Pennisetum sinese : A Potential Phytoremediation Plant for Chromium Deletion from Soil

: Chromium is one of the major pollutants in water and soil. Thus, it is urgent to develop a new method for chromium removal from the environment. Phytoremediation is a promising approach for heavy metal pollution recovery. As a perennial giant grass with a fast growth rate, Pennisetum sinese has been widely used as livestock feed, mushroom culture medium and biomass energy raw material. Interestingly, we have found a high adsorption capacity of P. sinese for chromium. P. sinese was treated with di ﬀ erent concentrations of chromium for 15 days. Results showed that P. sinese plantlets grew well under low concentrations (less than 500 µ M) of chromium (VI). The plantlet growth was inhibited when treated with high concentrations of chromium (more than 1000 µ M). Up to 150.99 and 979.03 mg · kg − 1 DW of chromium accumulated in the aerial part and root, respectively, under a treatment of 2000 µ M Cr. The bioaccumulation factor (BCF) of P. sinese varied from 10.87 to 17.56, and reached a maximum value at the concentration of 500 µ M. The results indicated that P. sinese showed strong tolerance and high accumulation capability under Cr stress. Therefore, the chromium removal potential of P. sinese has a great application prospect in phytoremediation.


Introduction
Along with the increasing discharge of industrial waste, more and more heavy metals are accumulated in soil and water. The indiscriminate release of heavy metals into the environment is becoming a major health concern worldwide, since heavy metals are hard to turn into non-toxic forms under natural conditions, and will therefore have long-lasting effects on ecosystems. A very low dose of heavy metals such as chromium (Cr), arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) can also cause serious health problems, because they are not only cytotoxic but also mutagenic and carcinogenic in nature [1]. In the past few decades, the serious contamination caused by Cr has become an increasing threat [2][3][4]. Cr is widely distributed in soil, water and biological materials [4,5]. Cr exists in several oxidation states, and the stable forms in nature include Cr (0), Cr (III) and Cr (VI). Cr in different valence states shows different characteristics in chemistry, toxicology and epidemiology, and Cr (VI) is more toxic than Cr (III) because of its higher mobility [6][7][8]. Although it is not essential for plants, Cr may have hermetic effects on plant growth-it can promote the growth of water hyacinths (Eichhornia crassipes), with an increased yield at low concentrations [2,[9][10][11]. However, Cr could have deleterious effects on plant physiological processes such as photosynthesis, water relations and mineral nutrition at high concentrations [11][12][13]. Thus, it is urgent to develop efficient techniques for Cr removal from the environment.
Heavy metal depletion can occur under natural conditions through leaching, plant absorption, erosion and deflation. The bottleneck for these natural processes is their low efficiency. In order to

Plant Growth Conditions
Mature P. sinese stem cuts were purchased from Fujian Agriculture and Forestry University, and the propagation was later performed in our lab. Stem segments containing two nodes were used in the cottage for propagation. To screen the growth traits, plantlets (about 10 cm height) were transplanted in 60 cm diameter pots (height 40 cm) containing 60 kg soil with a planting density of one plant per pot. One hundred pots were adjacently arranged in a square (about 40 m 2 ). In the second year after planting, the height, length of internode, effective tillers, leaf length, stem circumference and biomass were measured. To analyze the effects of chromium on P. sinese, plantlets (about 20 cm height) with six to seven leaves were transferred from the soil to a Hoagland nutrient solution and cultured in the greenhouse under natural conditions. The nutrient solution was constantly aerated through an air pump, and it was renewed every three days. To test the Cr tolerance capability, P. sinese were treated with 0, 50, 250, 500, 1000 and 2000 µM concentrations of potassium dichromate (K 2 Cr 2 O 7 ). To investigate the Cr absorption rate, P. sinese plantlets were cultured in a Hoagland solution containing 400 and 800 µM concentrations of potassium dichromate (K 2 Cr 2 O 7 ), respectively, after 48 h pre-culture in deionized water. Roots and shoots were separately collected after 0, 12, 24, 36, 48, 60 and 72 h.

