Isolation and screening of plant growth-promoting bacteria
A total of 11 strains were screened and isolated from the collected soil samples, and four strains with favorable IAA production, nitrogen fixation, inorganic phosphorus solubilization, iron carrier production, and metal tolerance were obtained, i.e., RH1, RH3, MO2, and MO4, as shown in Table 3.
Table 3
Qualitative assessment of the strains
Strain
|
Production IAA
|
Solving inorganic phosphorus
|
Investigated the nitrogen fixation
|
Iron carrier production
|
Metal tolerance at different concentrations(Cu、Cd、Zn、Cr)
|
RH1
|
+
|
+
|
+
|
−
|
+
|
RH3
|
+
|
+
|
+
|
−
|
+
|
MO2
|
+
|
+
|
+
|
+
|
+
|
MO4
|
+
|
+
|
+
|
+
|
+
|
Notes: +: strains with corresponding characteristics; −: strains without corresponding characteristics.
Quantitative IAA production capacity
IAA is a plant hormone. It is essential for growth and development and regulates root, stem and leaf growth, as well as the formation and development of various other tissues17. IAA produced by microorganisms is also able to promote plant growth, thus facilitating symbiosis with super-enriched plants18. As shown in Fig. 1, all strains tested in this study were capable of producing IAA: MO4 had the highest IAA production capacity of 26.98 mg/L, followed by MO2 and RH3 with IAA yields of 23.40 and 19.94 mg/L, respectively; RH1 had the lowest capacity of 13.94 mg/L.
Inorganic phosphorus solubility
Phosphorus is essential for plant growth. However, most of the phosphorus in the soil is in an insoluble form and is difficult for plants to access directly19. Fortunately, phosphorus-dissolving probiotic bacteria can dissolve the insoluble organic and inorganic phosphorus in the soil and convert it to water-soluble phosphorus, increasing the soluble phosphorus content of the soil and promoting phosphorus uptake by plants20. The phosphorus solubilizing ability of the strains studied here was evaluated according to the phosphorus solubilizing circles produced, as shown in Table 4. The test strains all had phosphorus solubilizing ability. RH3 had the strongest inorganic phosphorus solubilizing ability with a diameter of 0.66 cm and a phosphorus solubilizing activity of up to 1.33. The Bacillus sp. Z3-4 isolated from the inter-rhizosphere soil of Dianthus spp. had basically the same inorganic phosphorus solubilizing ability, and the diameter of its phosphorus solubilizing circle was 0.52–1.22 cm21. The best Bacillus belleus strain for solubilization of inorganic phosphorus (i.e., LPL-410.7) was isolated from different soil samples with a solubility index of 3.12, which shows that strain RH3 has an advantage in phosphorus solubilization22.
Table 4
Inorganic phosphorus solubilization capacity of each strain
Strain
|
Soluble phosphorus ring diameter(HD/cm)
|
Colony diameter(CD/cm)
|
Phosphorus solubilization activity(HD/CD)
|
RH1
|
0.81 ± 0.09c
|
0.63 ± 0.10c
|
1.29 ± 0.05a
|
RH3
|
0.66 ± 0.07c
|
0.50 ± 0.09c
|
1.33 ± 0.10a
|
MO2
|
1.36 ± 0.15b
|
1.18 ± 0.10b
|
1.15 ± 0.03b
|
MO4
|
1.74 ± 0.19a
|
1.57 ± 0.17a
|
1.11 ± 0.01b
|
Note: The data are presented as the means ± standard deviations, and different letters in the same column indicate significant differences (P < 0.05).
Iron carrier production capability
Iron carriers have a strong Fe3+ chelating ability. Plant inter-root microorganisms can synthesize such substances to extract iron from the soil and deliver it to plants23. As shown in Fig. 2, strains MO2 and MO4 both produced iron carriers with 14% and 35% iron carrier activity, respectively, whereas strains RH1 and RH3 did not produce any iron carriers.
