Introduction

Heavy metals are a group of metal or metalloids that are toxic to animals and human being at even lower concentrations. They tend to accumulate in a living organism i.e., plants and animals, so their uptake is ultimately a severe threat to human health. Heavy metal contaminated soil adversely affect the growth and development of plant and microorganisms1,2, which results in a reduction of crop productivity3.

Among various heavy metals, lead (Pb) has become a significant soil contaminant. Although Pb is a non-essential element, it gets absorbed by the crop plants and inhibits plant growth4. A large portion of Pb in the soil may come from fertilizers and automotive exhaust5. Furthermore, anthropogenic activities are also playing an imperative role in the buildup of Pb contamination in air, soil and water6. Plants uptake Pb from the soil solution by roots, and it gets accumulation in an insoluble form within the roots7. Higher Pb contamination in the soil causes low nitrogen assimilation in plants8, reduces the rate of seed germination and alterations in plant water relations9. Carotenoid, chlorophyll contents, carbon dioxide, assimilation rate and photosynthetic rate are also reduced in plants due to Pb exposure4. However, Pb transportation is usually limited from roots to other parts of the plant10. Casparian strip present in endodermis is the main barrier to lead transport across the endodermis into vascular tissue11.

So far, various strategies have been examined by many research groups to mitigate Pb toxicity in plants1,12. However, the reports suggested that the use of activated black carbon biochar is largely effective in reducing heavy metals induced stress in crops. The use of biochar to absorb organic contaminants and heavy metals in the soil is a promising and low-cost solution to heavy metal toxicity 13,14,15. Biochar is gaining the attention of the scientists16 as it can immobilize heavy metal and reduce their bioavailability to plant17. Activated carbon biochar is an appropriate organic amendment for the alleviation of heavy metals induced stress in plants due to its high absorption ability for metallic ions. It has been well documented that the microporous structure, ion exchange capacity, and active functional groups of biochar and play an imperative role in decreasing the mobility and bioavailability of heavy metals18. Furthermore, the use of compost as an organic amendment also enhances the productivity of crops. Application of compost facilitates rhizobacterial proliferation, improves soil aggregation, water holding capacity, and pH when applied in the soil19.

In addition to compost and biochar, augmentation of plant growth-promoting rhizobacteria (PGPR) also produce a wide variety of molecules, which improves plant growth and productivity20,21,22,23,24,25,26,27,28. These PGPRs increased the production of phytohormones or other molecules that protect plants from biotic and abiotic stress, increases mineral nutrition, modulating ethylene levels in plants and production of volatile organic compounds20,29. Furthermore, PGPRs also promotes beneficial symbioses and degrades the xenobiotic to protect the plants29,30.

Spinach (Spinacia oleracea L.) is an essential dietary vegetable. It plays a vital role in the supply of micronutrients and providing potassium, iron, folic acid, magnesium and manganese, and vitamins, i.e., K, C, B2, A31. Spinach has high antioxidant activity, mainly related to the presence of flavonoids, which is a major constituent of water-soluble polyphenols32. High omega-3 fatty acids, vitamins (E & B6), and dietary fiber found in spinach are essential for the improvement, regulation, and maintenance of the tissues in humans33. However, spinach is a very good accumulator of metals, especially Pb34.

That’s why current study was conducted with aim to examine the combined effects of ACC deaminase producing rhizobacteria and compost mixed biochar (CB) regarding immobilization of Pb in spinach cultivated in artificially induced Pb-contaminated soil. We hypothesized that combined use of ACC deaminase producing rhizobacteria and CB could be a more effective strategy over the sole application for the improvement in spinach growth under Pb stress.

Results and discussion

Soil pHs

One-way analysis of variance between different treatments shows a significant (p ≤ 0.05) decrease in soil pH value as compared to control. It was observed that PGPR1 and CB have a significant (p ≤ 0.05) main effect on soil pH (Fig. 1), and a significant ordinal interaction was found between PGPR1 and CB (Fig. 1A). Inoculation of PGPR2 and CB do not have either their significant main effect or their interaction but the interaction was ordinal for soil pH (Fig. 1B). Application of CB remained significant regarding the decrease in soil pH as compared to the control. It was observed that PGPR2 also differed significantly from control for decreasing the soil pH. Treatment CB + PGPR2 remained statistically alike with PGPR2 and CB but differed significantly as compared to the control (Fig. 1). No significant change was noted over control in the soil pH where PGPR1 and CB + PGPR1 were applied. However, Maximum decrease of 2.0% in the soil pH was noted than control where CB was applied as an amendment. The reduction in the soil pH by applying CB occurred due to organic secretions of PGPR and low pH of compost as compared to biochar and soil (Table 1). The presence of microbes also secretes organic acids which play an imperative role in the solubilization of immobilized nutrients and decrease in pH of rhizosphere 35. Furthermore, decomposition of organic material i.e., compost also releases acidic compounds in the soil 36. Enrichment of humic acid in the rhizosphere by application of compost is another allied reason for a decrease in soil pH 37. In addition to above, the presence of water-soluble carbon compounds in compost are readily degradable by microbial acidic secretions which also contribute in decreasing the pH of soil 38.

