Keywords

14.1 Introduction

Bacillus species represents one of the most important and dominant groups of microbes that exist in various environments with a high rate of survival in extreme and adverse environmental conditions (Islam et al. 2019). The gram-positive or facultative gram-negative spore-forming bacteria can survive for a long time under unfavourable environmental conditions (Radhakrishnan et al. 2017), and spore formation is one of the most important characteristics of Bacillus spp. The aerobic endospore-forming bacteria are ubiquitous in agricultural systems due to possession of certain physiological traits associated with their survival which include production of a multilayered cell wall, production of endospores that are stress-resistant, secretion of antibiotics, peptide signal molecules, and extracellular enzymes (Gardener 2004). The cell-wall-degrading substances (protease, cellulase, chitinase, glucanase, hydrogen cyanide, and lipopeptides) from Bacillus spp. damage the pathogenic organisms including bacteria, fungi, viruses, nematodes, and pests to control their populations (Radhakrishnan et al. 2017). Bacillus spp. also have great potential for applications in agriculture, industry, and medicine and are a good source of important metabolites and enzymes of various biotechnological interests. As an example, it was shown that B. thuringiensis H-14 is able to kill mosquito larvae within 24 h of treatment with the lowest concentration of spore-crystal suspension (0.05 mg/L) (Fun et al. 2016).

There is an increasing interest in Bacillus spp. on the aspects of agricultural and environmental biotechnology due to increasing demand for food production‚ recovery of degraded soils as well as the development of biofertilizers and biopesticides (Nascimento et al. 2020). The unfavourable biotic and abiotic stimuli affect normal plant metabolism, suppressing the growth and yield of plants, however, the stress factors on crops are mitigated by Bacillus-induced physiological changes (Radhakrishnan et al. 2017). Under unfavourable environmental conditions including heavy metal stress, Bacillus produces important substances such as siderophores and exopolysaccharides, which prevent the movement of toxic ions and adjust the ionic balance and water transport in plant tissues (Radhakrishnan et al. 2017). Bacillus can live both outside and within plant tissues, facilitating plant growth and development as well as protection from harsh environmental conditions through several mechanisms including N2 fixation, production of indole acetic acid (IAA), siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Additionally, phosphate and potassium solubilization and production of exopolysaccharides show antagonistic actions against pathogens and other pests. There are many bacterial species that are being applied as plant growth-promoting bacteria (PGPB ) to crop plants; however, members of the Bacillus group are more favoured for commercialization as PGPB due to some features including their ability to produce the heat and desiccation-tolerant endospores, which help to maintain high cell viability and prolong the shelf life of the bacteria in the carrier formulations (Akinrinlola et al. 2018). Bacterial colonization of roots provides a nutrient source, and the plants on the other hand, receive bacterial metabolites and other stimuli that enhance growth and stress resistance (Hashem et al. 2019). In this relationship, the microsymbiont (e.g. B. subtilis ) will form a thin biofilm layer on the root surfaces for long-term bacterial colonization of the host plants (Hashem et al. 2019). Additionally, the rhizosphere of the host plants is also heavily colonized by beneficial bacterial populations. Nowadays, plant growth-promoting Bacillus spp. are gaining prominence as biofertilizer , biopesticides, and bioremediator of certain mineral elements that are toxic to crop plants (Amir et al. 2005; Fun et al. 2016; Mia et al. 2010; Salwani et al. 2012; Tang et al. 2020). Salwani et al. (2012) revealed that the beneficial diazotrophic microsymbionts of leguminous cover crop Mucuna bracteata were not only from the conventional Alphaproteobacteria class (e.g. Brevundimonas sp.) but were also from the Betaproteobacteria class (e.g. Achromobacter sp. and Burkholderia sp.) and the Gammaproteobacteria class (e.g. Stenotrophomonas sp.). The authors also successfully obtained and identified Bacillus sp. from the root nodules of the host plants.

