Introduction

Globally, heavy metal pollution is a major issue which has seriously threatened the sustainability of environment. The toxic heavy metals emerging from sources like, industries, tannery, sewage wastes and other metal discharging industries have been found to adversely affect the density, diversity and physiological activities of soil microbiota (Xie et al. 2016). Among various heavy metals, cadmium, a highly lethal metal is widely used in batteries, for coating, electroplating, PVC stabilizers and as a component in alloy formation in various industries (Sharma et al. 2015). Due to lack of regulatory policy for controlled and safe discharge, cadmium is added to the environment via many anthropogenic activities like combustion of metal ores, burning of wastes and by the use of fossil fuels. However, after it accumulates in soils, Cd can be absorbed by plants leading eventually to the disruption of food chain and indirectly affects human health (Rahimzadeh et al. 2017). Chromium is yet another toxic metal which is used in plating, alloy formation, tanning of animal hides, for inhibiting water corrosion, textile dyes and mordants, pigments, ceramics, refractory bricks, pressure-treated lumber etc. The overuse and abuse of Cr has however hugely dented human health by acting as carcinogenic and mutagenic element (Oliveira 2012). Nickel which is even though present in very low concentrations in the environment has been found to cause serious human health problems when present beyond threshold concentrations. For instance, Ni can cause- (i) genotoxicity (ii) several types of carcinomas (iii) toxic impacts on the immune system and (iv) damage to other metabolically active tissues (Das et al. 2018).

Some of the distortive/destructive impact of heavy metals on microbes are-(i) alterations in cell surface morphology and growth behaviour (Rizvi and Khan 2017) (ii) cell membrane disruption (Chen et al. 2016) (iii) inhibition of enzyme activity (Alnuaimi et al. 2012) (iv) oxidative phosphorylation leading to lipid peroxidation (Mishra and Mishra 2015) and (v) denaturation of microbial proteins (Ayangbenro and Babalola 2017). However, with the advent of certain state of the art techniques such as SEM (Golding et al. 2016), EDX (Ackerman et al. 2016) and CLSM (Hao et al. 2013), it has now become possible to assess the finer details of metal toxicity to microbes (Gomes and Mergulhão 2017) and plants (Rizvi and Khan 2018) which a few years back were almost impossible. For example, the SEM reveals the morphological distortions while the EDX provides information on distribution of heavy metals within bacterial cells (Ramya and Thatheyus 2018). Similarly, the CLSM is considered a powerful tool which is used to assess the speciation/composition of metals and hence, the hazardous impact of toxicants on bacterial cells (Hao et al. 2013). However, microorganisms have developed various efficient mechanisms to endure the continuous and prolonged exposure to heavy metals. While surviving under metal stressed environment, metal tolerant microbes transform the toxic heavy metals to less toxic forms (Fashola et al. 2016) and adsorb/desorb (François et al. 2012) metals. Apart from these, the ability of bacteria to- (i) pump out metals and (ii) chelate and trap heavy metals by producing exopolysaccharides (EPS) are some other mechanisms adopted by microbes to detoxify and manage the contaminated sites (Haferburg and Kothe 2010). These features of metal tolerant bacteria are usually exploited to remove toxic metals from metal polluted environments. For example, the EPS among various active biomolecules secreted by a few bacterial species (Ates 2015) provides special protection to the producing organisms while growing under harsh environment (Nocelli et al. 2016). The EPS forms a complex by binding with the ionic forms of heavy metals and makes heavy metals inaccessible to microbes (Zhang et al. 2017). Thus, the toxicity of metals to microbes is restricted. Besides this, the EPS so generated by the bacterial cells also safeguard them from extreme environmental conditions like high temperatures, pH, starvation, and desiccation etc. (Gupta and Diwan 2017). Thus, secretion of EPS could be an adaptive feature which aids the bacterial communities to survive even in the environments heavily contaminated with metals. And hence, the metal chelating ability of the bacterial EPS is being largely employed for bioremediation purposes (Ojuederie and Babalola 2017). This strategy employed by metal tolerant bacteria could be a cost effective approach for metal clean up from contaminated soils.