Analysis of Growth Parameters
The lengths and fresh weights of the shoots and roots were recorded after harvest. Dry weights were measured after oven drying at 70 • C for 48 h. The root to shoot ratio was calculated in dry weight. Abiotic tolerance indices (TIs) were calculated as the increase in fresh weight of chromium-treated plants divided by the increase in fresh weight of untreated plants [38]. The relative growth rate (RGR) was calculated as RGR = (ln W2 − ln W1)/(t2 − t1), where W1 and W2 represent a plant's dry weights at time t1 and t2, respectively.

Determination of Mineral Elements
Dried samples were used to detect Cr and other mineral elements in P. sinese. 1.0 g of samples was digested by perchloric acid and hy++drogen nitrate (V/V = 1:1) in 90 mL of pure quartz vessels at a high pressure asher (HPA) (Bergh.of Speedwave-2, Reutlingen, Germany) [39]. After digestion through HPA, the content of chromium was 6determined through IRIS Interpid II XPS ICP (Thermo, USA). The ICP analysis was carried out on axially viewed plasma. The emission intensity measurements were performed under the following conditions: 1150 watt of radio frequency (RF) power, 2.4 mL·min -1 plasma flow rate, 2.4 mL·min −1 nebulizer flow rate, 25 psi nebulizer pressure and 0.5 L·min -1 auxiliary flow rate. The bioaccumulation factor (BCF) was calculated as C plant /C solution . C plant meant the concentration of Cr in P. sinese (mg·kg −1 DW), and C solution was the concentration of Cr in solution (mg·kg −1 DW). The transfer factor (TF) was calculated as C 1 /C 2 , wherein C 1 is the metal concentration in the aboveground tissues (mg·kg −1 DW) and C 2 is the metal concentration in the roots (mg·kg −1 DW).

Data Analysis and Statistics
Data analysis and statistics were performed by Microsoft Excel 2010. Three independent biological replicates were analyzed. All significant treatment effects were determined by using Dunnett's test at p < 0.05.

Results
3.1. Growth Analysis of P. sinese P. sinese is a perennial plant and grows well in the tropics, subtropics and temperate zone. Its optimal growth temperature ranges from 25 • C to 35 • C. P. sinese has no heading stage in the temperate zone, but a few seeds with low germination rate can be harvested in the subtropical zone. Consequently, stem cuts with strong axillary buds are the main parts for propagation ( Figure 1a). P. sinese is a typical C 4 plant, and thus shows a high conversion rate for solar energy [36]. P. sinese plants are erect and clustered (Figure 1b,c). Generally, the biomass of P. sinese tended to be stable in the second year after planting both in pot and field (Figure 1c). In this study, we detected the growth traits and yield of P. sinese cultured in pots at maturity in the second year after planting ( Table 1). The average plant height was about 4 m ( Table 1). Previous researchers reported that P. sinese could grow to 3-5 m and even to 7 m in a field [40]. This indicated that P. sinese can grow very quickly. Moreover, we obtained a theoretical yield of about 168 t/hm 2 FW in the pot experiment. This yield was close to the reported yield (150-255 t/hm 2 FW) in field cultivation [35]. The results indicated that P. sinese had a huge biomass.

Effects of Cr on P. sinese Growth
Tolerance to heavy metals is one of the most important characteristics for phytoremediation plants. To clarify the Cr tolerance capability of P. sinese, plant growth and tolerance were analyzed under treatments with different concentrations of Cr. The growth and development of P. sinese were affected by different concentrations of Cr. Compared to the control (0 μM Cr), plant growth was significantly stimulated under the concentrations lower than 250 μM Cr (Figure 2a,b). Plants showed a faster growth at the level of 250 μM Cr and 6-7% increases in length and biomass. The same stimulatory effect was also observed in the ratio of Root/Stem and RGR of plantlets under treatments lower than 250 μM Cr (Figure 2c,d). However, the root to shoot ratio and relative growth rate significantly decreased when the Cr concentration was further increased. When 1000 μM or more of Cr was applied, P. sinese plants began to show some toxicity symptoms, e.g., thin and yellow leaves with sporadic brown spots plus serious growth inhibition. By contrast, toxicity symptoms were not obvious and no plant wilted at Cr levels lower than 1000 μM. The tolerance indices (TIs) of P. sinese remained stable at Cr levels lower than 500 μM ( Figure 2e). Therefore, P. sinese showed relatively high tolerance to high Cr exposure.

