Soil contamination and degradation are important issues from both environmental and agricultural points of view. The continual anthropogenic activities, extensive use of chemical fertilizers, pesticides and rapid industrialization are responsible for increasing environmental pollution (Aktar et al., 2009; Singh et al., 2015, 2017; Tripathi et al., 2016a). These activities are major causes of soil degradation and contamination and thus pose a great challenge for food safety and public health (Qishlaqi and Moore, 2007; Tripathi et al., 2012, 2015, 2016b). Soils of mining sites have extremely high percentage of heavy metals such as chromium, lead, cadmium, and arsenic which cause destruction of soil properties such as pH, nutrient content and quality (Ladwani et al., 2012; Hansda et al., 2014); thereby, significantly affecting the fertility of surrounding soil, associated microbial community, vegetation status as well as pose several health risks at different levels of food chain through a well-established process of bioaccumulation (Wuana and Okieimen, 2011; Upadhyay et al., 2016).

Heavy metals are the most dominant form of contamination found in the waste dump sites of mines and their remediation is considered to be a technically difficult task due to their recalcitrant nature (Wuana and Okieimen, 2011). There are a number of traditional and conventional technologies which are widely acceptable to remediate metal contaminated soils. However, these approaches are time consuming and usually generate complex secondary waste. In this context, our major concern is to reclaim and restore soil properties through implementation of situation-specific technique(s) that permit the best possible decontamination at the lowest potential cost with modest or no side effects. The application of plant–microbe interaction for the remediation of contaminated soil is an important and well-adapted technology (Hansda et al., 2014). Plant growth is regulated by soil fertility which influences the physical (e.g., soil structure), chemical (e.g., soil pH, organic matter, C: N ratio) and biological (e.g., microbial diversity in soil) properties of soil. On the other hand, most of the time, adverse conditions of degraded soils results in the destruction of aboveground vegetation. Rhizobacteria generally colonize root and enhance plant growth in degraded soils, as they can alleviate biotic and abiotic stress by releasing phytohormones (e.g., IAA, ethylene), solubilizing minerals and producing iron chelating compounds (e.g., siderophores) (Ahemad, 2015). Several studies have reported the utilization of plant growth promoting (PGP) rhizobacteria for the bioremediation of heavy metals including Bacillus sp., Pseudomonas sp. etc. (Samuel et al., 2013; Sobariu et al., 2016; Ndeddy Aka and Babalola, 2016).

Among various heavy metals, chromium (Cr) is one of the most common pollutants originating mainly from industrial effluents, mining practices, petroleum refining industries and use of pesticides (Oliveira, 2012). In nature, chromium mainly exists in trivalent [Cr (III) form and found naturally in environment. Hexavalent (Cr (VI)] is mainly released in environment through various anthropogenic activities and imparts toxic effects on microorganisms, human beings and soil fertility (Oliveira, 2012; Tripathi et al., 2012; Ahemad, 2015). It is well documented that bacterial composition is greatly affected by soil type, its characteristics and stress factors which restrict the bacterial growth in soil (Upadhyay et al., 2016). Only a few bacteria may adapt to these stressed environments and proliferate. The transition into this physiological state and recovery were found to be correlated with the environmental stress equivalent to pH, temperature and bioavailability of minerals and nutrients (Olszewska et al., 2016).

The behavior of micro-organisms toward Cr(VI) was studied previously which demonstrated the significant alleviative impact of many microorganisms, including strains of Pseudomonas, Enterobacter, Bacillus, and Shewanella (Samuel et al., 2013; Ahemad, 2015). Microorganisms were found to reduce hexavalent chromium by different mechanisms either by utilizing the hexavalent chromium as final electron acceptor or by secreting certain soluble enzymes (Samuel et al., 2013; Ahemad, 2015). However, the availability of efficient hexavalent chromium reducing microorganisms is a crucial requirement for the bioremediation of soils contaminated with hexavalent chromium. With this background, the present study is envisaged to isolate and select the most resistant bacterial isolate from the contaminated soils with potential to reduce hexavalent chromium. In addition, the bacterial growth and morphological modifications were examined against various Cr(VI) treatments using modern techniques so that it can be utilized further for the effective bioremediation in associated with the enhancement in plant growth promotion when grown in contaminated soil.