Heavy metal may be found naturally in the soils or added to the soils through various anthropogenic activities such as exploitation of mines and smelters, fossil fuel combustion, metallurgical and electronics industries, indiscriminate waste disposal, and military training. Additionally, the agricultural practices involving excessive application of inorganic fertilizers and chemicals for optimum crop improvement are also contributing towards the escalated accumulation of heavy metal in soils (Kapahi and Sachdeva 2019; Oves et al. 2016). Examples of heavy metals which are contaminating soils and water bodies include As, Cd, Pb, Hg, Cr, Co, Cu, Hg, Ni, Zn, Se, U, Mn, and Ni (Weissmannová et al. 2019). The continuous release of heavy metals into the environment severely affects soil and water quality. Despite the fact that waste water may serve as an important source of essential nutrients for plants, many risks can be attributed to the use of such waste water for crop irrigation due to presence of toxic contaminants including heavy metal (Khalid et al. 2018). Many heavy metal are toxic even at very low concentrations, and some (e.g. As, Cd, Cr, Hg, Ni, Cu, Pb, and Zn) are not only cytotoxic but also mutagenic and carcinogenic in their nature (Dixit et al. 2015).

Heavy metal contaminated soils are generally deficient in essential nutrients, and if the contaminated soils are used for crop production, there is a huge risk of metal being transferred into food chain at higher concentration. It can cause severe human health problems (Oves et al. 2016). Certain heavy metal is essential for plant growth and health at certain concentrations. For example, Co is required in nitrogenase enzyme as metal activator for N2 fixation both in legume and non-legume crops, and Mo is needed as catalytic centre of many enzymes namely nitrate reductase. However, when the elements exceed the required quantities, they may cause serious injury or even lead to death of plants (Oves et al. 2016). Biological methods for remediation of heavy metal known as bioremediation are considered most eco-friendly, cost-effective, reliable, and have no adverse effects to the environment (Dixit et al. 2015). There is an increasing interest towards studying the role of microorganisms in biotransformation and detoxification of heavy metals and the production of plant growth promoting substances by them under stress conditions (El-Meihy et al. 2019; Nayak et al. 2018). Understanding various mechanisms of metal accumulation and plant growth promotion has numerous biotechnological implications for bioremediation of heavy metal and crop productions in heavy metal contaminated soils. Mitigating the effects of heavy metal and their subsequent remediation on plants is one of the hot topics of environmental research nowadays. Application of Bacillus species in this area of research has received considerable attention (El-Meihy et al. 2019; Li et al. 2019; Tang et al. 2020) probably due to the high rate of adaptability and survivability of the species under extreme environments (e.g. heavy metal stress tolerance and their multiple beneficial mechanisms of actions) (Radhakrishnan et al. 2017). However, very few study reports are available on the application of Bacillus in bioremediation of heavy metal in plants. Most of the relevant studies were carried out on the physiological mechanisms of plant growth promotion or heavy metal tolerance of the individual Bacillus spp. . Rhizobacteria viz. Bacillus spp. (B. megaterium, B. cereus, and B. pumilus), isolated from Ludwigia octovalvis, were capable of absorbing As and can be good candidates for bioremediation of toxic effects of that element on plants (Titah et al. 2018). More studies on heavy metal tolerant and plant growth promoting Bacillus and other bacteria to harness their potentials as bioremediation or phytoremediation agents are therefore still needed. In this chapter, potentials of Bacillus spp. on promoting growth of associated selected host plants and their ability to remediate heavy metal are discussed.

14.2 Production of Plant Growth Promoting (PGP ) Substances by Bacillus Species

14.2.1 Production of PGP Substances by Individual Bacillus Species Under Culture Conditions Supplemented with Heavy Metal

