Root- Nodulating Ensifer Adhaerens Ks23 of Pisum Sativum L. In Optimisation of Cadmium Biosorption Using Rsm Based Approach

The Cadmium tolerance by root nodulating bacteria Ensifer adhaerens KS23 inhabiting in Pisum sativum L. var. Arkel revealed linear relationship with inorganic salt cadmium sulphate (CdSO 4 ) upto 200 μg/ml, corresponding to growth and survival in solid as well as liquid Yeast Extract Mannitol (YEM) medium with LC50 value of 107.2 μg/ml and LC95 of 184.5 μg/ml. The results of phylogenetic and morpho-physiological analysis exhibited the genus E. adhaerens. KS23 was found to be the most promising among all the 20 isolates. The increase in Glutathione S-transferase (GST) activity by KS23 was 9.7 fold under Cd stress. Wherein, P and F values were <0.05 and 26.54 respectively and predicted r 2 value of 0.8192 and adjusted r 2 value 0.8908 were reasonable (i.e. <0.2) of the Box Behnken design. The data showed that 81.24% cadmium bio-removal achieved at pH 6.0, 30°C and 168 h of incubation while supplementing the YEM medium with 25 μg/ml cadmium. Further, its effect on plant growth and development exhibited due to production of IAA, secretion of siderophores, phosphate solubilisation and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity by E. adhaerens KS23. In addition to inherent PGP attributes, Cd tolerant E. adhaerens KS23 played dual role of biosorption of cadmium and upsurge in growth promotion of P. sativum which may provide a new root-nodulating bacterium inhabiting in P. sativum cultivated at high altitudes of Himalayan region. closely linked to H + unavailable for other cations. Based on the results, pH 6.0 for further


Introduction
Due to the fact that biostimulation, bio fertilization, biocontrol and sometimes by biosorption are multitask phenomenon and in this scenario, bacteria of a group of PGPR impart tolerance to biotic and abiotic stresses including heavy metals. Cadmium (Chi et al., 2020;Wang et al., 2020), Klebsiella pneumoniae and Citrobacter freundii for lead exhibited their potential in promoting the plant growth in heavy metals contaminated soil (Al-Garni, 2005). Soil can be contaminated by many polluting sources, such as composts, pesticides, emissions from municipal waste sites, metal smelting industry, etc. and leached out several heavy metals such as zinc (Zn), cadmium (Cd), lead (Pb) and copper (Cu) (Stylianou et al., 2007). Soil acts both as a reservoir as well as temporary storage of metal-ions, therefore, the heavy metals estimation is utmost important. Few root nodulating bacteria inhabiting in legumes observed in bioremediation and biosorption of cadmium and other heavy metals and pesticides (Sathvika et al., 2018;Edulamudi et al., 2019). The available literature revealed that inspite of its broad ecological niche (Katiyar et al., 2021), investigations on biosorption potential of cadmium by rootnodulating Ensifer adhaerens, even at low concentrations of Cd caused signi cant harm to the food chain ecosystem . However, other alternatives for their removal such as biosorbents by living or dead biomass, agricultural waste or industrial byproducts, are other options (Wang et al., 2009). The physiochemical degradation of these heavy metal pollutants is a cumbersome and onerous affair and also not eco-friendly. Root colonizing bacteria have the capacity to accumulate further amounts of metal in the host plant roots and limit the entry of it to other parts of the plant (Nagata et al., 2015). It is therefore, the removal of heavy metls by microbes has gained a great deal of interest.
Response Surface Methodology (RSM) is commonly used in rhizosphere bio-engineering the biosorption of iron, lead and cadmium to newly isolated bacteria using a Box-Behnken design (Choińska-Pulit et al., 2018). The root-nodulating bacteria procured from standing crop of Pisum sativum L. var. Arkel cultivated in eld soil contaminated with heavy metals with cadmium as dominant contaminant of soil. The plant growth promotion attributes such as IAA, siderophore, HCN production, production of ACC-deaminase and assessment of heavy metal (Cd II) biosorption (Zhang and Shu, 2006) by the selected bacterial isolates and determination of Glutathione S-tranferase activity and optimization of variables, such as pH, temperature, initial concentration, these parameters have been aimed for maximum biosoprtion (Actual Removal E ciency) of Cadmium (Cd) and their removal in farmer's eld soil.