Effects of Cr on P. sinese Growth
Tolerance to heavy metals is one of the most important characteristics for phytoremediation plants. To clarify the Cr tolerance capability of P. sinese, plant growth and tolerance were analyzed under treatments with different concentrations of Cr. The growth and development of P. sinese were affected by different concentrations of Cr. Compared to the control (0 µM Cr), plant growth was significantly stimulated under the concentrations lower than 250 µM Cr (Figure 2a,b). Plants showed a faster growth at the level of 250 µM Cr and 6-7% increases in length and biomass. The same stimulatory effect was also observed in the ratio of Root/Stem and RGR of plantlets under treatments lower than 250 µM Cr (Figure 2c,d). However, the root to shoot ratio and relative growth rate significantly decreased when the Cr concentration was further increased. When 1000 µM or more of Cr was applied, P. sinese plants began to show some toxicity symptoms, e.g., thin and yellow leaves with sporadic brown spots plus serious growth inhibition. By contrast, toxicity symptoms were not obvious and no plant wilted at Cr levels lower than 1000 µM. The tolerance indices (TIs) of P. sinese remained stable at Cr levels lower than 500 µM ( Figure 2e). Therefore, P. sinese showed relatively high tolerance to high Cr exposure.

Cr Accumulation in P. sinese
Plants cultured for fifteen days were used for Cr distribution analysis. The Cr concentration in P. sinese increased along with the increasing Cr levels in the nutrient solution (Figure 3a,b). The highest Cr concentrations accumulated in shoots and roots were 150.99 and 979.03 mg•kg −1 DW, respectively, under a treatment with 2000 μM Cr in the solution. The Cr concentrations in the roots were higher than in the shoots. Compared with the control, the differences reached significant levels in all treatments at 5% level (P = 0.05). More Cr accumulated in roots than in shoots indicated that the roots show a stronger affinity for Cr. The bioaccumulation factor (BCF) of P. sinese exposed to Cr ranged from 10.87 to 17.56, reaching a maximum value at the Cr concentration of 500 μM. The BCF value decreased along with the concentration increase ( Figure 3c). The data might indicate that the bioaccumulation depends on the duration of exposure, and the level in the environment. Generally, in all treatments, TF values obtained from the aerial and underground part of the plant were below 0.2 ( Figure 3d). Hyperaccumulators are species capable of accumulating metals at levels 100 times greater than those typically measured in the shoots of common, non-accumulator plants [41]. These plants should accumulate metals at levels of up to 0.1-1.0% of the dry weight based on the plant biomass produced, depending on the specific metal being taken up [42]. Therefore, P. sinese showed a high Cr accumulation and was close to hyperaccumulation level.

Cr Accumulation in P. sinese
Plants cultured for fifteen days were used for Cr distribution analysis. The Cr concentration in P. sinese increased along with the increasing Cr levels in the nutrient solution (Figure 3a,b). The highest Cr concentrations accumulated in shoots and roots were 150.99 and 979.03 mg·kg −1 DW, respectively, under a treatment with 2000 µM Cr in the solution. The Cr concentrations in the roots were higher than in the shoots. Compared with the control, the differences reached significant levels in all treatments at 5% level (p = 0.05). More Cr accumulated in roots than in shoots indicated that the roots show a stronger affinity for Cr. The bioaccumulation factor (BCF) of P. sinese exposed to Cr ranged from 10.87 to 17.56, reaching a maximum value at the Cr concentration of 500 µM. The BCF value decreased along with the concentration increase (Figure 3c). The data might indicate that the bioaccumulation depends on the duration of exposure, and the level in the environment. Generally, in all treatments, TF values obtained from the aerial and underground part of the plant were below 0.2 (Figure 3d). Hyperaccumulators are species capable of accumulating metals at levels 100 times greater than those typically measured in the shoots of common, non-accumulator plants [41]. These plants should accumulate metals at levels of up to 0.1-1.0% of the dry weight based on the plant biomass produced, depending on the specific metal being taken up [42]. Therefore, P. sinese showed a high Cr accumulation and was close to hyperaccumulation level.