Contamination of soil with chromium can modulate the soil microbial diversity and metabolic activity. In addition, nutrient deficiency was also reported in metal contaminated soils which often causes stress for plants (Qishlaqi and Moore, 2007; Ladwani et al., 2012). Till now, several PGP rhizobacteria have been identified such as Azotobacter, Bacillus, Pseudomonas, Azospirillum, and Serratia for their potential to enhance plant growth by solubilizing minerals, producing iron chelating compounds (siderophores), phytohormones and biocontrol compounds and enhancing phytoremediation of metal contaminants (Hansda et al., 2014; Ndeddy Aka and Babalola, 2016; Sarathambal et al., 2017). Chromium [Cr(VI)] exerts toxic effect on microorganisms by different mechanisms. Chromium resistant bacteria can overcome toxic effects of Cr(VI) and have been utilized for bioremediation of contaminated sites (Samuel et al., 2013; Sobariu et al., 2016). It has been reported that several metal resistant PGP rhizobacteria enhance metal translocation and improve metal uptake efficiency (Hansda et al., 2014).

Isolation and utilization of PGP rhizobacteria for rhizoremediation of heavy metal contaminated soils have gained worldwide attention because it is an environmental friendly and cost-effective approach. Therefore, the current study deals with the isolation and characterization of chromium-resistant-PGP bacteria capable of reducing Cr(VI) to Cr(III). The PGP characteristics were screened and the potential PGP isolate was further screened for chromium resistance and tolerance potential. The reduction of Cr(VI) to Cr(III) was also studied and subsequently the morphological and physiological responses were checked to study the resistant pattern of bacterial strain in the presence of various chromium concentrations to evaluate its impact on bioremediation potential.

The results of the present study confirm those from previously reported literature on Bacillus sp. regarding its ability to produce PGP substances and promote plant growth (Shukla et al., 2012; Cherif-Silini et al., 2013; Ndeddy Aka and Babalola, 2016). Production of a plant growth regulator, such as IAA, is an important characteristic of PGP rhizobacteria (Saharan and Nehra, 2011). In the present study, production of IAA was reported by the test isolate and the concentration of IAA (Table 1) produced was in agreement with other studies (Karnwal, 2009; Shukla et al., 2012). However, the intrinsic potential of bacteria to produce growth hormone such as IAA in the plant rhizosphere greatly relies on the accessibility of precursors and uptake of microbial IAA by plant (Arshad and Frankenberger, 1991).

The potential of the bacteria to solubilize the insoluble tricalcium phosphate in vitro is an additional significant method for accomplishing plant growth promotion (Saharan and Nehra, 2011). The present study shows that the isolate MNU16 solubilizes maximum phosphate (285.13 ± 1.05 μg/ml) after 120 h of incubation, and with increasing incubation time, the pH of the medium declined to 4.1 from an initial pH of 6.4 as shown in Figures 1A,B. The result draws support from the previous studies which highlighted the solubilization of phosphate by producing several types of organic acids by different Bacillus species and other bacterial species along with the subsequent drop in medium pH (Shukla et al., 2012; Cherif-Silini et al., 2013). Besides, phosphate solubilization and IAA production, the isolate possess another important characteristic of PGP activity, i.e., ACC deaminase activity, in the bacterial isolates which are known to mitigate plant stress by reducing ethylene concentration in plants (Karthikeyan et al., 2012). ACC deaminase activity is an important property that diminishes plant stress caused by adverse environmental stresses such as presence of high metal concentration. ACC deaminase converts ACC, which serves as an ethylene precursor in plants, to ammonia and α- ketobutyrate and thus reduces the plant stress caused by higher ethylene concentration (Karthikeyan et al., 2012). A significant concentration of α- ketobutyrate was produced by the selected isolate in the present study (Table 1). It proves that bacteria possess the attribute of phytoremediation by facilitating plants to attain higher and dense roots in chromium contaminated soils.

Siderophore molecules exert both growth promotion and biocontrol mechanisms and have significantly high affinity for iron (Saharan and Nehra, 2011). Consequently, the low availability of iron in the surrounding environment of plant rhizosphere could suppress the growth of pathogenic microorganisms (Patil et al., 2014). In the present study, the bacterium isolated shows a highly orange halo zone in plate assay which confirms siderophore production. Therefore, it was studied quantitatively for estimation of siderophore, which was significantly lower than the values previously reported for PGP strains (Patil et al., 2014; Dutta et al., 2015).