Materials And Methods
Procurement of root-nodulating bacteria and culture conditions The root nodulating bacterial isolates KS09, KS23 and KR16 were procured from Department of Microbiology, Grukul Kangri (Deemed to be University), Haridwar-249404 Uttarakhand (India). The isolates were obtained from our previous study (Katiyar et al., 2021) and maintained on Yeast Extract Mannitol Agar supplemented with congo red (crYEMA) medium at 4 °C as described by Dubey and Maheshwari, (2012).

Enrichment and effect of cadmium concentration
The cadmium-bacteria mixed culture was centrifuged at 3500×g for 5 min, and the supernatant was collected in a clean tube and acidi ed with 1 M HNO3. Finally, the concentration of cadmium ions remaining in the supernatant was measured at 228.8 nm of wave length using a UV-Vis spectrophotometer (Shimadzu-1601) in triplicates. The cadmium removal e ciency was calculated by the following equations: where Co and Ce was the initial concentrations and equilibrium concentration of cadmium (mg/l), respectively.

Physio-chemical soil analysis
Atomic Absorption Spectrometry (Thermo sher iCE 3300 AAS) was used in determining the content of heavy metals in the previously digested soil samples from pre-sowing and post-harvest soil of farmer's eld (Srinagar, Garhwal, India). The nitrous oxide, acetylene gas, and compressor were all xed, and the compressor was turned on, with the liquid trap blown to remove any trapped liquid. The AAS control and the extractor were both turned on. Purifying wire was used to clean the slender tube and nebulizer piece, and an arrangement card was used to clean the burner opening. The light was switched on and the cathode beam was changed to hit the arrangement card's goal zone. The ne was positioned in a 10 ml graduated chamber lled with deionized water and the rate of yearning was calculated.
Based on the absorbance obtained for the unknown sample, the different metal concentrations in the sample solution were calculated from the calibration as given by Radu and Diamond (2009).

Effects of treatments on vegetative parameters
The isolates KS09, KS23 and KR16 were selected for seed bacterization (Weller and Cook, 1983). Healthy seeds of P. sativum var. Arkel were washed with distilled water and air-dried. The seeds were then sown in earthen pots of six inches height and eight inches diameter. The four treatments were given, Control: sterile seeds; T1: seeds + KS09; T2: seeds + KS23; T3: seeds+ KR16. After thirty days of sowing (30 DAS), early vegetative parameters such as root/shoot length and fresh and dry weight of root/shoot were recorded.

Glutathione S-transferase activity (GST)
The estimation of GST produced was determined as given by Habig et al. (1974). The sample was homogenised in phosphate buffer (pH 6.5 @ 100 mM) and centrifuged at 9,000 g for 30 minutes. The absorbance was measured at 340 nm (Shimadzu UV-Vis 1601, Japan) wave length. It was measured by monitoring the reduction of GSH concentration at 412 nm after ts reaction with 1-chloro-2, 4dinitrobenzene (CDNB): GSH + C 6 H 3 (NO 2 ) 2 Cl (CDNB) →C 6 H 3 (NO 2 ) 2 GS + H + + Cl -. U is de ned as a unit of enzymatic activity, which reduces 1.0 µmol/1000 ml GSH per mg protein in one minute at 37 °C, after subtracting non-enzymatic reaction. The protein was quanti ed by the Coomassie blue colorimetric assay using bovine serum albumin as the standard. Concentration of glutathione Stransferase was expressed in units/ml protein.

Designing of experiment for biosorption studies
A set of 150 ml Erlenmeyer asks were used in the single factor test, which contained 50 ml of Yeast mannitol Broth with different metal concentrations. The effects of contact time (12-144 h), pH (2-10), temperature (15-40 °C), biomass dosage (0.002-0.016 g) and initial cadmium concentration (5-100 μg/ml) on the removal e ciency of cadmium were studied (Wang et al., 2014;Qasemi et al., 2018). The bacteria were harvested after respective time of culturing at different parameters for subsequent experiments.

Optimization of biosorption conditions
In order to accurately predict the optimum biosorption conditions for Cd by isolate KS23 and minimize the number of experiments, the Box-Behnken design (BBD) based on RSM was designed. The RSM consists of a set of experimental methods designed to the evaluation of correlation between a number of controlled experimental factors and obtained responses according to one or more selected criteria. Contact time (A), pH (B), and initial cadmium concentration (C) were screened as key factors affecting removal e ciency and the appropriate range of independent variables was determined. According to the BBD (Design Expert software, V.13.0, 2020) (Raymond and Montgomery, 2009), three levels and three factors were employed to determine the optimal biosorption variables to improve biosorption e ciency.