Dynamic of Cr Accumulation
The results of Cr absorption in P. sinese are shown in Figure 4. The total amount of Cr in the plants increased gradually with the extension of treatment time. The plants experienced a rapid absorption and then a slow absorption process under different treatment concentrations (Figure 4a,b). Under treatment with 400 μM Cr, the change of Cr contents in the plants followed an "S" distribution along with the extension of treatment time, and the saturation of Cr accumulation in the plants occurred at 60 h (Figure 4a). Under a treatment with 800 μM Cr, Cr accumulation followed a linear trend in the first 48 h, and then Cr absorption was obviously slowed. Cr absorption was not saturated until 72 h at 800 μM Cr (Figure 4b). The results indicated that the Cr absorption rate and the time to reach Cr accumulation saturation are related to the treatment concentration of Cr.

Dynamic of Cr Accumulation
The results of Cr absorption in P. sinese are shown in Figure 4. The total amount of Cr in the plants increased gradually with the extension of treatment time. The plants experienced a rapid absorption and then a slow absorption process under different treatment concentrations (Figure 4a,b). Under treatment with 400 µM Cr, the change of Cr contents in the plants followed an "S" distribution along with the extension of treatment time, and the saturation of Cr accumulation in the plants occurred at 60 h (Figure 4a). Under a treatment with 800 µM Cr, Cr accumulation followed a linear trend in the first 48 h, and then Cr absorption was obviously slowed. Cr absorption was not saturated until 72 h at 800 µM Cr (Figure 4b). The results indicated that the Cr absorption rate and the time to reach Cr accumulation saturation are related to the treatment concentration of Cr.

Dynamic of Cr Accumulation
The results of Cr absorption in P. sinese are shown in Figure 4. The total amount of Cr in the plants increased gradually with the extension of treatment time. The plants experienced a rapid absorption and then a slow absorption process under different treatment concentrations (Figure 4a,b). Under treatment with 400 μM Cr, the change of Cr contents in the plants followed an "S" distribution along with the extension of treatment time, and the saturation of Cr accumulation in the plants occurred at 60 h (Figure 4a). Under a treatment with 800 μM Cr, Cr accumulation followed a linear trend in the first 48 h, and then Cr absorption was obviously slowed. Cr absorption was not saturated until 72 h at 800 μM Cr (Figure 4b). The results indicated that the Cr absorption rate and the time to reach Cr accumulation saturation are related to the treatment concentration of Cr.

Effects of Cr on Element Accumulation
P. sinese seedlings can grow normally at a Cr concentration of less than 500 µM. Cr in the range of 500 µM to 1000 µM significantly inhibited the growth of P. sinese seedlings but no plant wilted. Furthermore, the contents of part nutrient elements in roots and leaves were determined to analyze the effects of Cr on element absorption. The Cr concentrations of 50, 250, 1000 µM were used according to plant growth performances. Compared with those at 0 µM Cr, the contents of Fe, Mg and Mn increased both in the roots and the shoots under treatments with Cr, but the increase of Mg and Mn was not significant (Figure 5a-c). The contents of Ca, P and K slightly decreased in the roots but increased in the shoots under treatment with Cr (Figure 5d-f). P was significantly higher than the control in the shoots (Figure 5f). The content of S in the roots increased significantly (by 11.1%) under Cr treatment at 50 µM, but it showed a lower content in the shoots under three Cr treatments (Figure 5g). Cr at more than 250 µM significantly affected the Cu content in the roots, but there was no obvious effect in the shoots under all Cr treatments (Figure 5h). Overall, Cr treatment promoted the absorption of Fe, Mg, Mn, and Cu, and slightly inhibited the uptake of Ca, P and S. Cr showed more influence on the accumulation of Fe and Cu compared to other elements.