To date, no iron carriers or compounds have been found to serve as microbial iron carrier standards for standard curve plotting, and the quantitative assay used here was a relative quantification reflecting the ability of different strains to produce iron carriers.
Identification of superior inter-root growth-promoting bacteria
Morphological identification
The colony morphology of strains RH3 and MO4 on solid LB medium is shown in Fig. 3(a)(b). The colonies of both strains appear creamy white. The MO4 colony appears to be much smoother and opaque than the RH3 colony. The RH3 colony is round and raised and larger than the MO4 colony (diameter 1.5-2.0 mm); the MO4 colony has a more regular edge, is smoother and has a diameter of 1.0-1.5mm. The results of Gram staining are shown in Fig. 3(c)(d). RH3 was red (i.e., Gram-negative) after Gram staining, and MO4 was purple (i.e., Gram-positive).
Growth curve plotting
The growth curves of the four strains are shown in Fig. 4. Each strain had a long growth lag of about 8 h. The times to reach the logarithmic growth phase and the stable phase were 24 and 46 h for RH1, 12 and 46 h for MO2, 16 and 46 h for RH3, and 20 and 48 h for MO4, respectively. The pH during growth was stable at 6.21 to 7.45. Bacillus subtilis isolated and screened from the roots of red clover and other plants grew for 48 h to the stabilization phase and then gradually entered the decay phase24.
16S rDNA sequence identification
After sequencing, RH1 and RH3 were identified as Rhizobium strains and MO2 and MO4 were identified as Microbacterium strains.
The 16S rDNA gene fragments of RH1 and RH3 were approximately 1500 bp in length. The 16S rDNA of RH1 and RH3 strains were compared using the National Center for Biotechnology Information database, and the strains with high homology were used as reference bacteria for phylogenetic analysis. RH1 and RH3 were highly homologous (100%) to chromosome 76 of a Rhizobium pusense strain. The 16S rDNA fragments of MO2 and MO4 were about 1500 bp long. The 16S rDNA was compared with that in the National Center for Biotechnology Information database, and the reference strains with high homology were used for phylogenetic analysis. MO2 and MO4 were highly homologous (100%) to a chromosome of Microbacterium oxydans strain VIU2A (complete genome CP031338.1). The phylogenetic trees of RH3 and MO4 were constructed using MEGA11 software (Fig. 5 and Fig. 6).
Effect of strains on the migration of heavy metal forms in soil
RH3 and MO4 strains were subjected to shake flask experiments using soil samples from the e-waste dismantling site. The changes in heavy metal migration revealed by the shake flask tests are shown in Fig. 7, and the proportions of each form of heavy metal in the soil samples are shown in Fig. 8. The five heavy metals in the soil samples (i.e., Cu, Zn, Cr, Cd, and Pb) were analyzed separately.
According to Fig. 8(a)-which shows the morphological distribution of Cu in the soil-compared to the corresponding proportions in the bacteria-free group (CK), F1 + F2 (F1: the weak acid extraction form; F2: the reducible form) increased by 7% and F3 + F4 (F3: the oxidizable form; F4: the residual form) decreased by 7% after the action of RH3. This can be attributed to the activation of Cu in the soil by RH3, which converted some oxidized and residual forms into reducible and weak acid extraction forms. Compared with the corresponding proportions in the CK group, the proportion of F1 + F2 increased by 4% and that of F3 + F4 decreased by 3% after the action of MO4. This may also be attributed to the activation of Cu by MO4, which converted the oxidized form and a small amount of the residual form into weak acid extraction and reducible forms. RH3 activated Cu more effectively than MO4. It was demonstrated that inter-root microbial-mycorrhizal mixtures can be used to remediate heavy metal-contaminated soil. The results showed that the concentration of soluble Cu increased significantly and accumulated in plant roots and shoots25.
