Bioaugmented Phytoremediation of Metal-Contaminated Soils and Sediments by Hemp and Giant Reed

Abstract

We assessed the effects of EDTA and selected plant growth-promoting rhizobacteria (PGPR) on the phytoremediation of soils and sediments historically contaminated by Cr, Ni, and Cu. A total of 42 bacterial strains resistant to these heavy metals (HMs) were isolated and screened for PGP traits and metal bioaccumulation, and two Enterobacter spp. strains were finally selected. Phytoremediation pot experiments of 2 months duration were carried out with hemp (Cannabis sativa L.) and giant reed (Arundo donax L.) grown on soils and sediments respectively, comparing in both cases the effects of bioaugmentation with a single PGPR and EDTA addition on plant and root growth, plant HM uptake, HM leaching, as well as the changes that occurred in soil microbial communities (structure, biomass, and activity). Good removal percentages on a dry mass basis of Cr (0.4%), Ni (0.6%), and Cu (0.9%) were observed in giant reed while negligible values (<100‰) in hemp. In giant reed, HMs accumulated differentially in plant (rhizomes > > roots > leaves > stems) with largest quantities in rhizomes (Cr 0.6, Ni 3.7, and Cu 2.2 g plant^-1). EDTA increased Ni and Cu translocation to aerial parts in both crops, despite that in sediments high HM concentrations in leachates were measured. PGPR did not impact fine root diameter distribution of both crops compared with control while EDTA negatively affected root diameter class length (DCL) distribution. Under HM contamination, giant reed roots become shorter (from 5.2 to 2.3 mm cm^-3) while hemp roots become shorter and thickened from 0.13 to 0.26 mm. A consistent indirect effect of HM levels on the soil microbiome (diversity and activity) mediated by plant response (root DCL distribution) was observed. Multivariate analysis of bacterial diversity and activity revealed not only significant effects of plant and soil type (rhizosphere vs. bulk) but also a clear and similar differentiation of communities between control, EDTA, and PGPR treatments. We propose root DCL distribution as a key plant trait to understand detrimental effect of HMs on microbial communities. Positive evidence of the soil-microbe-plant interactions occurring when bioaugmentation with PGPR is associated with deep-rooting perennial crops makes this combination preferable over the one with chelating agents. Such knowledge might help to yield better bioaugmented bioremediation results in contaminated sites.

Keywords: Arundo donax (L.); Cannabis sativa L.; bioaugmentation; heavy metals; phytoremediation; plant growth-promoting rhizobacteria; plant uptake and accumulation; plant-root-microbes interactions.

FIGURE 1

Mean values of Cr, Ni, and Cu concentration (mg kg^–1) in plant components for hemp (roots, stems, leaves, and flowers) and giant reed (rhizome, roots, stems, and leaves) as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) concentration values among treatment for each HM/crop combination.

FIGURE 2

Summary of mean values of Cr, Ni, and Cu uptake (μg HM plant^–1) in plant components for hemp and giant reed as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) uptake values among treatment for each HM.

FIGURE 3

Whole-canopy net photosynthetic rate (Pn, μmol m^–2 s^–1) and transpiration rate (E, mmol m^–2 s^–1) of giant reed (A) and hemp (B) as affected by PGRP and EDTA application (black arrows). DAT, day after transplanting.

FIGURE 4

Diameter class length (DCL, mm cm^–3) distribution of hemp (A) and giant reed (B) whole root systems and (C,D) relative contribution (%) of the very fine, fine, and coarse roots to the total root length density (RLD). Coefficients and statistics obtained from the regression of extreme value model are reported in the table where the statistical significance of the DCL curve parameters (a–d) were assessed through testing their standard errors using the t-statistics at P < 0.05. Different letters in graph (C,D) denote statistical differences (Tukey’s test, P = 0.05) among treatment for each root diameter classes.

FIGURE 5

Distance-based redundancy analysis (dbRDA) plots showing shifts in enzyme activities of RS (A,C) and BS (B,D) of hemp (A,B) and giant reed (C,D) among treatments. Arrow indicates environmental variables with significance level (* < 0.05, ** < 0.001, *** < 0.001). Species scores corresponding to the dbRDA plots (coordinates for enzymes included in model) are reported in the scatter plots on the right. Letters within ellipses denote significant differences (Bonferroni’s test, P = 0.05) in EA similarity matrices among fertilizers as assessed by permutational multivariate analysis of variance (PERMANOVA).

FIGURE 6

Mean values of Simpson’s index (D) in BS and RS of giant reed (Left) and hemp (Right) as affected by treatments and sampling time (T_z, time zero sampling; T_f, at the end of experiment). Different letters denote statistically different (Tukey’s test, P = 0.05) D-values among treatment in BS and RS for single crops.

FIGURE 7

Distance-based redundancy analysis (dbRDA) plots showing shifts in microbial diversity (OUT) of RS and BS of hemp and giant reed among treatments and sampling time. Arrow indicates environmental variables with significance level (* < 0.05, ** < 0.001, *** < 0.001). Letters within ellipses denote significant differences (Bonferroni’s test, P = 0.05) in OUT’s similarity matrices among treatments/sampling time combination as assessed by permutational multivariate analysis of variance (PERMANOVA).