Effects of Cr on Element Accumulation
P. sinese seedlings can grow normally at a Cr concentration of less than 500 μM. Cr in the range of 500 μM to 1000 μM significantly inhibited the growth of P. sinese seedlings but no plant wilted. Furthermore, the contents of part nutrient elements in roots and leaves were determined to analyze the effects of Cr on element absorption. The Cr concentrations of 50, 250, 1000 μM were used according to plant growth performances. Compared with those at 0 μM Cr, the contents of Fe, Mg and Mn increased both in the roots and the shoots under treatments with Cr, but the increase of Mg and Mn was not significant (Figure 5a-c). The contents of Ca, P and K slightly decreased in the roots but increased in the shoots under treatment with Cr (Figure 5d-f). P was significantly higher than the control in the shoots (Figure 5f). The content of S in the roots increased significantly (by 11.1%) under Cr treatment at 50 μM, but it showed a lower content in the shoots under three Cr treatments ( Figure  5g). Cr at more than 250 μM significantly affected the Cu content in the roots, but there was no obvious effect in the shoots under all Cr treatments (Figure 5h). Overall, Cr treatment promoted the absorption of Fe, Mg, Mn, and Cu, and slightly inhibited the uptake of Ca, P and S. Cr showed more influence on the accumulation of Fe and Cu compared to other elements.

Discussion
Cr is not an essential element for plant growth [9,10]. Several reports pointed out that Cr is a stimulator for plants, promoting growth and increasing biomass at low concentrations, but high concentration Cr reduces the seed germination rate, inhibits plant growth and results in a significant biomass decrease [43][44][45]. In fact, the phenomenon whereby low concentrations of a heavy metal promote plant growth is defined as hermetic effect and frequently occurs in treatments with heavy metals [46][47][48]. Because of the hermetic effect, P. sinese plantlets grew better and obtained a higher biomass than the control plants at low concentrations (<250 μM), and the biomass and the RGR were significantly increased compared to the control plants ( Figure 2). A mineral composition analysis revealed that the total contents of Cu, Mn, Fe and Mg in Cr-treated P. sinese plantlets were increased compared to those in the control plants (0 μM Cr). So did the K content in the shoots (but not in the roots). The results indicated that Cr presented a certain impact on the absorption of other minerals in

