in the shoot of Zea mays by siderophores production (Jiang et al., 2008). Coinoculation of Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 also increased the TEs translocation in the shoot by 69% (Pb) and 82% (Zn), while Cu remained constant (Babu et al., 2014). Cd uptake was enhanced up to 16% by application of 2 g kg−1 Act12 in shoots collected from FC soil. However, Cd was below the detection level in TG shoot samples. The uptake was significantly (p < 0.05) influenced by the inoculation of Act12. As shown in Table 1, DTPA extractable Cd in FC (36.4 mg kg−1) is 66 times greater than TG (0.549 mg kg−1). This resulted in higher shoot uptake in FC shoot samples and the concentration was even below the detection in TG samples. Similarly, Streptomyces sp. F4 also improved the uptake of Cd from culture medium and soil (Haferburg et al., 2008). Cao et al. (2016) also reported 86.5% increase in shoot Cd uptake by Amaranthus hypochondriacus after being inoculated with Act12. The lowering of Cd in TG shoots is attributed to adsorption on the MSC surfaces, added to soil as a nutritional supplement (Babu et al., 2014). Farm yard manure also lowered Cd concentration in wheat grains, but the response varied with types of the soil (Dahlin et al., 2016). Cu uptake in the shoot increased up to 8% (2 g kg−1 Act12) in FC, while decreased by 52% (0.5 g kg−1 Act12) in TG shoot as compared to their respective controls. Cu in the shoot of Brassica juncea increased
* Values indicate mean of one sample with three replications.
ranged from 97.5% to 102.2%, 96.2–101.5%, 96.4–101.5% and 98.1–102.2% for Cd, Cu, Pb and Zn, respectively. The recovery of the standard soil sample ranged from 93.2% to 101.5%, 97.8–105.5%, 96.4–104.8% and 94.5–102.3% for Cd, Cu, Pb, and Zn, respectively. The results obtained for the standard wheat and soil samples were within the acceptable ranges. Data were subjected to one-way ANOVA at p < 0.05 for independent variable analysis and significant differences among the mean values were calculated using SPSS 22.0 (IBM SPSS, Somers, NY, USA). All graphs were drawn using Origin-Pro (7.5 version).
3. Results and discussion 3.1. Characteristics of the studied soils and medical stone compost The main characteristics of soil (FC and TG) and MSC are illustrated in Table 1. The higher Zn, Pb and Cd content in FC and TG soil is attributed to the mining and smelting activities in these areas over a longtime period (Mahar et al., 2016; Xu et al., 2012). Total TEs concentration in FC and TG soil were in decreasing order of Zn > Pb > Cd > Cu and Pb > Zn > Cd > Cu, respectively. Trace elements content in FC and TG soil was higher than the National Environmental Quality Standards for Agricultural Soil (Environmental Quality Standards, China, GB15618-1995). Bioavailability of trace elements in soil is a dynamic process limited by chemical, biological, and environmental factors (Ali et al., 2017). The DTPA-extractable proportion of TEs is considered as bioavailable 389
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Fig. 1. Effect of Streptomyces pactum (Act12) on shoot uptake (mg kg−1 DW) of TEs in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
from 4.47 to 4.76 mg kg−1 in FC samples, while, it raised from 1.16 to 1.74 mg kg−1 in TG samples after the inoculation of Act12. Similarly, Achromobacter xylosoxidans also increased Cu uptake in Brassica juncea grown on the mines contaminated soil in São Domingos, Portugal (Ma et al., 2009). Furthermore, the addition of MSC is also reported to reduce the bioavailability of the TEs, especially Cu uptake by Brassica juncea due to adsorption and immobilization in the soil (Rizwan et al., 2016; Wang et al., 2016). Poor translocation in Brassica juncea shoots could be due to confinement of TEs in the vacuoles of root and Act12 cells to render them and reduce toxicity in the plant (Babu et al., 2013; Hanikenne and Nouet, 2011). Babu et al. (2013) and Rosselli et al. (2003) also reported that Alnus firma growing in polluted soil contained the low concentration of TEs in shoots.