Some of Bacillus spp. namely B. sphaericus strain UPMB10 are able to produce phytohormone like auxin and gibberellin which resulted in increased root growth in banana and oil palm under hydroponics and pot culture conditions (Mia et al. 2010; Amir et al. 2005). A large body of studies reflected that Bacillus species can produce plant growth promoting substances even under heavy metal stress conditions (Wu et al. 2019). Rizvi et al. (2019) also stated that heavy metal tolerant B. subtilis strain BM2 was able to synthesise variable concentrations of indole acetic acid (IAA) when cultured under different concentrations of Pb and Ni. However, the highest concentration of IAA was produced by the bacterial strain (BM2) when cultured in metal free medium (51.6 μg/mL), which kept declining with increasing concentrations of the added metal. At 400 μg/mL Pb and Ni, IAA secretion was reduced by 58 and 47%, respectively. Despite the toxicity and inhibitory effects of the metal, the strain maintained reasonable amount of IAA production potentiality. The strain was also able to produce substantial amount of ACC deaminase and siderophore when cultured in media supplemented with high concentrations of metal. However, the siderophore production by the strain was not detected on CAS agar with metal supplementation but detected under liquid medium with the metal supplementation. The cause of siderophore not detected in CAS agar but in liquid medium with metal supplementation remains unknown, but likely due to the presence of HDTMA (hexadecyltrimethyl-ammonium bromide) in the solid agar (Rizvi et al. 2019).

Bacterial IAA induce root elongation and development to overcome heavy metal toxicity and improve plant growth, while ACC deaminase, on the other hand, metabolizes the ethylene precursor ACC, thereby alleviating the effects of ethylene stress due to the metal and helps in plant survival under stress conditions (Mishra et al. 2017). Siderophore produced by Bacillus and other plant growth promoting bacteria is one the most important substances that can directly alleviate heavy metal toxicity by binding/chelating heavy metal ions and increasing the supply of iron to plants (Nayak et al. 2018). Therefore, maintenance in the production of plant growth promoting substances by Bacillus spp. under heavy metal stress could play an important role in improving growth of the host plants in addition to reduce the toxic effects of the metal on the plant.

14.2.2 Production of PGP Substances by Bacillus Species in Association with Plants

Nitrogen is an important essential nutrient which is required for growth and development of plants, taken up as NO3-, NH4+, and N2 (via biological nitrogen fixation process) (Bellogín et al. 2014). Atmospheric nitrogen fixing bacteria form symbiotic association on the roots of plants where they convert nitrogen into form that can be used by plants, a process mainly restricted to leguminous plants. However, several non-symbiotic bacteria have been recognized including many Bacillus spp. as free living nitrogen fixers in many plants of agronomic importance. Various species of Bacillus were reported to fix atmospheric nitrogen which include B. cereus, B. marisflavi, B. megaterium, Paenibacillus polymyxa, and P. massiliensis (Ding et al. 2005; Halim et al. 2016; Nayak et al. 2018; Tang et al. 2020). Others were able to solubilize phosphates like B. cereus, B. thuringiensis , B. megaterium, B. safensis, P. cineris, and B. subtilis (Akinrinlola et al. 2018; Babu et al. 2013; Huang et al. 2020; Zaidi et al. 2006). Phosphorus, which mostly occurs in the soil in the form of non-soluble compounds and thus not always available for plant consumption (Bellogín et al. 2014), is converted to soluble form that can be absorbed and utilized by plants in the process of phosphate solubilization. Besides that, PGPB inoculation can also stimulate and increase uptake of other nutrients such as K, Ca, Fe, Cu, and Zn which may be achieved through acidification of the rhizosphere (e.g. by production of organic acids), and in general, decrease in soil pH increases solubilization of nutrients (Bellogín et al. 2014). Some Bacilli have iron-chelating properties through the production of siderophores that are known to solubilize iron in the rhizosphere from minerals and organic compounds (Radhakrishnan et al. 2017). The siderophores produced bind insoluble Fe3+ and reduce it to soluble Fe2+, which can then be utilized by plants.

