## 3 Results and Discussion
### Growth, Lignocellulosic Composition and Metal Uptake by Vetiver Grass Grown in Stamp Sand
The present study did not specifically investigate the effects of copper speciation. The lack of plant available phosphorus could also contribute to the decreased root length of veiver grown in stamp sand [48]. There were differences in Cu content in the stamp sand and control soil over the 60-day period, due to Cu uptake by veiver as well as Cu leaching out of the column. However, the changes were not significant for the stamp sand or control soil. Vetiver roots contained increased concentrations of Cu by day 60 (Figure 2a). The increase in Cu content of the roots was greater than 14-fold, which was significant for the experimental biomass (\(p=0.0002\)). The average Cu accumulation in the root tissue (n = 6) for the experimental group was 538 mg/kg and for the control group it was 60 mg/kg over the 60 days. The shoots of veiver grass did not accumulate significant concentrations of Cu over the course of the study, in either the control or experimental group (Figure 2b). Our results are consistent with previous reports showing that veiver grass is capable of tolerating Cu toxicity, but accumulation of Cu in shoots and roots is limited [10]. The efficiency of metal removal by veiver grass was quantified using bioconcentration factor (BCF) and translocation factor (TF). The BCFshoot, BCFroot, and TF were 0.0106 (\(\pm\) 0.0098), 0.3781 (\(\pm\) 0.1814), and 0.0175 (\(\pm\)0.0064), respectively. Bioconcentration factors and translocation factor for Cu indicate that Cu accumulates mainly in the root tissue. Vetiver has been reported to be able to accumulate large amounts of metals in their root, but prevent translocation to the shoot to protect against toxicity in aerial structures [49; 50]. Figure 2: Copper accumulation in veiver grown (biotic columns) in control soil versus stamp sand in (**a**) root and (**b**) shoot. “Control” = veiver grown in control soil; “Experimental” = veiver grown in stamp sand. (**c**) Total suspended solids (TSS) in leachate (mg) between biotic and abiotic columns. Error bars show standard deviation (n = 6). (*) indicates \(p\)-value = \(<\)0.05, using \(t\)-test with Welch’s correction and \(\alpha\) = 0.05. The leachates collected from day 30 to day 60 was used for measuring differences in particulate leaching between the biotic and abiotic columns. There was a significant difference between the TSS leached from biotic and abiotic columns (\(p=0.0187\)) (Figure 2c). This result indicated that the root structures significantly reduced leaching of TSS, which would potentially reduce Cu leaching from the columns in the presence of vetiver. The aerial shoots of vetiver from both treatment groups of the uptake study (n = 6) were harvested to evaluate lignocellulosic composition. Lignin content significantly decreased (\(p=0.0006\)) (Figure 3), fermentable carbohydrates comprising the hemicellulose fraction significantly increased (\(p=0.0125\)) (Figure 3), and crystalline cellulose content significantly increased (\(p=0.0002\)) (Figure 3) in experimental shoots. These changes in the lignocellulosic composition of Cu-treated vetiver favor fermentability and compensate for the reduction in biomass caused by metal exposure. Significant reductions in acid insoluble and acid soluble lignin were found. Monosaccharides that comprise the hemicellulose and crystalline cellulose content both increased significantly. Lignin production is carried out by phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol-NADPH-dehydrogenase (CAD), which are regulated by extracellular Cu-dependent lacases and peroxididases [51; 52]. [PERSON] et al. (2012) found that there is a peak Cu concentration at which lacase activity switches from being promoted to repressed and/or terminated [53]. This threshold concentration reflects the Cu tolerance of the organism; at lower concentrations Cu acts as a cofactor and at higher concentrations Cu toxicity is observed. Copper tolerance of lacase enzymes among basidiomycetes can vary 100-fold in fungal cell walls [54].