Identi cation and characterization of bacterial strain
On subjecting the selected isolate to molecular identi cation using 16S rRNA gene sequencing, the data revealed the strains to be Rhizobium leguminosarum KS09 (MW575402), Ensifer adhaerens KS23 (MW019954) and Rhizobium phaseoli KR16 (MW621971). A phylogenetic tree was constructed for each strain by using MEGA X (Kumar et al., 2018) (Supplementary Figure S1, S2 and S3).

Enrichment and effect of cadmium on isolates
After 7 days of incubation, the isolate was supplemented with varying concentrations of CdSO 4 at 28 °C. Minimum inhibitory concentrations (MIC) of E. adhaerens KS23 was calculated using probit analysis. The probit analysis revealed that LC50 value of KS23 against Cd was 107.2 μg/ml and LC95 value of KS23 against Cd was 184.5 μg/ml for a maximum concentration of CdSO 4 upto 200 μg/ml (Figure 1).

Physio-chemical soil analysis
Pre-sowing Soil analysis revealed that Cd and Mn present in the soil sample were signi cantly maximum in concentration in comparison to that of Cu, Cr and Pb, which were present in lower concentration (%) than that of Cd and Mn. Cd and Ni were present in amounts within the permissible limits (Table 1). However, there was a signi cant reduction in the concentration especially of Cd (II) which was well above permissible limit of 0.8 mg/kg (Saha et al., 2010). There is a decrease in cadmium concentration statistically from 4.84 μg/kg to 1.62 μg/kg, which provides evidence of Cd biosorption by isolate E. adhaerens KS23 and thus improved the quality of soil.

Effects of treatments on vegetative parameters
After thirty days of sowing the seeds with different treatments, the treatment T2 (seeds+ KS23) was found to be signi cant (p>0.05) in achieving effective enhancement in root/shoot length as well root/shoot fresh and dry weight. T2 showed a maximum increase of 34.83 % in root length, a 37.67 % increment in shoot length over control. Similarly, T2 also enhanced root fresh weight (34%), shoot fresh weight (37%), root dry weight (24%) and shoot dry weight (33%) over controls respectively. This shows that E. adhaerens KS23 is capable of enhancing plant productivity effectively (Figure 2 a-c).

Glutathione S-transferase (GST) activity
For the lowest concentration of Cd used in the previous tests, the GST activity was determined ( Figure 3). It is well understood that as the concentration of Cd (II) rises, corresponding to the activity of GST, but on further incubation of 144 h, the GST activity declined. E. adhaerens KS23 was the most effective showing the 9.7 fold increase in the GST activity, followed by KS9 and KR16 that caused 7.6 and 7.3-fold increase in GST activity(p<0.05) (Figure 4).

Effect of Temperature
Under the above optimal conditions for maximum cadmium biosorption viz.-contact time-116 h, biomass dosage 0.01 g/50 ml, cadmium initial concentration 25 μg/ml, pH 6.0 were observed ( Table 2). Optimization of temperature (15, 20, 25, 30, 40 °C), the removal e ciency was investigated and it was revealed that the removal e ciency increased corresponding to increase in temperature was recorded and on further incubation, decrease in activity was recorded. Taking into account of removal e ciency of 81.24%, the maximum removal e ciency occurred at 25 °C.

Effect of intial pH
The effect of pH (2, 3, 4, 5, 6, 7, 8, 9 and 10) determined, when the other experimental conditions were set as follows: contact time 120 h, biomass dosage 0.01 g, cadmium initial concentration 10 μg/ml and temperature 35 °C, cadmium adsorption by Ensifer adhaerens KS23. A slow increase in removal e ciency observed at lower pH but the removal e ciency linearly increased from pH 5 to pH 6. At pH 4, the removal e ciency maximally observed to be 48.23 %. At low pH, the cell surface sites are closely linked to H + ions, which is unavailable for other cations. Based on the results, pH 6.0 was applied for further investigations.

Effect of initial cadmium concentration
The initial cadmium concentration played an important role in the removal e ciency. At higher concentration of cadmium, bacterial growth declined from 125 μg/ml concentration to the complete inhibition at 200 μg/ml. In this work, different initial cadmium concentration 5, 10, 25, 40, 60, 80, 100 μg/ml were prepared when other experimental parameters were performed as follows: contact time 120 h, biomass dosage 0.012 g, pH 6.0, and temperature 25 °C. The removal e ciency reached the maximum of 81.24% at a concentration of 25 μg/ml.