14.2.3 Role of Phytohormone (IAA) and Enzyme (ACC Deaminase) in Mitigating the Heavy Metal Stress in Plants

The IAA production by plant-associated bacteria plays an important role in plant-bacterial interactions. IAA synthesized by beneficial bacteria can increase the number of root hairs and lateral roots as well as the total root surface area, leading to an enhancement of root exudation and mineral uptake from the soil (Kong and Glick 2017). Number of studies have also suggested that PGPB producing IAA may play essential role in improving metal phytoremediation, however, most did not provide definitive proof of the direct involvement of the IAA (Kong and Glick 2017). In a previous study, two PGPB strains B. paralicheniformis YSP151 and Brevibacterium frigoritolerans YSP40 isolated from Brassica juncea in a Pb-contaminated mine soils were used to inoculate similar host plants grown in a metal-contaminated soil (Yahaghi et al. 2018). Results from the trial showed enhanced growth and Pb uptake by the inoculated host plants and both bacterial strains were reported to have high IAA producing ability. Multiple plant growth promoting traits may together be responsible for survival and enhanced metal tolerance of the host plants grown in heavy metal contaminated soils. Some bacteria can stimulate plant growth either by synthesizing more IAA or by degrading excess synthesized IAA when it is detrimentally higher than normal levels (Kong and Glick 2017).

Increase in ethylene synthesis is mostly associated with various environmental stresses such as heavy metal stress, extreme temperatures, drought, high salinity, organic pollution, insect damage, radiation, wounding, and various pathogens including viruses, bacteria, and fungi. Much of the plant growth inhibition that occurs due to environmental stresses is the result of increased levels of stress induced ethylene, which affects the plant’s response to the stressor (Kong and Glick 2017). Some PGPB have great potential to decrease ethylene production that in turn enhance plant growth. This potential is due to the production of enzyme ACC (1-Aminocyclopropane-1-Carboxylate) deaminase. ACC is the precursor to ethylene synthesis and ACC deaminase enzyme hydrolyses ACC into a-ketobutyrate and ammonia (Ashraf et al. 2017). The ammonia generated add to the nitrogen source of the plants. ACC deaminase producing PGPB can facilitate plant growth and development by converting the ACC into α-ketobutyrate and ammonia, thereby reducing the levels of plant ethylene and providing some protection against growth inhibition caused by heavy metal and other stress factors (Kong and Glick 2017).

14.3 Bacillus Species and Heavy Metal Transformation, Detoxification, and Mobilization

Bacteria are known to enhance plant growth and survival under heavy metal stress conditions due to their ability of consuming and converting complex waste into simple non-toxic products/compounds (Tiwari and Lata 2018). Some heavy metal tolerant bacteria are also involved in enzymatic oxidation or reduction of toxic metal to a non-toxic or less toxic forms. Application of Bacillus spp. into heavy metal-contaminated soils can reduce the deleterious and toxic effects of heavy metal on growth of crop plants. For example, the Cr and As resistant B. firmus strain TE7 isolated from tannery effluent was able to reduce Cr(VI) to Cr(III) and oxidize As(III) to As(V) (Bachate et al. 2013). It reduced 100 mg/L Cr(VI) and oxidized 150 mg/L As(III) within 60 hours in nutrient broth and 10 hours in minimal medium. The hexavalent chromium [Cr(VI)] is the most toxic form of chromium, and arsenite [As(III)] the most toxic form of arsenic, and thus, their respective reduction and oxidation have great environmental importance as they affect their toxicity and mobility (Bachate et al. 2013). Furthermore, two B. cereus strains (B. cereus D and 332) were recently shown to have high Cr tolerance and reduction ability, and B. cereus D achieved 87.8% Cr(VI) removal in 24 h (Li et al. 2020). According to the authors, Cu (II) significantly increased the removal rate of the Cr (VI). Inoculation of B. cereus WSE01 was reported to increase the level of leaf enzymatic activity in Myriophyllum verticillatum, which, on the other hand, mitigate the effects of oxidative damage caused by reactive oxygen species resulting from heavy metal stress. Li et al. (2019) found that metal-resistant B. thuringiensis HC-2 was able to reduce the concentrations of Cd and Pb by extracellular adsorption and increasing the pH of the solution. Treatments of metal contaminated substrates with B. thuringiensis HC-2, biochar, and biochar combined with B. thuringiensis HC-2 significantly reduced water-soluble Cd and Pb concentrations by 34–56% and 31–54%, respectively and also increased the pH and NH4+ concentration in solution, in comparison to the values in a control.

