Combined effects of Bacillus sp. M6 strain and Sedum alfredii on rhizosphere community and bioremediation of cadmium polluted soils

Abstract

Concerns regarding inevitable soil translocation and bioaccumulation of cadmium (Cd) in plants have been escalating in concomitance with the posed phytotoxicity and threat to human health. Exhibiting a Cd tolerance, Bacillus sp. M6 strain has been reported as a soil amendment owing to its capability of reducing metal bioavailability in soils. The present study investigated the rhizospheric bacterial community of the Cd hyperaccumulator Sedum alfredii using 16S rRNA gene sequencing. Additionally, the Cd removal efficiency of strain Bacillus sp. M6 was enhanced by supplementing with biochar (C), glutamic acid (G), and rhamnolipid (R) to promote the phytoremediation effect of hyperaccumulator S. alfredii. To the best of our knowledge, this is the first time the amendments such as C, G, and R together with the plant-microbe system S. alfredii-Bacillus sp. M6 has been used for Cd bioremediation. The results showed that soil CaCl₂ and DTPA (Diethylenetriamine penta-acetic acid) extractable Cd increased by 52.77 and 95.08%, respectively, in all M6 treatments compared to unamended control (CK). Sedum alfredii with Bacillus sp. M6 supplemented with biochar and rhamnolipid displayed a higher phytoremediation effect, and the removal capability of soil Cd (II) reached up to 16.47%. Moreover, remediation of Cd polluted soil by Bacillus sp. M6 also had an impact on the soil microbiome, including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and cadmium transporting ATPase (cadA) genes. Quantitative PCR analysis confirmed the Bacillus sp. M6 strain increased the abundance of AOB and cadA in both low Cd (LC) and high Cd (HC) soils compared to AOA gene abundance. Besides, the abundance of Proteobacteria and Actinobacteria was found to be highest in both soils representing high tolerance capacity against Cd. While Firmicutes ranked third, indicating that the additionof strain could not make it the most dominant species. The results suggested the presence of the hyperaccumulator S. alfredii and Cd tolerant strain Bacillus sp. M6 supplemented with biochar, and rhamnolipid, play a unique and essential role in the remediation process and reducing the bioavailability of Cd.

Keywords: Bacillus sp. M6; Sedum alfredii; biochar (BC); bioremediation; cadmium; rhamnolipid; soil microbial community.

FIGURE 1

The bacterial structure of electron microscopy by using Bacillus sp. M6. And amendments (a) M6 strain 2 μm scale; (b) M6 + rhamnolipid 2 μm scale; (c) M6 strain 500 nm scale; (d) M6 + rhamnolipid 500 nm scale; (e,f) M6 + biochar.

FIGURE 2

Changes in pH and available cadmium in LC and HC soils. (A) Changes in extractable Cd of DTPA and CaCl₂ in HC soils; (B) changes in extractable Cd of DTPA and CaCl₂ in LC soils; (C) changes in soil pH; (D) correlation between soil pH and CaCl₂-extracted Cd. The arrow bar above the line shows standard error. The different letters show significant differences while same letters represent no significant differences among the treatments. CK, control; M6, M6 strain; C, M6 + Biochar; G, M6 + Glutamic acid; R, M6 + rhamnolipid; FY, Fuyang; ZJ, Zhuji; LC, low cadmium; HC, high cadmium; DTPA, Diethyene triaminepenta acetic acid.

FIGURE 3

Abundance of soil functional genes amoA AOA and amoA AOB in (A) HC (B) LC soils collected from two different soils. (C) The abundance of cadA genes in two soils. Data points represent means the standard deviations (n = 3). The various lowercase letters indicate significant differences among all treatments at p < 0.05. CK, control; M6, M6 strain; C, M6 + Biochar; G, M6 + Glutamic acid; R, M6 + rhamnolipid; FY, Fuyang County; ZJ, Zhuji County; LC, low cadmium; HC, high cadmium.

FIGURE 4

The cluster dendrogram of Bray-Curtis dissimilarity of samples. CK, control; M6, M6 strain; C, M6 + Biochar; G, M6 + Glutamic acid; R, M6 + rhamnolipid; F, Fuyang County; Z, Zhuji County.

FIGURE 5

LEfSe cladogram indicating the phylogenetic distribution of bacteria lineages under different treatments in HC (A) and LC (B) soils. In the figure, red, green, dark blue, purple, and light blue circles, respectively represent the flora of C, CK, G, M6, and R treatments which are significantly higher than those of the other four treatments. LDA > 3 is taken to represent the significant difference, and the circles from inside to outside, respectively represent the classification level of domain, phylum, class, order, family, genus, and species. CK, control; M6, M6 strain; C, M6 + Biochar; G, M6 + Glutamic acid; R, M6 + rhamnolipid; FY, Fuyang County; ZJ, Zhuji County; LC, low cadmium; HC, high cadmium; LEfSe, Linear discriminant analysis effect size; LDA, Linear discriminant analysis.

FIGURE 6

Cd content in stems and leaves of Sedum alfredii (A) and total Cd and available Cd content in LC soil (B), Cd content of Sedum alfredii and total soil Cd content in HC (C). The data is the means and standard deviations of three replicates (n = 3). The significant differences among all treatments (p < 0.05) are presented by lowercase letters. LC, low cadmium; HC, high cadmium.