Cr and lindane uptake in Lactuca sativa (Aparicio et al., 2015). The Pb uptake was significantly (p < 0.05) increased as high as 21% and 15% (2 g kg−1 Act12) in shoot in FC and TG soil, respectively. Although, the addition of Act12 to FC soils showed 25% and 16% decrease (0.5 and 1 g kg−1 Act12) in Pb uptake in Brassica juncea root, while higher dose (2 g kg−1 Act12) increased the uptake. It is believed that Pb is immobile in plants and the translocation is low in the Brassica juncea root system. Diminution in Pb uptake was also reported in Phaseolus vulgaris and pine (Krupa and Kozdrój, 2007). In another study, Streptomyces sp. MC1 also immobilized Cr in the soil, while improved maize growth (Polti et al., 2011). Soil microbes reduce the TEs phytotoxicity by employing biosorption and bioaccumulation (Ma et al., 2011). It is believed that microbial adsorption in FC soil might have occurred, which reduced (25, 16%) the Pb uptake in Brassica roots (Babu et al., 2014). The concentration of Pb is higher in soil but the root translocation is lower than Cd ions. TEs translocation in roots depends on the selection of specific ions by the plants and soil physicochemical characteristics (Ansari and Malik, 2007). Cd uptake in root was significantly (p < 0.05) improved by 17% and 33% in the FC and TG (2 g kg−1 Act12) samples, respectively. The DPTA extractable Cd (36.4 mg kg−1) is higher in the FC soil, which was 95 times more translocated than in the TG root samples. The shoot/root growth and Cd uptake were amplified by Bacillus megaterium H3 in Pennisetum purpureum. IAA, organic acids, and siderophores production are believed to promote the growth, reduce stress symptoms and increase TEs tolerance as well as translocation in plants (Li et al., 2016). In another study, Amaranthus hypochondriacus grown in spiked soil, the root Cd uptake was enhanced by 23.6% after Act12 inoculation, which confirmed the results obtained in this study (Cao et al., 2016).
3.2.2. Uptake of TEs by Brassica juncea root The accumulation of TEs in Brassica roots collected from FC and TG soils is shown in Fig. 2. Root data revealed that TEs uptake was significantly (p < 0.05) influenced in both soils by Act12. Act12 enhanced the root and shoot development, which in turn promoted TEs uptake in Brassica juncea shoot (Gopalakrishnan et al., 2013). The Zn uptake was significantly (p < 0.05) increased by 28% and 14% (2 g kg−1 Act12) in FC and TG root samples compared to their controls. The higher Zn uptake in FC roots is attributed to the higher degree of Zn contamination in FC soil. The samples were collected near the main smelter installation, where the extractable Zn (584 mg kg−1) was above the permissible limit. Dary et al. (2010) and Sheng et al. (2008) also reported an increase in Zn, Pb and Cd uptake due to high biomass production after inoculation of seed/soil with plant growthpromoting microbes. Similarly, Streptomyces sp. M7 also increased the 390
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Fig. 2. Effect of Streptomyces pactum (Act12) on root uptake (mg kg−1 DW) of TEs in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
Cu uptake in the FC and TG roots was higher than their respective controls. The translocation was improved by 10% and 17% in FC and TG at 2 g kg−1 Act12, respectively in their respective soil samples. Combine inoculation of Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 improved the root TEs translocation as much as 39% (Cu), 50% (Pb) and 38% (Zn) (Babu et al., 2014). These findings confirmed the role of Act12 in phytoextraction of TEs in polluted soil. TEs uptake in TG shoot is reduced while the accumulation in roots is increased after the inoculation of Act12. The plant-associated microbes have the ability to immobilize the TEs in root and reduce their uptake through binding, accumulation or dilution in the host plant (Babu et al., 2013; Rajkumar et al., 2012). Our findings are in line with the previous scientific reports (Ali et al., 2017). Fig. 3. Effect of Streptomyces pactum (Act12) on the shoot and root dry biomass (g pot−1) of Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
3.3. Effect of Streptomyces pactum on growth attributes of Brassica juncea The effect of Act12 on the shoot and root dry biomass, chlorophyll (a and b) and carotenoid content in Brassica juncea grown on contaminated soils were assessed. Mining activities have adverse effects on the plant physiology, which can be minimized by biotechnological approaches (Alvarez et al., 2017).