The MIC of the bacteria for chromium was obtained by agar well diffusion method in Muller Hinton agar medium with Cr(VI) concentration ranging from 50 to 1000 mg/L and the bacterial isolate MNU16 was found to be highly resistant for chromium with a concentration of 900 mg/L (Supplementary Figure S1). In addition, the present isolate was also found to be resistant for various other metals (Supplementary Figure S1). The previous studies reported the tolerance range of chromium resistant microorganism from 100 to 4000 mg/L Cr(VI) (Thacker et al., 2007; Zhu et al., 2008). The isolated Bacillus sp. MNU16 falls within the above reported range of tolerance. From the molecular characterization and phylogenetic analysis of the test isolate, it was found that the test isolate MNU16 showed maximum similarity with the B. subtilis species (Figure 1C). Not many isolates from the genus Bacillus have been known to tolerate and reduce Cr (VI) (Ibrahim et al., 2011; Mangaiyarkarasi et al., 2011). This difference in resistance profile may be because of the variation in medium composition used for studying the chromium toxicity which influences the toxicity via ‘masking’ effect (Caravelli et al., 2008).

In the present study, B. subtilis MNU16 shows chromium reduction to a concentration of 13.23 mg/L (Figure 1D) from an initial concentration of 50 mg/L within 72 h of incubation. The reduction efficiency of Cr(VI) for Bacillus sp. MNU16 was evaluated and shown in Figure 1D and the percentage (%) reduction of Cr(VI) was recorded upto 75% by the bacterium Bacillus sp. MNU16 (Table 1). The results are in accordance with the previous study which also showed reduction of chromium by Bacillus sp. (Samuel et al., 2013) and the Cr(VI) reduction percentages lies within the previously reported studies (Maqbool et al., 2015). This inherent ability of many bacterial species to resist and reduce chromate both under field and laboratory conditions have been previously studied for remediation of chromium contaminated environments (Ibrahim et al., 2011; Maqbool et al., 2015). However, the exact mechanism that is responsible for the reduction of Cr(VI) to Cr(III) is not completely known, and various explanations have been given previously (Ahemad, 2015). The possible mechanism behind the conversion of Cr(VI) to Cr(III) can be either when chromate serve as a final electron acceptor to acquire energy, or the bacteria may secrete some waste product which can possibly reduce Cr(VI) to Cr(III) or by the secretion of several enzymes that might act on Cr(VI) and reduce its toxicity by converting it into Cr(III) (Ahemad, 2015).

Microorganisms when subjected to heavy metals become susceptible to the toxicity caused by reactive oxygen species. Due to the PGP and metal resistance properties, the isolates can be used to remediate metal contaminated soil. The PGP activities of microorganisms are one of the reasons behind the survival of microorganism in metal contaminated soils. The siderophore producing microorganisms have the potential to bind with metals present in surrounding and therefore enhance the bioavailability of metals in plant rhizosphere (Sarathambal et al., 2017). In general, the phytoremediation process of contaminated soils will enhance with the increase in plant growth and metal bioavailability (Ahemad, 2015).

Before studying the physiological response of Bacillus sp. MNU16 by FCM, the bacterial response in the presence of Cr(VI) was also studied in the liquid medium by studying the growth pattern of isolate under various chromium concentrations. The growth profile of Bacillus sp. MNU16 is shown in Figure 1E. It was clearly observed from growth profile (Figure 1E) that the absorbance at 300 mg/L chromium concentration was less which indicated that chromium showed highest toxicity at concentration 300 mg/L as compared to other concentrations. The toxic effect of metal was effective in the liquid medium (Shakoori et al., 2000) as the mobility of metals is comparatively more in liquid medium as compared to solid medium. The reduced growth observed in the presence of higher concentrations of Cr(VI) was attributed to the reduction of metabolic rate of bacterium by Cr(VI) (Pal and Paul, 2004).

The application of electron microscopy to examine the morphology of cells and modification in cell structure due to exposure to various contaminants can provide novel dimensions. The results obtained from TEM analysis of B. subtilis MNU16 demonstrated the allocation of FDP inside and outside cells which might be due to the exposure of hexavalent chromium treatments. Similarly, study conducted by Batool et al. (2014) reported similar pattern of FDP and cell disruption in cells of P. aeruginosa and Ochrobactrum intermedium treated with different concentrations of Cr(VI). Further, in the present study, the deposition of EDP inside the B. subtilis MNU16 might be due to the accumulation and intracellular reduction of Cr(VI) as shown in Figures 2C,D. The study of TEM also showed that the toxicity of Cr(VI) is maximum at 300 mg/L Cr(VI) concentration as shown in Figure 2D. The intracellular reduction mechanism of Cr(VI) was reported previously on the strains of Acinetobacter sp. and Shewanella oneidensis which explains the deposition of EDP inside the cells (Daulton et al., 2007; Srivastava and Thakur, 2007). Thus the presence of these EDP inside cells clearly revealed the modification mechanisms of Cr(VI) into Cr(III) in the form of hydroxyl and carboxyl groups (Bruins et al., 2000; Bencheikh et al., 2007).