Analysis of the Response Surface
These plots could help to understand better, both main and interaction effects of variables, such as temperature and pH value on cadmium removal e ciency ( Figure 5 [a-d]). At 25 μg/ml, the removal e ciency of Cd (II) decreased from79 % to 68 % with the increasing initial pH from 5 to pH 7, while the removal e ciency of Cd increased from 77 % to 79 % with the increasing of contact time from 48 h to 144 h. At pH 6, the removal e ciency of Cd (II) decreased from 72 % to 56 % with the increasing of initial concentration from 10 to 40 μg/ml and the removal e ciency increased from 79 % to 81 % with the increase of contact time from 48 h to 144 h. The removal e ciency of Cd decreased from about 81 % to 78 % with the increase in initial concentration of Cd from 10 to 40 μg/ml, while that of Cd increased from 72 % to 77 % slightly with the increase of pH 5.

Discussion
Sorption is a method of mass transfer by which a material is moved from the liquid phase to a solid's surface and/or chemical interactions are attached to the substance. Sorption may be used as a low-cost alternative to traditional processes due to the wide surface area, high sorption potential and surface reactivity of sorbents being the suitable candidates for environmental regeneration, restoration and recovery of heavy metals (Congeevaram et al., 2007;Bestawy et al., 2013;Andreazza et al., 2011). Biosorption is a passive adsorption mechanism in which heavy metal is oxidative and in cell surface elements, ions are passively adsorbed to that of (Ayangbenro and Babalola, 2017;Shamim, 2018).
Bacterial symbiosis with leguminous plants offers tolerance against various stresses such as drought, salinity, heavy metals and other contaminants to the host plant wherein remediation is primarily achieved through the processes of biosorption or bioaccumulation (Limcharoensuk et al., 2015). Interstingely, the root nodulating E. adhaerens has been reported in biosorption of polychlorinated phenyls (Chen et al., 2015) but heavy metals bioaccumulation and sorption by microorganisms such as rhizobia is still in infancy (Oves et al., 2017).
First we, identi ed the heavy metal tolerant bacterial strain E. adhaerens KS23 and optimised conditions (pH and temperature) for maximum bioaccumulation taking into account the toxic effects of heavy metals and the biosorption ability of soil microbiota. Strain E. adhaerens KS23, a characterized bacteria from contaminated soils despite the presence of Cd (II) in excess of permissible limits (Saha et al., 2010) proved to be tolerant to heavy metals toxicity upto 184.5 μg/ml as evident by MIC values (Figure 1). Many factors, such as metal-ion concentration, biomass volume, contact time and pH value, etc., affect the removal e ciency of heavy metals by microorganisms (Bueno et al., 2008). It can be used to design a study, build experiments, and models study the in uence of such factors on one or more dependent variables in order to clarify the variables' connection and selecting the optimal experimental conditions (Homayoonfal et al., 2015). Kotoky et al., (2019) elaborated that Glutathione-S-transferase (GST) activity is primarily responsible for heavy metal tolerance in bacteria, therefore, an increase in activity indicates positive biosorption of heavy metals by bacteria thus contributing to bioremediation. Similarly, the 9.7 fold increase in GST activity in our study reveals effective heavy metal biosorption by E. adhaerens KS23. The appropriateness of temperature directly affects the role of microbial adsorbents, so it is a very important factor affecting the removal e ciency of cadmium adsorption. In a similar study by Zhang et al. (2013), upto 80 % of Hg 2+ was adsorbed by Gst pm -4 from Proteus mirabilis when the pH was adjusted to 6.0 as also evidenced in E. adhaerens KS23 wherein, the maximum adsorption of Cd 2+ by Gst a mediated from E. adhaerens KS23 was 81%, respectively.
The quantity of transition metals accumulated within cells, due to the variable in genetic makeup of microbe and external physiological conditions, such as pH and temperature. In this regard, Banerjee et al. (2015) also observed that the maximum metal accumulation occurrs at pH 6 and temperature 35 °C, which were ideal conditions for microbial growth and development. In our analysis, E. adhaerens KS23 accumulated the most metal at pH 6 and 30 °C signi cantly (Table 4). Similar to our study on Ensifer adhaerens and Pisum sativum L. demonstrated a fascinating phenomenon, proving bene cial and defensive and majorly reduced heavy metal stress in polluted soil. Khan et al. (2009) found that legume symbiotic rhizobia can act as a heavy metal chelator as well as a plant growth promoter in a contaminated eld.
From the soil analysis, heavy metals contaminants in soil con rmed that the soil was contaminated by the heavy metals due to leaching from the nearby dumpsite and vehicular emissions. In such circumstances, high temperature in uence the growth of the microorganism by involvement of the catalytic reactions. The decrease in the removal e ciency was adversely affected to that of high temperature. In such situations, the functional groups and the uidity of the cell membrane showed maximum biosorption e ciency was recorded at 25°C by E. adhaerens KS23. These observations get support from previous studies; pH is the most important factor affecting the biosorption capacity of microorganisms for heavy-metals (Choińska-Pulit et al., 2018). The change of pH can not only affect the nature of cell surface charge, the protonation or deprotonation of functional groups and the permeability of cell membranes, but also the valence of cadmium in aqueous solutions (Kazy et al., 2006;Liu et al., 2016) but role of other factors cannot be ruled out.
The removal e ciency due to E. adhaerens gradually decreased corresponding to the increasing cadmium concentration. Previous studies had shown that at low ion concentration, the ratio of the moles of metal ions to the available surface area was low, and a large number of binding sites remained in the solutions (Leyva-Ramos et al., 2005). On the contrary, at high concentrations of Cd (II), lack of su cient free binding sites adversely affected the removal e ciency (Erkaya et al., 2014). Therefore, the center point of Cd (II) concentration chosen for further course of investigators and it was 25 μg/ml and yielded maximum biosorption by E. adhaerens KS23. On the other hand, high initial concentrations of metal ions could adversely affect microbial biomass (Kazy et al., 2006).
The 3D surface plots are graphical diagrams of regression equations showing two factors, while all other factors maintained at xed levels (Hadiani et al., 2018). In fact, one of the important parameters that affected the adsorption of soluble metal ions by various biosorbants is the pH of the biosorption medium (Farhan and Khadom, 2018;Isam et al., 2019). The pH changes had an effect on the competition of binding sites and the activity of functional groups on the microbial cell wall (Javed et al., 2019). Further, the potential of cationic metal biosorption increased with rising pH of the sorption system upto pH 7, but not in a linear relationship being decreased on further rise in pH 8. At very high pH and temperature, it is likely to precipitate of metal complexes (Guibaud et al., 2006), which also affected biosorption. Therefore, an increase in biosorption e ciency of root-nodulating bacteria E. adhaerens KS23 have dual role to play, thus alleviating stress caused by heavy metal Cd (II) to the soil and increases the growth and productivity of P. sativum L.