Discussion
Cr is not an essential element for plant growth [9,10]. Several reports pointed out that Cr is a stimulator for plants, promoting growth and increasing biomass at low concentrations, but high concentration Cr reduces the seed germination rate, inhibits plant growth and results in a significant biomass decrease [43][44][45]. In fact, the phenomenon whereby low concentrations of a heavy metal promote plant growth is defined as hermetic effect and frequently occurs in treatments with heavy metals [46][47][48]. Because of the hermetic effect, P. sinese plantlets grew better and obtained a higher biomass than the control plants at low concentrations (<250 µM), and the biomass and the RGR were significantly increased compared to the control plants ( Figure 2). A mineral composition analysis revealed that the total contents of Cu, Mn, Fe and Mg in Cr-treated P. sinese plantlets were increased compared to those in the control plants (0 µM Cr). So did the K content in the shoots (but not in the roots). The results indicated that Cr presented a certain impact on the absorption of other minerals in P. sinese. In this work, hexavalent Cr was supplied with potassium. Due to the lack of the same dose of potassium application treatment, we were unable to determine whether the potassium supplied affected the promotion of plantlet growth. However, in treatments with CrCl 3 (data not shown in this paper) and K 2 Cr 2 O 7 as a Cr source, plant growth and Cr accumulation did not show any obvious differences at the same Cr concentration. This indicated that the effect of K on P. sinese growth promotion was minimal comparing to the Cr effect. Mg contents in roots treated with Cr were higher than those of the control plants. The results indicated that Cr treatment somehow contributed to the absorption of Mg. The inhibitory effects of high concentrations of Cr on plant growth were possibly due to the destruction of normal physiological metabolism or the interference with the absorption of other nutrient elements.
Cultivated P. sinese was initially used as forage and mushroom culture medium because of its huge biomass. Recently, researchers have also found that it showed some advantages in resistance to abiotic stresses. P. sinese had a certain ability to resist alkali stress [37]. P. sinese also showed relatively high tolerance to Cd stress and could be applied to repair the cadmium pollution in the environment [49,50]. In this study, we found that the growth of P. sinese plantlets was not inhibited by relatively high concentrations of Cr, indicating that P. sinese had a relatively high tolerance to Cr. High Cr accumulation showed that P. sinese had a high enrichment capability for Cr. With the concentration increase in the Cr treatment, the accumulation of Cr in P. sinese plants also increased, and the plant obtained the largest enrichment of 979.03 mg·kg −1 DW under the treatment of 2000 µM Cr. According to the usual standard, hyperaccumulators can accumulate a certain ion at levels 100 times greater than those measured in the shoots of common, non-accumulator plants [41], and the content of metal in plant tissues can range from 0.1% to 1.0% (DW) depending on the specific metal being taken up [42]. The Cr content in P. sinese was close to that of hyperaccumulators. Moreover, the TF of Cr varied between 0.28 and 0.53 in Hemarthria compressa [51]. Similarly to pumpkin and green vegetables [45,52], the TF of P. sinese for Cr was at relative low levels under hydroponic conditions, but it reached 0.317 under soil culture conditions (not shown in this paper).
The excess of Cr the in environment is severely harmful to the human health and a serious source of stress for plants. Under Cr stress, plant growth and development can be inhibited, and crops may be threatened by reduced production [53][54][55]. Phytoremediation is considered a green and sustainable method for pollutant removal. Thus, some hyperaccumulator plants were screened and had great application value in Cr phytoremediation [27][28][29][30][31][32][33][34]56]. Regretfully, the identified hyperaccumulator plants are mainly small herbs, whose average annual biomass is relatively limited. When they are used as Cr phytoremediation plants, the efficiency of Cr removal from Cr contaminated soils is not high. P. sinese showed a huge advantage in the growth rate and biomass (more than 200 t FW·hm −2 per year), although the level of Cr accumulation in tissues is lower than in hyperaccumulator plants. Taken together, Cr can be quickly removed from the soil through planting P. sinese in Cr contaminated soils and recycling Cr in collected plants. Thus, P. sinese has a great potential for retrieving Cr from the soil. It can be applied in phytoremediation for Cr polluted soils.

Conclusions
Cr is a main source of heavy metal contamination, since it is widely present in the environment. Screening phytoremediation plants are very important, and using them as a sustainable method for Cr removal is very helpful for restoring the natural conditions of Cr-polluted soils. Under treatment with different concentrations of Cr, concentrations lower than 500 µM did not significantly inhibit the growth and development of P. sinese seedlings, although Cr affected the absorption of some necessary elements, suggesting a relatively high tolerance of P. sinese to high Cr exposure. Cr accumulation in the shoots and roots can be as high as 150.99 and 979.03 mg·kg −1 DW, respectively, when culturing in a Hoagland solution containing 2000 µM Cr, which indicates the high Cr accumulation capability of P. sinese. In addition, P. sinese is a perennial giant grass with high photosynthetic efficiency and growth rate, and its biomass can reach more than 200 t FW·hm −2 per year, which is far greater than that of reported Cr hyperaccumulation plants. Therefore, P. sinese can be used to retrieve Cr from soils and has great application prospects in phytoremediation.