mining site in Ronneburg, Germany (Schütze et al., 2014). Similarly, a Cd-resistant Streptomyces tendae F4 secreted siderophores and significantly promoted the growth of Helianthus annuus (Dimkpa et al., 2009). Another study, conducted in Cd contaminated soil inoculated with Act12 also reported 45% and 20% increase in the shoot and root dry weight, respectively (Cao et al., 2016). Act12 promoted the plant development in the TEs rich soil of FC and TG due to enough nutrition available to the Brassica plant. Root dry biomass of Brassica juncea was significantly (p < 0.05) increased by 79% and 33% at 1 g kg−1 Act12 (FC) and 2 g kg−1 Act12 (TG) as compared to their respective controls. Streptomyces develop the association with the plant roots and their impact was also pronounced in the root. Shoot and root development was almost high in the TG soil
3.3.1. Effect of Streptomyces pactum on shoot and root dry biomass The inoculation of contaminated soil with Act12 significantly (p < 0.05) increased the shoot and root dry biomass of Brassica juncea as compared to control in both soils (Fig. 3). The maximum increase in shoot dry weight was 65% and 36% in case of FC and TG at 2 g kg−1 Act12, respectively, as compared to their respective controls. Antioxidants and siderophores promoted the shoot development in the TEs stress condition. Streptomyces mirabilis is also reported to improve biomass of Sorghum bicolor grown on soil collected from a former uranium 391
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as compared to FC soil, showing the adversaries of TEs on plant growth and development in mines polluted sites (Babu et al., 2013). The dry biomass of rapeseed grown on Cd and Pb spiked soil inoculated with Fusarium sp. CBRF44 was 47% and 32% higher than control, respectively (Shi et al., 2016). Penicillium aculeatum PDR-4 and Trichoderma sp. PDR-16 inoculation of sorghum grown in mine tailings produced 37–95% more biomass due to higher P availability (Babu et al., 2014). Similarly, Bacillus thuringiensis GDB-1 inoculation of mines contaminated soil also increased the shoot dry biomass (170%) and root length (141%) as compared to control. Plant growth is enhanced by the inhibition of ethylene production in the roots by Act12 by producing ACC deaminase in TEs stress. Plant dry biomass was positively correlated with Act12 application rates, might be due to the release of specific sugars and amino acids into the plant rhizosphere (Babu et al., 2014; Cao et al., 2016). In addition, 2.5% MSC also promoted root growth, increased the availability of major cations and provide a substrate for Act12 growth.
3.3.2. Effect of Streptomyces pactum on chlorophyll and carotenoid Act12 application significantly (p < 0.05) increased the chlorophyll (a and b) and carotenoid content in Brassica juncea leaves collected from FC soil, while remained non-significant in TG soil as shown in Fig. 4. TEs adversely affect the chlorophyll and carotene content in plants. TEs mediated ROS production can damage the biosynthesis of chlorophyll and carotene production in plant cells and the severe condition can lead to chlorosis in the plant (Xian et al., 2015). Chlorophyll a, b and carotenoid content were significantly increased in treatments which received Act12 inoculum as compared to control. Chlorophyll a, b and carotenoid content were enhanced by 68.2%, 94.5% and 88.3% in FC (2 g kg−1 Act12) and 9%, 7% and 11% in TG (1 g kg−1 Act12) leaves samples. Meanwhile, there was a maximum of 18% reduction at 0.5 g kg−1 Act12 in chlorophyll in TG samples. Published reports have confirmed the harmful effects of TEs on carotene production in Ulva prolifera (Jiang et al., 2013) and Spirogyra setiformis (Çelekli et al., 2015). Previously, siderophores and ACC producing bacteria have been reported to enhance the chlorophyll by inhibiting ethylene production (Dimkpa et al., 2009; Ma et al., 2011). Pseudomonas aeruginosa KUCd1 and Enterobacter aerogenes NBRI also enhanced the chlorophyll, shoot and root dry biomass in Cucurbita pepo and Brassica juncea (Kumar et al., 2009). Pseudomonas inoculation improved chlorophyll (a, b), carotenoids as high as 69% and 65% in Festuca rubra plant grown on abandoned Pb/Zn mines in Biscay, Spain (Burges et al., 2016). Our results confirmed the role of Act12 to enhance chlorophyll and carotene content in Brassica juncea grown on mines contaminated soil.