Figure 1
figure 1

pH values of soil treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for soil pHs.

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Table 1 Pre-sowing analyses of soil and organic amendments.
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Soil ECe

One-way ANOVA showed that different treatments remained significant (p ≤ 0.05) for the decrease in soil ECe value over control. The result shows that PGPR1 and CB have a significant (p ≤ 0.05) main effect on soil EC (Fig. 2), and a significant ordinal interaction was found between PGPR1 and CB (Fig. 2A). Similarly, PGPR2 and CB show a significant main effect on the soil ECe (Fig. 2), with ordinal interaction (Fig. 2B). Addition of CB remained significant regarding the reduction in soil ECe over control. Inoculation of PGPR2 also remained significant from control for the decrease in soil ECe. It was noted CB + PGPR2 remained statistically alike with PGPR2 but differed significantly from control. For reduction in soil ECe, PGPR2 was significantly different as compared to PGPR1 (Fig. 2). No significant change was noted over control in the soil ECe where PGPR1 and CB + PGPR1 were applied. However, a maximum decrease of 17.6% in the soil ECe was noted from control where CB + PGPR2 was applied as an amendment. The results of the current study contrary to the other documented results regarding biochar and soil ECe. The reduction in the soil ECe in the current study might be due to high oxidation of biochar when applied by mixing in compost. Inoculation of PGPR2 might also speed up the oxidation of CB. Application of biochar significantly increases soil cation exchangeability39. Higher cation exchange capacity (CEC) increases the accumulation of ions in the rhizosphere that increases the soil ECe. However, for improvement in the soil CEC slow oxidation of biochar is a necessity40. In addition to the above, growth promoting PGPR increases root surface area. This improvement in the surface area of roots facilitates the plants for the uptake of nutrients41.

Figure 2
figure 2

Ece value (dSm−1) values of soil treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B) for soil ECe (dSm−1).

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Soil organic matter (OM)

Application of different treatments remained significant (p ≤ 0.05) for an increase in OM value over control. Inoculation of PGPR and CB have significant interaction on soil OM (Fig. 3). Significant ordinal interaction was found between CB and PGPR2 (Fig. 3A). Furthermore, PGPR1 and CB also have a significant (p ≤ 0.05) ordinal interaction for soil OM (Fig. 3B). Application of CB significantly enhanced the organic matter in the soil over control. Inoculation of PGPR2 also performed significantly better than control in improving the soil OM. It was noted CB + PGPR2 also remained statistically alike with PGPR2 but differed significantly from control for the improvement in soil OM (Fig. 3). Inoculation of PGPR1 and addition of CB + PGPR1 did not differ significantly for the soil OM over control. Maximum decrease of 41.2% in soil OM was noted from control where PGPR2 was inoculated as an amendment. The improvement in the soil OM was due to better proliferation of PGPR2 and high organic carbon contents of compost mixed biochar (Table 1). Low level of OM in PGPR1 inoculated soil might be due to poor proliferation of PGPR1. Biochar is an activated form of carbon. It is produced at high temperature and limited or no oxygen that causes carbon sequestration 42. An application of biochar increases the soil aggregation that plays an important role in the soil OM buildup43,44,45. Furthermore, biochar indirectly promotes the soil microbial activities, biomass and growth46,47. In addition to the above use of compost was another important factor for enhancing the soil organic matter. Recently it has been documented that the application of organic amendments i.e., compost organic matters significantly affects the soil organic on a long term basis48.

Figure 3
figure 3

OM (%) values of soil treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for soil OM (%).

