Mechanistic Elucidation of Plant Growth Promoting Bacteria in Biosorption and Phytoremediation of Heavy Metals

Abstract

The sustainable agricultural, global food security and maintenance of ecological regimes, native bacterial species can be scrutinized for their capability to tolerate heavy metals stress along with plant growth promotion. Four experiments were performed to assess the step-by-step ability of isolated PGPB for heavy metals (HMs) tolerance. The first investigation rhizospheric soil samples were collected from five different districts: Larkana, Qambar Shahdadkot, Shaheed Benazirabad, Naushahro Feroze, and Jacobabad, Sindh Pakistan. Twenty bacterial strains were isolated, screened and characterized for plant growth promoting activities and initial heavy metals tolerance potential. These isolates were confirmed to produce phosphate solubilization, hydrogen cyanide, indole acetic acid, zinc, ammonia, and siderophore Additionally, they also showed positive results to produce various extracellular enzymes i.e., ACC-deaminase, cellulase, catalase, amylase, protease, pectinase enzyme and exopolysaccharides. Among all, bacterial strain PM21 exhibited maximum ACCD (1.56-1.75 µM/mg protein/h), exopolysaccharides (2.73-2.98 mg/mL), and indole acetic acid (IAA) production (99-119 µM/mL) under normal and metal stressed conditions. The studied bacterial strains (PM21, PM22, PM23, PM24, and PM25) were identified via 16S rRNA gene sequencing technique. These bacterial strains were identified as Bacillus anthracis (PM21), Bacillus safensis (PM22), Enterobacter cloacae (PM23), Bacillus sonorensis (PM24) and Bacillus thuringiensis (PM25). The presence of nifH gene responsible for nitrogenase activity was confirmed in two strains (PM21 and PM23) through its polymerase chain reaction (PCR) amplification. Further, PCR amplification of acds gene in all strains verify their ability of heavy metal tolerance. These promising bacterial strains were proceeded further to check heavy metals biosorption potential. Assessment of mechanisms and abilities of plant growth promoting bacterial stains (PM21, PM22, PM23, PM24, and PM25) were conducted in second study for biosorption of cadmium (Cd), chromium (Cr), and nickel (Ni). Growth curve analysis of five bacterial strains, based on best performance, were plotted at different levels of Cd, Cr and Ni stresses. Bacteria were challenged with high concentrations of Cd 100- 800, Cr 100-300, and Ni 100-500 mg/L. In batch biosorption experiments, the maximum adsorption value was obtained for Bacillus anthracis PM21 for the applied heavy metals at the optimum pH i.e., 8 for Cd, 6 for Cr, and 4 for Ni respectively. The xvii maximum adsorption values for Cd, Cr, and Ni were recorded after 60 min for all the bacterial strains i.e., PM21, PM22, PM23, PM24, and PM25. The maximum adsorption capacities (qe) of PM21 were observed 5-35 mg/g for Cd, 4-24 mg/g for Cr and 3-24 mg/g for Ni under 200 mg/L heavy metals. All the applied models supported the results of Cd and Cr biosorption with highest correlation coefficient (R 2 ) values as compared to Ni. The pseudo-second order kinetic model accurately represented the biosorption processes of biosorbents, indicating that heavy metal biosorption was primarily chemisorption. The participation of functional groups in metal ion adsorption was anticipated by FTIR surface characterization of bacteria. The scanning electron microscope (SEM) results revealed that application of 200 mg/L of Ni showed damaging effects on cell surface morphology. While in case of Cd and Cr (200 mg/L) the cells maintained their shape and size. The existence of CzcD gene responsive for Cd and Cr resistant, in four strains (PM21, PM22, PM24, and PM25), was confirmed by its PCR amplification. Based on plant growth promoting and biosorption potential strain PM21 was further applied on legume plant species, Sesbania sesban L. under heavy metals (Cd and Cr) stress to study seed germination of Sesbania sesban L. In third study, the seed germination test of Sesbania sesban L. was evaluated by the application of isolated strain PM21 in the absence and presence of cadmium and chromium. The PM21 inoculation to seedlings enhances seed germination (97.01%), length of roots (59.51%), length of shoot (5.03%), chlorophyll a (20%), b (16%) and total chlorophyll content by 18%, under Cd stress as compared to control. According to the findings, B. anthracis PM21 was able to withstand metal stress by maintaining antioxidant activity homeostasis, which had a beneficial impact on S. sesban L. growth and biomass. All the physiological, biochemical and growth parameters proposed that PM21 could better remediate Cd as compared to Cr. After germination experiment the strain PM21 was also applied to remediate Cd spiked soil with Sesbania sesban L. greenhouse study. The fourth study was conducted to investigate the plant growth promoting rhizobacterial assisted phytoremediation of cadmium (Cd). Phytoremediation potential of S. sesban L. was explored in Cd contaminated soil inoculated with Bacillus anthracis PM21. Application of B. anthracis PM21 significantly enhanced the studied plant attributes under normal and Cd stress conditions. Application of bacillus anthracis xviii PM21 increased morphological and physiological parameters as compared to uninoculated ones. Application of B. anthracis PM21 significantly (p≤0.05) enhanced Cd uptake in root, shoot translocation factor, bioconcentration factor 118.6, 73.4 mg/kg, 0.61, and 0.36 respectively. After assessment of data acquired in the four current studies, the analysis of comparison with the reported data was performed with the best of our knowledge. The data showed following parameters with significant increase which were bioconcentration factor, seed germination (%), root length, chlorophyll a, b. For the first time, plant growth promoting Bacillus anthracis PM21 have been evaluated for heavy metal tolerance potential. The tolerance level of Bacillus anthracis PM21 was reported as 800 and 300 mg/L cadmium and chromium, respectively. To best of our knowledge, Bacillus anthracis PM21 showed maximum tolerance towards Cd and Cr. So, its efficiency to tolerate dual metal stress under in-vitro conditions has been reported in our study. Furthermore, Bacillus anthracis PM21 was also applied for the first time in biosorption and phytoremediation that enhanced plant growth of Sesbania sesban L. under heavy metals stress at different levels.