Fig. 5. (a, b). Effect of Streptomyces pactum (Act12) on leaf antioxidant enzymatic activities in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
3.4. Effect of Streptomyces pactum on leaf antioxidant activities Biotic and abiotic stresses lead to the formation and accumulation of ROS (O2, H2O2 and OH·), generated by different metabolic pathways. Excessive production of ROS may lead to oxidative stress, DNA and protein damage, membrane permeability, lipid peroxidation, loss of cell function and even cell death (Ding et al., 2007). To scavenge ROS and avoid oxidative stress posed by TEs, plants develop antioxidant defense enzymes such as POD, PPO, PAL, and CAT (Cheng et al., 2016; Wang et al., 2014). Application of Act12 has significantly affected antioxidant biomarkers in FC and TG plants as shown in Fig. 5. Enhancement of antioxidants improves the detoxification of TEs by plants (Cao et al., 2016). POD is involved in multiple catalytic activities and its activity changes in response to TEs stress (Fig. 5a). It is involved in photosynthesis, protein metabolism and reduces TEs toxicity (Chen et al., 2015; Muratova et al., 2015). POD activity is influenced by high Zn concentration in FC and TG soil (Weisany et al., 2012). POD activity significantly (p < 0.05) declined by 44% and 20% in FC and TG leaf samples after inoculation with 0.5 g kg−1 Act12. Induced POD might be due to the protective measurements adopted by Brassica juncea against oxidative damage pronounced by ROS accumulation in TE stress. The higher TEs content stimulated the POD activation in both soils. Our findings are in line with Siddiqui and Meon (2009), who reported 30% POD reduction in Capsicum annuum with Pseudomonas aeruginosa inoculation. PPO play a vital role in oxidation of phenols to chinone and used as biomarkers for metal-induced oxidative stress in plants (Weisany et al., 2012). PPO activity in Brassica juncea leaves also showed significant (p < 0.05) diminution with the application of Act12 (Fig. 5a). This diminution was more (34% and 37%) in the treatments receiving 0.5 g kg−1 Act12 as compared to control. Hence, it can be concluded that Act12 showed more potential to enhance the defense mechanism in Brassica juncea to resist against the TEs stress. Similarly, phenanthrene and Cd contamination induced the PPO activities in tomato (Ahammed
Fig. 4. Effect of Streptomyces pactum (Act12) on leaf chlorophyll (a & b) and carotenoid (mg g−1 FW) in Brassica juncea. Data represent the mean of three replicates and error bars are the standard deviation. Different letters over the same bar represent significant differences between the treatments at p < 0.05.
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extraction (451, 43.3 µg plant−1) was far high in FC and TG plant tissues (2 g kg−1 Act12) as compared to the other TEs (Pb, Cd, and Cu). MEA of Cd was also high in FC (79.8 µg plant−1), while it was below detection level in the TG plant tissues. Similarly, MEA values for Pb was high in TG (21.1 µg plant−1 at 2 g kg−1 Act12) as compared to FC plant tissues. The MEA value for the Cu was very low compared to the other elements in this study. MEA results confirmed that Act12 promoted the uptake of TEs in Brassica juncea shoot collected from FC and TG soil. Meanwhile, an unusual decreasing trend in the Cu MEA confirmed the adsorption of the TEs on the surface of microbial cells, the effect of soil physicochemical characteristics and the plant's selection for specific ions under certain conditions (Ansari and Malik, 2007).
et al., 2013). Plant PAL activity is increased after Act12 application in all treatments except at a dose of 0.5 g kg−1 Act12 (FC soil). PAL activity was significantly (p < 0.05) amplified by 8% and 27% in FC and TG, due to reduced formation of ROS in Brassica juncea as implicated from POD data (Fig. 5b). Act12 proved to alleviate the TEs stress by increasing the PAL production in their respective treatments. Soil microbes boast plant survival in TEs contaminated soil by promoting antioxidant activities (Rajkumar et al., 2012). Higher antioxidant enzymatic activities were also reported in Ricinus communis and Helianthus annuus collected from polluted soil (Ma et al., 2010). CAT is an ubiquitous enzyme, which is involved in the removal of toxic peroxides (Cheng et al., 2016). CAT quenches H2O2 to H2O and molecular oxygen. CAT activity was also significantly (p < 0.05) increased in treatments receiving Act12 (Fig. 5b). The higher Act12 rates have a positive effect on the CAT levels in leaf as compared to lower doses. CAT activity was increased up to 16% and 12% after Act12 application at 1 g kg−1 (FC) and 2 g kg−1 (TG), respectively. The increased CAT level can be assumed an adaptive mechanism developed by Brassica juncea in the mines and smelter polluted soil (Reddy et al., 2005). Reduction in CAT activity at 0.5 g kg−1 Act12 (FC) might be attributed to inactivation of the enzyme by TEs stimulated ROS, decrease in enzyme synthesis as well as changes in the assembly of its subunits (Verma and Dubey, 2003). Similarly, Cd spiked soil was inoculated with Act12 and reported 11.9% and 29.8% increase in CAT and SOD activities in Amaranthus hypochondriacus, respectively (Cao et al., 2016). Our findings suggest that Act12 is more effective in the single metal polluted soil than the multi-metals polluted mines soil. Further experiments are needed to investigate the detailed effects of Act12 in TEs polluted soil.