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Soil N, P and K concentration

One-way ANOVA showed that treatments remained non-significant in improving the soil N (Figs. S1, S2) and P (Figs. S3, S4) contents but significant for K. It was noted that main effect of PGPR and CB (Fig. 4) were also significant for K concentration. However, non-significant ordinal interaction was found between PGPR1 and CB (Fig. 4A) as well as PGPR2 and CB (Fig. 4B). The application of CB + PGPR2 remained significant for improvement in soil K concentration over control. No significant change was observed among CB + PGPR2 and PGPR2; however, PGPR2 also differed significantly as compared to control for K concentration in the soil. The addition of CB in soil was also significantly different from control for K concentration in the soil (Fig. 4). No significant change was observed among control, PGPR1 and CB + PGPR1 for K concentration in the soil. The maximum increase of 34% in K concentration of the soil was noted where CB + PGPR2 was applied as compared to control. The increase in K concentration of soil was due to the presence of K in compost and biochar. In sole inoculation of PGPR2, solubilization of K by organic secretions might be the major cause of a significant increase in soil K concentration. Application of biochar decreases the leaching losses of nutrients49. High surface area and ion exchangeability of biochar make it most suitable amendment for improving the soil fertility status50,51. Furthermore, organic acids i.e., oxalic acid, tartaric acid, citric acid, malic acid, and succinic acid secretions of PGPR decrease the soil pH and chelate K by producing siderophores that increases its bioavailability to the plants52,53. Compost is enriched with the mineralized form of K which is water extractable and governs the soil fertility status54. Improvement in the root physiology by the application of organic manure combined with biochar also improves the nutrient’s availability to the plants55.

Figure 4
figure 4

Soil K (%) values of soil treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for soil K (mg kg−1).

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Root and leaves fresh and dry weight

One way ANOVA showed that treatments were non-significant for leaves fresh (Figs. S5 and S6) and dry (Figs. S7, S8) weight but significant for root fresh (Fig. 5) and dry (Fig. 6) weight under Pb stress. Significant (p ≤ 0.05) main effect (Fig. 5) but non-significant ordinal interaction was observed between PGPR1 (Figs. 5A and 6A) and PGPR2 (Figs. 5B and 6B) with CB for root fresh and dry weight respectively. It was noted that CB + PGPR2 differed significantly for root fresh and dry weight over control. No significant change was noted among CB, CB + PGPR2 and PGPR2; however, PGPR2 also differed significantly as compared to control for the root fresh and dry weight. The addition of CB in the soil was also significantly different from control for improvement in root fresh and dry weight under Pb stress. Furthermore, the sole inoculation of PGPR2 was significantly different for improving root fresh and dry weight from PGPR1 (Figs. 5 and 6). No significant change was noted from control where PGPR1 and CB + PGPR1 were applied for root fresh and dry weight. The maximum increase of 47 and 31% in the root fresh and dry weight was noted where PGPR2 was inoculated as compared to control, respectively. Lead is one of the heavy metal pollutants which is not essential for the plant growth and remains accumulated in the roots. The higher amount of Pb decreases the root growth, thus induces negative effects on the plants. In the current study, increase in the dry and fresh weight of the leaves and roots of the spinach crops might be due to the siderophores production, phosphate solubilization and by providing the systematic resistance against heavy metal stress through plant growth-promoting rhizobacteria56,57. Secretion of indole acetic acid by PGPR improves the elongation of roots58. According to Mohite59 IAA promotes the growth of adventitious roots that play an important role in the uptake of nutrients. Biochar and compost application increase the plant biomass production due to the improvement in the uptake of nutrients in the plants and/or soil physicochemical properties60,61. The high porosity of biochar makes it an important organic amendment that decreases the losses of nutrients62.

Figure 5
figure 5

Fresh root weight (g) values of spinach treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for fresh root weight (g) of spinach plant.

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Figure 6
figure 6

Dry root weight (g) values of spinach treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for dry root weight (g) of spinach plant.

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N, P and K concentration in plant

One-way ANOVA showed that treatments did not differ significantly for N (Figs. S9, S10) and P (Figs. S11, S12) concentration but remained significant (p ≤ 0.05) for K concentration in the plants. Inoculation of PGPR1 showed significant main effect whereas CB does not have a significant main effect, although there was a significant interaction between CB and PGPR1 (Fig. 7A). It was observed that PGPR2 showed non-significant while PGPR1 showed significant, ordinal interaction with CB (Fig. 7A,B). Application of CB and PGPR2 differed significantly for K concentration in the plants over control. No significant change was noted among CB, CB + PGPR2 and PGPR2; however, CB + PGPR2 and PGPR2 also differed significantly as compared to control for K concentration in the plant (Fig. 7). In addition, treatment CB + PGPR2 remained significant as compared to CB + PGPR1 for K concentration in the plants. No significant change was noted from control where PGPR1 and CB + PGPR1 were applied for K concentration in the plants. The maximum increase of 10.5% in K concentration was observed where CB was inoculated as compared to control, respectively. The improvement in K concentration of the plant might be due to the plant growth promoting rhizobacteria which promoted the uptake and availability of the nutrients by recycling63, solubilization64 of nutrients and siderophores production56. Besides, the imperative role of plant growth-promoting rhizobacteria, the BC, has a high water holding capacity, ion exchange capacity, high surface area that make it an effective amendment for enhanced uptake of water and nutrients in the plants65,66,67. Depending upon the feedstock, biochar itself carries a significant amount of mineral nutrients68. The application of compost also increased the nutrient status in the soils which consequently increased the uptake of nutrient in the crops61. According to Schulz et al.69, composted biochar significantly increased total organic carbon (TOC) that plays an imperative role in the uptake of nutrients. Danish and Zafar-ul-Hye24 suggested that the performance of PGPR for nutrients uptake in the crops can be enhanced when they are applied in combination with timber waste biochar70.