The synthesis of metallothioneins (MTs) by microbes is yet another important strategy adopted by useful soil microflora to contain/restrain heavy metal toxicity. Metallothioneins (metal binding proteins) comprising of small polypeptide chains with approximately 30% cysteine residues have a strong affinity for metals and therefore, chelates heavy metals like Zn, Ni, Cu, Pb, Cd, Hg etc. through thiolate bonds of cysteine (Diopan et al. 2008). While binding with metals, the MTs provide protection to bacterial cells against any oxidative damage caused due to heavy metal toxicity and metal transportation and regulation (Adam et al. 2014). Since the metallothioneins (MTs) belong to a family of metal-binding proteins, they regulate homeostasis within the cells while simultaneously protecting the cells from deadly effects of metals and combating the stress developed due to the presence of superoxide radicals (Vignesh and Deepe 2017). Metallothioneins also very effectively bind and sequester toxic metals like Cd, Hg etc. Due to their incredible role in substantial mitigation of oxidative stress and protection of bacterial cells from the harsher impacts of metals, MTs have safely emerged as a key component of bacterial cells which allows them to survive actively under metal stressed environments while maintaining the overall regulation of homeostasis within the cells. Also, the metalloregulatory nature of MTs makes them efficient molecules that can attach/attract xenobiotic compounds and thus confer a shield around bacterial cells to thwart metal toxicity (Si and Lang 2018). The production of a brown to black pigment (melanin) by some bacterial strains for example A. chroococcum (Banerjee et al. 2014) is yet other mechanism by which the toxicity of metals to microbes could be reduced (Cordero et al. 2017). The melanins have been found to efficiently chelate the metal ions and while doing this, protect bacterial cells from various biochemical (El-Naggar and El-Ewasy 2017) and thermal stresses (Hu et al. 2015) including the heavy metal stress (El-Naggar and El-Ewasy 2017). Melanin pigment due to its ability to absorb a broad spectrum of electromagnetic waves can also provide protection to bacterial cells against ultraviolet radiations. Apart from these, melanin has been reported to safeguard bacterial cells from multiple chemical stresses and high temperatures (Rao et al. 2017). Melanins also possess a remarkable feature of chelating heavy metal ions which can be exploited for detoxification/removal of metal ions from metal contaminated sites (Thaira et al. 2018).

The secretion of EPS, MTs and melanin by metal tolerant bacteria and the ability of these biomolecules to make heavy metals unobtainable for uptake by microbes/plants (microbes assisted phytoremediation) has received little attention as a viable metal cleanup strategy. Considering such a huge gap in this bioremediation area and to test this hypothesis of metal removal strategy, a detailed and systematic study was designed to achieve the following specific objectives (i) to understand how cadmium, chromium and nickel affect bacterial cells employing SEM, EDX and CLSM and (ii) exploring the mechanistic basis of EPS, MTs and melanin in heavy metal removal from derelict environment.

Material and methods

Strain selection, characterization and metal tolerance

The bacterial strain CAZ3 was isolated from rhizosphere of chilli (Capsicum annum) grown in heavy metal contaminated soils. In order to test the ability of this strain to survive under metal stress and hence to determine the tolerance limit, the strain was exposed to 0–2400 µg mL−1 each of cadmium, chromium and nickel added to nutrient agar (NA) and nutrient broth (NB) medium. The metal amended plates and tubes were incubated in a bacteriological incubator (York Scientific Industries Pvt. Ltd. India) maintained at 28 ± 2 °C for 48 h. Strain CAZ3 exhibiting maximum tolerance to Cd, Cr and Ni was characterized morphologically and biochemically (Holt et al. 1994). Following this, the strain was identified to species level by 16S rDNA sequence analysis done by Macrogen, Seoul, South Korea. The two primers used were: universal primers, 785 F (5′-GGATTAGATACCCTGGTA-3′) and 907 R (5′-CCGTCAATTCMTTTRAGTTT-3′). Later, strain CAZ3 was identified to species level using BLASTn analysis, the resulting nucleotide sequence was submitted to GenBank sequence database and accession number was assigned. The evolutionary relationship of strain CAZ3 was traced by preparing a phylogenetic tree using MEGA 6.0 software.