The resistance pattern of the test isolate Bacillus sp. MNU16 was further studied by the flow cytometric technique in the presence of different chromium concentrations. FCM method has been previously studied for analyzing bacterial physiology when exposed to various stresses (Ionescu et al., 2015; Olszewska et al., 2016) as well as for screening antibiotic susceptibility in bacteria and fungi (Wright et al., 2006). This technique is more suitable for studying physical and chemical characteristics of bacteria due to its ability to rapidly and efficiently analyze large number of microorganisms with better accuracy. The current study reports the use of PI, an exclusion dye used for the analysis of cell viability and complexity by FCM analysis of B. subtilis MNU16, during chromium treatments to evaluate its effect on the membrane permeability and eventually on cell viability. The relative sizes and internal complexity of bacterial cells were estimated by flow cytometric analysis by estimating the forward scatter (FSC) and side scatter (SSC) of light, respectively, and shown in Figures 4A–I. The results showed that with increasing chromium concentration, a shift in FSC and SSC of bacteria was observed (Figure 4). The forward scatter (FSC) is affected by the size of bacteria and the intensity of scatter depends on the refractive index of the particle and flow medium; although, for particles having size in the range of bacteria were found to be correlated with the total volume of bacterial cell (Baatout et al., 2007). The side scatter of light (SSC) is proportional to cell granularity or internal complexity. The reflected and refracted light was measured at several interfaces of bacterial cell where alteration in refractive index was observed and also aids in detecting the presence of inclusion bodies generated due to the environmental stresses (Baatout et al., 2007).

The mean FL2 graph showing the relative fluorescence due to PI staining of B. subtilis MNU16 grown in the presence of various chromium concentration given in Figure 5B, describing the comparison of cell count and fluorescence intensity at various Cr(VI) concentrations. It is clear from the Figure 5C that high cell count and less fluorescence intensity was observed in the control sample where no stress was provided and eventually a decrease in cell count was recorded when growing the cells in the presence of different Cr(VI) concentrations.

The study shows that a significant population of bacterial species is present in the medium at high concentration of chromium which shows B. subtilis MNU16 has chromium resistance potential. The noticeable increase in PI fluorescence intensity with increasing Cr(VI) concentration (Figure 5) shows the enhancement in loss of membrane integrity with the increasing concentration of chromium. Furthermore, in the presence of chromium treatments, variation in cell death was observed by varying the concentration of chromium with the help of FM. The presence of maximum number of cells stained with PI showed the highest cell death in the sample which is correlated with the data obtained from the PI florescence of FCM as shown in Figure 5. These results are confirmed by the data obtained by florescence imaging of bacteria that gives fluorescence when PI intercalates with bacterial DNA which shows the increase in membrane permeability due to membrane damage.

Furthermore, the results obtained from the study confirm the fact that the most crucial target for any environment stress is cell membrane whether it is pH, heavy metal or temperature (Mangaiyarkarasi et al., 2011; Olszewska et al., 2016). This suggests that differences in properties of cell membrane can possibly be an essential factor in establishing the metal tolerance potential between various bacterial strains. On the other hand, the nature of Cr(VI) toxicity as a function of membrane composition and structure in determining tolerance of metal and other environmental stresses still needs to be clarified.

The main focus of the study was to isolate an efficient chromium resistant PGP rhizobacterium having the potential to tolerate chromium stress which can be utilized for bioremediation purpose. The selected B. subtilis MNU16 strain shows significant PGP characteristics highlighting resistance to high concentration of chromium and able to decrease the toxicity of Cr(VI) by reducing it to Cr(III), a less toxic form. The resistance pattern against chromium was studied by analyzing the cell viability percentage by FCM and FM. FCM presents the probability of performing both qualitative and quantitative evaluation established on the simultaneous measurements of structural and practical parameters of single cell. The study concluded that the isolated PGP rhizobacterial strain with significant Cr(VI) reduction potential provides new possibilities to design strategies for improving the efficacy of rhizoremediation (microbial assisted phytoremediation) of contaminated soils.

NU, KV, JS, RM, SS, MM, and DT designed experiments. NU, KV, RM, and JS performed experiments. NU, KV, VK, RR, DC, RM, and DT, analyzed data and wrote the manuscript. DT, DC, and SS critically evaluated the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.