According to Fig. 8(b)-which shows the morphological distribution of Zn in the soil-compared with the corresponding proportions in the CK group, the proportion of F2 increased by 1% and that of F3 decreased by 1% after the action of RH3. This can be attributed to the activation of Zn by RH3, which converted a small amount of the oxidized form into the reducible form. Compared to the corresponding proportions in the CK group, the proportion of F1 + F2 increased by 1% and that of F3 + F4 decreased by 1% after the action of MO4. Similarly, MO4 activated Zn in the soil sample, converting a small amount of oxidized and residual forms to weak acid extractable and reducible forms. RH3 and MO4 had the same activating effect on the Zn in the soil. Someone investigated the ability of e-waste tolerant bacteria to mobilize heavy metals in soil. The results showed that the mobilization of Zn from the soil to the plant rhizomes significantly reduced the toxicity of Zn in the soil after inoculation26.
According to Fig. 8(c)-which shows the morphological distribution of Cr in the soil-compared to the corresponding proportions in the CK group, the proportion of F1 + F2 decreased by 6% and that of F3 + F4 increased by 5% after the action of RH3. This can be attributed to the Cr-fixing effect of RH3, which converted some of the weak acid extractable and reducible forms into residual and oxidized forms. After MO4 was applied to the soil, the proportion of F1 + F2 was reduced by 2% and that of F3 + F4 was increased by 1% compared to the corresponding proportions in the CK group. This can be attributed to the Cr-fixing effect of MO4, which converted some of the weak acid extraction and reducible forms into residual and oxidized forms. RH3 fixed Cr in the soil more effectively than MO4. This is similar to27, where Pseudomonas was used to almost completely transform the most toxic water-soluble Cr component of soil into other stable components.
According to Fig. 8(d)-which shows the morphological distribution of Cd in the soil-compared to the proportions in the CK group, the proportion of F1 + F2 increased by 1% and that of F3 + F4 decreased by 2% after the action of RH3. This can be attributed to the activation of Cd by RH3, which converted part of the oxidized form and a small amount of the residual form into weak acid extraction and reducible forms. Compared to the proportions in the CK group, the proportions of F1 + F2 increased by 4% and the proportions of F3 + F4 decreased by 4% after the effect of MO4. This can be attributed to the activation of Cd by MO4, which converted the oxidized and residual forms into weak acid extractable and reducible forms. MO4 activated Cd more effectively than RH3. In one study, researchers used Bacillus strains screened and isolated from oily soil to restore weathered soil from a landfill. The removal rate of Cd by Bacillus strains was as high as 70%, which is of great significance for the biological remediation of highly weathered oil-contaminated soils28.
According to Fig. 8(e)-which shows the morphological distribution of Pb in the soil-compared to the corresponding proportions in the CK group, the proportion of F1 + F2 decreased by 19% and that of F3 + F4 increased by 19% after the action of RH3. This can be attributed to the activation of Pb by RH3, which transformed the weak acid extraction and reducible forms into residual and oxidized forms. After MO4 application to the soil, the proportion of F1 + F2 was reduced by 21% and that of F3 + F4 was increased by 20% compared to the corresponding proportions in the CK group. This can be attributed to the Pb-fixing effect of MO4, which converted the weak acid extracted form and the reducible form into residual and oxidized forms. MO4 fixed Pb more effectively than RH3. Someone used Pasteurella to recover Pb-contaminated soil; the content of soluble exchangeable Pb decreased by 18%-33.3%. The PB was precipitated and fixed with carbonate29.
According to the principle of a constant total soluble exchange form and a constant stable form applied in the European Community Bureau of Reference sequential extraction method30, some of the treatment groups showed unequal increases or decreases in the proportions of forms. This was probably because RH3 and MO4 mobilized small amounts of heavy metals from the soil into the culture supernatant, thus changing the total amount of heavy metals in the soil. It is worth mentioning that the total amount of heavy metals in the soil decreased after the action of the strains. Therefore, such bacterial strains represent an important biological resource, the use of which in combination with super-enriched plants will be useful for the remediation of soils contaminated with heavy metals.