14.4 Physiological and Biochemical Mechanisms of Heavy Metal Tolerance of Bacillus

The PGPB may contribute in reducing metal phytotoxicity through biosorption and bioaccumulation (Ma et al. 2016). Metal biosorption by bacteria comprised of two steps, which are passive and active biosorption (Ma et al. 2016). Passive biosorption of metal usually by living and dead or inactive cells take place in the cell wall due to number of metabolism-independent processes. Here, metal ions are adsorbed rapidly to the cell surface by reactions between metal and functional groups on the cell surface. Metal binding mechanisms like ion exchange, complexation, coordination, sorption, chelation, and precipitation may be involved. Active biosorption (bioaccumulation), on the other hand, is referred to the uptake of metal by living cells through a much slower active metabolism-dependent transport of metal into bacterial cells. In heavy metal detoxification, studies carried out using different species, B. licheniformis NSPA5, B. cereus NSPA8, and B. subtilis NSPA13, indicated a significant level of Pb biosorption, with B. cereus having the maximum of 87–90% (Syed and Chinthala 2015). The negatively charged functional groups such as hydroxyl groups, phosphate groups, carbonyl groups, etc. that are present in biomolecules of microbial cell wall surfaces bind readily to heavy metal ions (Ojuederie and Babalola 2017). One of the most essential constituent in bacterial cells having ion sequestration capability is exopolysaccharide (EPS) (Ojuederie and Babalola 2017) mainly composed of complex high molecular weight organic macromolecules. Exopolysaccharides are known to protect bacteria against environmental stresses such as heavy metal toxicity.

Other abiotic and biotic stress factors with adverse effects on growth and survival of crop plants are mitigated by Bacillus-induced physiological changes, which include the activation of antioxidant and defence systems (Huang et al. 2020), regulation of water transport, nutrient uptake, and enhancement of photosynthetic pigments (Babu et al. 2013), leading to increased crop tolerance and productivity.

14.5 Molecular and Genetic Basis for Plant Growth Promotion and Heavy Metal Tolerance Ability of Bacillus Species

Bacillus association triggers plant resistance and immunity against different stresses (including heavy metal stress) by altering stress-responsive genes, proteins, phytohormones, and related metabolites (Radhakrishnan et al. 2017). Studies on genomic analysis of species of Bacillus have revealed rich genetic elements involved in important plant growth promoting and heavy metal tolerance activities. For example, the genome of B. megaterium STB1 possess genes related to rhizosphere colonization, xenobiotic degradation, pathogen antagonistic activities and several other genes for multiple stress resistance (Nascimento et al. 2020). Genes associated with different plant growth promoting activities are found in many species of Bacillus under heavy metal stress. Ding et al. (2005) selected 29 isolates in a study aimed at identifying the possible nitrogen-fixing Bacilli from plant rhizospheres based on their ability to grow on nitrogen-free medium, out of which seven had nifH gene belonging to Bacillus and Paenibacillus genera. It was the first report of nitrogen fixation in B. marisflavi and P. massiliensis and the first of the nifH gene from B. megaterium and B. cereus. Also, the whole genome of B. aryabhattai AB211 was sequenced, with main focus on genomic elements related to plant microbe interaction (Bhattacharyya et al. 2017). Genome comparisons between the strain AB211 and other related strains of B. aryabhattai revealed about 3,558 genes which were conserved among all the genomes, with most genes involved in plant growth promotion activities found within core genes of all the genomes used for comparison. The findings showed possible common plant growth promoting traits shared among the strains of B. aryabhattai. Functional annotation of genes in the B. aryabhattai strain AB211 revealed the presence of many PGP genes which include those responsible for phosphate solubilization, siderophore production, exopolysaccharides production, and IAA production, most of which were experimentally verified in the study. Earlier findings by Halim et al. (2016) also proven that Paenibacillus durus ATCC 35681 is a free-living nitrogen-fixing bacterium, and the complete genome of the strain was successfully sequenced too. Interestingly, the isolate can also be found in a symbiotic relationship with plant roots.