Conclusion
The study revealed an eco-friendly, cost-effective sorbent for heavy metals was demonstrated using RSM based approach. The PGP microsymbiont bacterium Ensifer adhaerens KS23 demonstrated a high potential for cadmium (II) adsorption. In this analysis, the effects of contact time, pH, temperature, dosage of biomass and initial concentration of Cd (II) on its adsorption by E. adhaerens KS23 were studied in single factor experiments by using RSM-based BBD in optimizing cadmium adsorption. A signi cant factor in the biosorption process has also been considered as a relationship between contact time and pH value. ANOVA analysis model (p < 0.02, r 2 = 73.35%) showed a su cient consistency between the observed and predicted values. E ciency of E. adhaerens KS23 under optimal conditions accounted for 81.249 %. Cd (II) removal e ciency demonstrated the model's accuracy. The in uence of temperature have a little impact on the e ciency of removal, which is of great practical importance, particularly in adverse conditions for the subsequent application of biological adsorbents to the natural environment. This study offers a potent and effective tool in the form of a bacteria (E. adhaerens). This has prompted to investigate the potentials of enhancing the biosorption of Cd (II) heavy metal accumulation in soil used for recovery of its fertility for rasising crops especially in those regions where heavy metal pollution is a matter of consideration.
Thus, the bene t of combining heavy-metals tolerant E. adhaerens KS23 may play a key role in host adaptation to a changing soil environment in contaminated soil.

Declarations
Author's Contributions PK executed the experiments and drafted the manuscript. SK and DKM planned, corrected and nalized the manuscript before submission. RCD checked and nalised the manuscript before submission.

Figure 2
Effect of treatments on vegetative parameters after 30 DAS (a) root and shoot length; (b) root and shoot fresh weight; (c) root and shoot dry weight.

Figure 4
Increase in GST activity of selected isolates under Cd stress increase in initial concentration of Cd from 10 to 40 μg/ml, while that of Cd increased from 72 % to 77 % slightly with the increase of pH 5.