Figure 7
figure 7

Plant K value (%) values treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for plant K value (%).

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Chlorophyll and Pb concentration in plant

One-way ANOVA showed that treatments did not differ significantly for chlorophyll contents (Figs. S13, S14) and Pb concentration in leaves (Figs. S15, S16). However, the effect of treatments was significant (p ≤ 0.05) for Pb concentration in the roots of plants (Fig. 8). It was observed that PGPR and CB do not have significant interaction whereas PGPR show significant main effect (Fig. 8A) for Pb concentration in roots. Sole application of CB + PGPR2 differed significantly for less uptake of Pb in the roots as compared to control. No significant change was noted among CB and PGPR2; however, CB and PGPR2 also differed significantly better as compared to control for less uptake of Pb by the plants’ roots. Inoculation of PGPR2 was significant as compared to PGPR1 for decreasing Pb concentration in the plant’s roots (Fig. 8). However, PGPR1 and CB + PGPR1 did not differ significantly as compared to control for Pb concentration in the roots. Maximum decrease of 43% in Pb concentration was noted where CB was inoculated as compared to control, respectively. Under heavy metal toxicity, production of ethylene is significantly increased in the roots that induced adverse effects on the growth of plants. Production of ACC deaminase breaks this endogenous stress generating ethylene into α-ketobutyrate and ammonia that mitigate heavy metal stress 71,72. Similar, kind of improvement was also noted by Zafar-ul-Hye et al.73 when they inoculated wheat while ACC deaminase was producing PGPR under toxicity of heavy metal. Furthermore, application of compost provided energy to rhizobacteria and improved the transfer of oxygen which played an important role in the immobilization of metallic ions in the soil74. Song and Greenway75 argued that binding of heavy metals with exchange sites reduced their bioavailability to plants. Active function groups on the surface of biochar adsorb heavy metals electrostatically, causing their immobilization in the soil through cation exchange mechanism76. Presence of CO3−2 and hydroxides on biochar surface also played an imperative role in the immobilization of divalent heavy metals77,78. Through organic chelating agents and secretions, PGPR changes the redox potential in the rhizosphere. Change in redox potential and acidification of rhizosphere by PGPR decreases the bioavailability of heavy metals to the plants79,80.

Figure 8
figure 8

Root Pb value (µg g−1) of spinach treated with CB, PGPR1, PGPR2 and their combination with CB. Means of three replicates having different small letters express significant differences at p ≤ 0.05 compared with Duncan’s test. Interaction graph of PGPR1 and CB (A); PGPR2 and CB (B), for plant K value (%) for Root Pb value (µg g−1) of spinach plant.

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Conclusion

It is concluded that the application of PGPR2 i.e., Bacillus amyloliquefaciens with compost mixed biochar can alleviate the Pb toxicity by the improving the nutrients uptake The combined use of ACC deaminase producing PGPR2 Bacillus amyloliquefaciens and compost mix biochar can improve the Spinacia oleracea L. root growth and K uptake in the plants under Pb stress. However, more investigation is needed at a field level to introduce combined use of Bacillus amyloliquefaciens and compost mix biochar as an efficacious amendment against mitigation of Pb toxicity in the crops.