Similarly, genes for the resistance, mobilization, detoxification, or transformation were detected in many Bacillus species. For example, genomic analysis of B. megaterium STB1 revealed abundance of genes for heavy metal resistance and transport, which include those encoding arsenate (arsC) and chromate (chrR) reductases and several others for Zn, Cd, Cu, Co, Mn, Ni, Cr, and As (Nascimento et al. 2020). One of the arsenate reductase genes in the genome occur in a cluster that contain other arsenate transport and resistance related genes. BLASTn analysis showed that the cluster is rare, and that close homologs were only found in B. weihaiensis Alg07 chromosome and B. oceanisediminis 2691 pB01. These bring in new insights into the capabilities and roles of Bacillus spp. as potential and important plant growth promoting bacteria with ability of not only improving plant growth but also enhancing the tolerance and survivability of the host plants under heavy metal stress conditions.

14.6 Enhancement of Plant Growth and Heavy Metal Tolerance of Plants in Association with Different Species of Bacillus

Representatives of plant-beneficial bacteria are widely spread among gram-negative and gram-positive bacteria, with Pseudomonas and Bacillus attracting main attention (Borriss 2014; Radhakrishnan et al. 2017). Despite the great progress in Pseudomonas research, its commercial use in agriculture is unfortunately limited due to difficulties in preparing stable and long-living bioformulations compared to Bacillus (Borriss 2014). Bacilli are increasingly and interestingly used commercially in agriculture to enhance yield of crops and reduce use of harmful agrochemicals (Ahmad and Saghir 2010; El-Meihy et al. 2019; Minaxi et al., 2012). When the plant growth promoting attributes of Bacillus sp. RM-2 were tested in both laboratory and field conditions, the isolate significantly increased growth (P<0.05) and yield parameters and nutrients uptake of cowpea plants (Minaxi et al., 2012). Metal tolerant B. subtilis BM2 was able to mediate the phytotoxic impact and significantly reduced Ni and Pb uptake in winter wheat and also improved plant growth (Rizvi et al. 2019). The BM2 strain was able to increase the grain yield significantly by 49% and 50% under 870 mg/kg Ni and 585 mg/kg Pb, respectively. The exceptional ability of the strain to produce plant growth promoting substances like siderophore, IAA, ammonia, and ACC deaminase under the metal stress condition might have played important role in achieving the overall performance and enabling continuous growth and survival of wheat even under the metal stress condition (Rizvi et al. 2019).

The Bacillus spp. are as well employed in the field of phytoremediation aimed at improving the uptake of heavy metal by hyperaccumulator plants (Babu et al. 2013; Ndeddy Aka and Babalola 2016; Tang et al. 2020). Recently, B. cereus strain WSE01, that can tolerate up to 1500 mg/L Mn, was shown to increase the growth and leaf enzymatic activities in Myriophyllum verticillatum under 400 mg/L Mn stress condition. The bacterium was also able to increase the Mn content in the stems and leaves of the inoculated plants by 36.4% and 54.7% , respectively, compared to non-inoculated plants (Tang et al. 2020). In a study to assess bacteria with potential to enhance growth and metal accumulation in hyperaccumulator Alnus firma (Babu et al. 2013), the bacterium identified as Bacillus thuringiensis GDB-1 had better capacity and increased significantly the biomass, chlorophyll content, and accumulation of As, Pb, Cu, Ni, and Zn in the A. firma seedlings. A summary of different species of Bacillus with plant growth promoting and heavy metal tolerant activity applied for growth enhancement and bioremediation of heavy metal in plant is presented in Tables 14.1 and 14.2.