4. Conclusion In this study, phytoremediation was practiced on TEs rich mines polluted soils, inoculated with Streptomyces pactum. Results showed that the TEs (Zn, Pb, Cd, and Cu) uptake in shoot and root of Brassica juncea was pronounced in FC. The shoot uptake of TEs in TG samples was decreased (Cd and Cu) as compared to the control, but increased with increasing Act12 dose in each treatment. Root uptake of TEs was reportedly increased compared to the untreated pots. Plant physiological characters like shoot and root dry weight were improved in FC and TG samples. Chlorophyll (a and b) and carotenoid contents were significantly improved after the inoculation of Act12 in the mines contaminated soil. The antioxidant enzymatic activities of Brassica juncea were enhanced, showing the improvement of the plant defense mechanism against the TEs induced ROS. Overall, the goal of phytoremediation of the TEs in the mines polluted soil was assessed. Further investigation of phytoremediation mechanism of TEs and the proper dose of Act12 as well as suitable hyperaccumulator plant species are suggested for future studies.
3.5. Phytoextraction indices of trace elements The efficiency Act12 assisted phytoremediation by Brassica juncea in mines and smelter polluted soil was evaluated by TF and MEA as shown in Table 2. TF assesses the ability of a phytoextractor to translocate the absorbed TEs from the root to harvestable biomass (Shi et al., 2016). The mines and smelter contaminated soil from the FC and TG sites were inoculated with Act12. TF value of TEs (Zn, Pb, Cu, and Cd) in Brassica juncea was lower than the critical value for an ideal hyperaccumulator ( > 1.0) in FC and TG plant samples. However, TF for Zn was high in both cases. Previous results have also revealed that Act12 can better improve the phytoremediation efficiency of Cd spiked soil (Cao et al., 2016). But, our results showed that the efficiency can reduce in aged and multiple trace elements contaminated soil of mines and smelters. MEA represents the amount of the TEs extracted within the plant biomass, grown on the polluted substrate. MEA results showed that Zn
Authors contribution The present study is part of Amjad Ali's Ph.D. thesis. Dr. Zengqiang Zhang and Dr. Ronghua Li played an important role to design this experiment. Di Guo, Ping Wang, and Amanullah Mahar helped in soil and plant analysis, while Feng Shen and Zhen Wang helped in soil sampling and assisted to draw the figures. Dr. Quanhong Xue provided the inoculum for this experiment. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests.
Table 2 Translocation Factor (TF) and Metal Extraction Amount (MEA) of Brassica juncea after Act12 inoculation in mine polluted soils. MEA FC (µg plant−1 shoot)
TF (FC) Treatments Control 0.5 g kg−1 Act12 1.0 g kg−1 Act12 2.0 g kg−1 Act12
Zn
Pb *
0.54 ± 0.01 0.51 + 0.02 0.52 + 0.02 0.45 + 0.01
a a a b
0.22 0.31 0.33 0.28
Cd + + + +
0.07 0.01 0.02 0.06
b ab a ab
0.26 0.27 0.24 0.26
Cu + + + +
0.02 0.01 0.00 0.01
ab a b ab
0.09 0.09 0.08 0.08
Zn + + + +
0.00 0.01 0.00 0.01
a a a a
257 ± 14 389 ± 28 405 ± 22 451 ± 34
Pb b a a a
6.03 ± 3.4 9.29 ± 0.9 11.8 ± 1.3 15.3 ± 2.4
c bc ab a
Cd
Cu
41.8 ± 12 b 65.5 ± 3.2 a 64.0 ± 6.0 a 79.8 ± 7.7 a
0.95 ± 0.4 b 1.42 ± 0.10 a 1.53 ± 0.16 a 1.70 ± 0.22 a
Cd
Cu
0 0 0 0
1.16 ± 0.31 0.62 ± 0.14 0.78 ± 0.42 1.14 ± 0.20
MEA TG (µg plant−1 shoot)
TF (TG) Treatments
Zn
Control 0.5 g kg−1 Act12 1.0 kg−1 Act12 2.0 g kg−1 Act12
0.39 0.40 0.43 0.49
Pb + + + +
0.05 0.02 0.02 0.02
b b ab a
0.12 0.14 0.17 0.19
+ + + +
0.00 0.02 0.01 0.00
c b a a
Cd
Cu
0 0 0 0
0.04 0.02 0.02 0.03
Zn + + + +
0.01 0.00 0.00 0.00
a b b ab
22.7 ± 5.6 25.9 ± 3.0 34.3 ± 3.7 43.3 ± 1.7
Pb c bc ab a
8.54 ± 1.3 11.1 ± 1.3 16.9 ± 2.2 21.1 ± 1.1
d c b a
* Values indicate mean of one sample with three replications. Different letters within the same column represent significant differences at p < 0.05.
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a a a a
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