Materials and methodology

For the production of biochar, the waste material of vegetables and fruits were collected from the SabziMandi, Multan. These waste materials were air-dried under suitable sunlight for two weeks until the moisture content remained < 15%. The waste material was chopped into small pieces, then filled in the electric pyrolyzer and heated at 450° C temperature for 120 min under anaerobic condition. The pyrolyzer was allowed to cool down at an average temperature. The prepared biochar sample was removed from the pyrolyzer, grinded and further allowed to pass through 2 mm sieve. Biochar was applied at 0.5% (5 g kg−1) soil according to the treatment plan in the pots. The Prepared compost, manufactured by Buraq Agro Chemicals, Industrial State Area, Multan was applied at 0.5% (5 g kg−1) soil according to the treatment plan. Two rhizobacterial strains previously identified as PGPR1 Alcaligenes faecalis and PGPR2 Bacillus amyloliquefaciens were obtained from the Soil Microbiology and Biochemistry Laboratory, BZU, Multan. The respective inoculum of rhizobacteria was prepared in Dworkin and Foster (DF) media present in 250 ml Erlenmeyer flasks81. Each flask containing DF media was inoculated with respective strains for 72 h at the laboratory temperature. The spinach seeds were inoculated (inoculum density 0.5 nm) with respective bacterial inoculum (5 ml 100 g−1 seeds) and mixed with sterilized clay, peat and sugar solution at the time of sowing.

A pot experiment was conducted in the warehouse at the experimental farm of the Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan. The effect of biochar, compost and plant growth-promoting rhizobacteria was evaluated by designing the experiment on the spinach grown on lead-contaminated soil. Six treatments were arranged with three replications in Complete Randomized Design (CRD). The pre-experimental soil characteristics are provided in Table 1. Each pot was filled with 7 kg of soil. A basal dose of K2O, P2O5 and N, was applied at the rate of 130, 90 and 110 kg per hectare, in the form of SOP, DAP and urea, respectively for the spinach crop82. The total of phosphate and potash fertilizer were applied at the time of sowing while N was applied in three splits. After two weeks of germination, Pb stress was applied artificially. Lead sulphate (PbSO4) was applied for introducing 250 mg Pb kg−1 soil83.There were six treatments as i.e. control, PGPR1 (Alcaligenes faecalis), PGPR2 (Bacillus amyloliquefaciens), compost + biochar (CB) (1:1), CB + PGPR1 and CB + PGPR2.

Bouyoucos hydrometer method was applied for sand, silt and clay percentage determination84. Soil saturated paste was prepared by adding distilled water in a plastic beaker containing 300 g of soil sample. The pH meter was calibrated using a buffer solution of strength 4, 7 and 9.2 pH. Then the pH of saturated paste was determined on it. Already prepared soil saturated paste for pH was extracted by a vacuum pump to get a clear extract. Electrical conductivity (EC) meter was calibrated with KCl solution (0.01 N), and ECe of the sample was measured in dS m−1. Walkley–Black85 method was used to measure the organic matter in the soil. Kjeldahl’s distillation method was used to measure the total N in the soil. For that H2SO4with digestion mixture (FeSO4: K2SO4: CuSO4, 1:10:5) was used for digestion. The evolved NH3 was absorbed in boric acid solution in a receiver having methyl red and bromocresol green indicators. The content was titrated with H2SO4, and N was calculated in percentage86. Sodium bicarbonate solution was used to extract 5 g soil sample by shaking on a mechanical shaker. 8 ml of colour developing reagent and 2 ml of aliquot was taken in 50 ml flask. Extractable soil phosphorus was measured at 880 nm wavelength with spectrophotometer87. Ammonium acetate method was used to extract soil potassium. The extractable soil potassium was calculated by using a flame photometer. In a 50 ml conical flask 10 g soil sample was taken. The soil sample was extracted with 20 ml of 0.01 M CaCl2 + 0.01 M TEA + 0.005 M DTPA solution. Extracting solution pH was adjusted up to 7.3 and shook for 120 min88. The atomic absorption spectrophotometer was used for measuring the Pb concentration89.

The samples were weighted two weeks later when they had been dried up by an electrical balance. Nitrogen was analyzed by using 2 ml of digested plant sample, 1 ml of 0.17 mm Na nitroprusside in 1% (w/v) phenol, 1.0 ml of a solution containing 0.125 N NaOH, 0.25 M Na2HPO4 in 0.03% (w/v) NaOCl. Test tube containing the above solution was mixed vigorously on a vortex mixer and incubated in a water bath at 37 °C for 30 min. The absorbance was measured at 625 nm on a spectrophotometer. For determination of phosphorus, the plant samples were digested in an acid mixture of HNO3 and HCIO490. The ammonium vanadate and ammonium molybdatewere added as colour developing reagents in the aliquot. The phosphorus was determined at 470 nm wavelength by using spectrophotometer after calibrating with P standards91. For determination of potassium, the digested sample aliquot was fed to the flamephotometer92. The reading of filtrate and standards was noted on atomic absorption spectrophotometer.

Data were analyzed by following standard statistical procedure 93. One way and two-way ANOVA were applied by using SPSS 20. Treatments were compared using Duncan's test for differentiation at p ≤ 0.05.