Table 14.1 Plant growth enhancement and bioremediation of heavy metal in crop plants as influenced by plant growth promoting Bacillus species
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Table 14.2 Influence of Bacillus species on growth enhancement of hyperaccumulator plants and phytoremediation potentials of soils contaminated with heavy metal
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In our recent study, one of the most promising and potential heavy metal tolerant plant growth-promoting isolate, identified as CCB-MBL5001 Bacillus cereus 2M1, was tested for its ability to alleviate metal stress and improve the growth of rice seedlings under Pb stress. Sterilized rice seeds (treated with or without 2M1 inoculation) were sown in sterile petri plates before uniformly germinated seeds were transferred aseptically to test tubes containing Yoshida medium supplemented with 0, 100 and 150 mg/L Pb. For both inoculated and non-inoculated seedlings, all growth parameters were noted to decline as affected by increasing concentrations of metal tested. However, better improvement in plant growth parameters were observed for seedlings inoculated with the bacteria compared to the control (non-inoculated, supplied with similar Pb concentrations) (Table 14.3). On the effects on photosynthetic pigments, the bacterial inoculated treatments significantly increased chlorophyll a, chlorophyll b, and total chlorophyll contents in the rice seedlings despite the occurrence of high metal stress (Fig. 14.1). Increase in the chlorophyll contents due to bacterial inoculation was not only found under Pb stress but also under non-stressed (control) conditions. Toxicity of heavy metal can affect photosynthesis by causing distortion in the ultrastructure of chloroplast, preventing the synthesis of photosynthetic pigment in chlorophyll content and enzymes of Calvin cycle. To assess the extent of oxidative stress and degree of damage caused by the heavy metal (Pb), the electrolyte leakage was estimated as presented in Fig. 14.2a. Stress resulting from the tested metal was shown to cause oxidative stress in the leaves of the host plants, which increased in response to increasing concentrations of the metal. However, there was significant decrease in the electrolyte leakage in 2M1 inoculated plants, which is a clear indication in the stability of the membrane . The % EL was decreased by 58% under the highest concentration of Pb, and the lower percentages of EL in the inoculated rice plants proved metal stress alleviation by the microbe to the plant. Plants are always being exposed to several stress factors in the field which can include heavy metal, that can affect their growth, development, and productivity. The adverse effects generally induce the accumulation of reactive oxygen species (ROS), which can cause severe oxidative damage including electrolyte leakage to plants. The balance between the production and detoxification of ROS is usually sustained by enzymatic and nonenzymatic antioxidants. The catalase (CAT) and superoxide dismutase (SOD) enzymes activities determined in the leaves of rice seedlings were higher in plants inoculated with 2M1 compared to their uninoculated counterparts (Fig. 14.2b and c). It was also observed that the inoculated controls without any heavy metal had the least activities. Increase in CAT and SOD activities by bacterial inoculation indicated strong response towards coping with oxidative stress generated by exposure to the heavy metal.

Table 14.3 Growth parameters of rice seedlings inoculated with locally isolated heavy metal tolerance B. cereus 2M1 under Pb stress condition
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Fig. 14.1
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Chlorophyll content in rice seedlings inoculated with locally isolated heavy metal tolerant B. cereus 2M1 after 15 days of growth under Pb stress and aseptic conditions

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Fig. 14.2
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Electrolyte leakage (a) CAT activity (b) and SOD activity (c) in rice seedlings inoculated with locally isolated heavy metal tolerant B. cereus 2M1 after 15 days of growth under Pb stress and aseptic conditions

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14.7 Conclusions and Future Perspective

Bacilli have multiple beneficial traits, which mediate better growth and development of crop either directly or indirectly through their different plant growth-promoting activities and heavy metal tolerance enhancement. This has great application towards growth enhancement and bioremediation of heavy metal in crop plants. Information made available in this chapter are testament of great potential of Bacillus spp. in growth enhancement and bioremediation of heavy metal in crop plants. However, most of the experiments were carried out under control conditions (lab or pot experiments), with paucity of information from field trials. Though the Bacillus group is one of the most commercially exploited bacteria in the agrobiotechnology industry, its potential has still not been realized sufficiently, and thus, the emphasis should be towards translating the relevant technologies from laboratory to the real world situation in the field (Saxena et al. 2019). More Bacillus-plant physiological studies are required to have better understanding of the Bacillus spp. mediated mitigation mechanisms against heavy metal stresses in plants.