Assessment of disparaging impact on bacterial strain and localization/distribution of metals by SEM and EDX

Cytotoxic damage and morphological changes were determined by growing bacterial cells in NB amended with 50 µg Cd mL−1and 100 µg mL−1 each of Cr and Ni. Furthermore, cells were visualized under SEM (model JSM 6510 LV; JEOL, Japan). For SEM analysis, two milli litre cultures of metal treated and untreated cells of strain CAZ3 were centrifuged at 10,000 rpm for 10 min. The cell pellets were washed twice with phosphate buffered saline (PBS) and fixed in primary fixative (2.5% glutaraldehyde and 2% paraformaldehyde) overnight at 4 °C. The pellets were again rinsed with PBS three times for 5 min. each. Following washing, the cell pellets were dehydrated using 30, 50, 70, 90 and 100% ethanol. Then, two micro litre suspensions of the dehydrated pellets were mixed with 8 µL of double distilled water on a glass cover slip and observed under SEM. The localization and percentage of heavy metals inside bacterial tissues was determined by EDX spectroscopy.

Bacterial cell death determined by confocal laser scanning microscopy (CLSM)

Bacterial cells grown in NB medium treated with (25, 50 and 100 µg mL−1 each of Cd, Cr and Ni) or without heavy metals (control) were visualized under CLSM (Model LSM-780, Zeiss, Germany) to assess bacterial cell death caused due to heavy metals. For this, bacterial cell pellets were washed three times with PBS for 15 min. and the pellets were dissolved in PBS. A-100 μL of bacterial cell suspension of metal treated and untreated cells was mixed with 10 μL of propidium iodide (PI) prepared in PBS (1 mg mL−1). The bacterial suspension mixed with the fluorescent dye was incubated for 10 min. at room temperature. The resulting suspension was centrifuged (at 5000 rpm for 10 min.) to remove the unbound dye, if any. Furthermore, the cell pellets were re-suspended in 500 μL of PBS and a thin smear was prepared on a glass slide. The samples were then observed for PI stained dead cells with an excitation/emission maxima of 493/636 nm for PI. Samples were prepared in dark to avoid photobleaching of the dye.

Impact of heavy metals on bacterial growth

The influence of heavy metals on growth was determined by culturing a 10 µL of strain CAZ3 into nutrient broth containing 0 to 400 µg mL−1 (at a dilution factor of two) of Cd, Cr and Ni. The growth pattern was observed in a 96-well micro titer plate, incubated at 28 + 2°C for 24 h. Following incubation, the growth was measured at 620 nm at a 2 h interval and growth curve was constructed.

Heavy metal removal mechanisms adopted by metal tolerant bacterial strain

Extraction of exopolysaccharides (EPS)

The production of EPS by strain CAZ3 under conventional and metal stressed conditions was determined by the method as suggested by Mody et al. (1989). For EPS quantification, the bacterial strain was cultured in 50 mL NB supplemented with 5% glucose (as C source) and treated with 0, 25, 50 and 100 µg mL−1 each of Cd, Cr and Ni. The bacterized flasks (100 ml capacity) were incubated at 28 ± 2 °C for 5 days on a rotary shaker incubator (Rivotek) at 120 rpm. The culture broth was spun at 8000 rpm for 30 min. and three volumes of chilled acetone were added to one volume of supernatant to extract the EPS. The extracted EPS was then washed three times with distilled water and acetone and later transferred to a filter paper (No. 42). The filter paper was dried overnight at room temperature and weighed for estimating the amount of EPS released by the bacterial strain.

Characterization of EPS

The structural alteration, metal location and disturbances/variations in functional moieties of dried powdered EPS extracted from metal treated and untreated (control) strain CAZ3 was determined by SEM, EDX, and FTIR respectively. For SEM and EDX, a small fraction of the powdered EPS was mounted on a stub provided with a double-sided carbon tape. After mounting, the samples were coated with gold coating in a sputter coater prior to visualization under SEM. Following the coating procedure, the EPS samples were visualized under SEM (model JSM 6510 LV; JEOL, Japan) and surface morphology of EPS was recorded. Moreover, the percentage of heavy metals entrapped within the EPS was detected by EDX detector coupled to SEM. The peaks (respective of each metal) appearing in the EDX analysis, gave a clear picture of the elemental composition of EPS extracted from metal loaded and unloaded cells of strain CAZ3. The functional groups of powdered EPS were further assayed by FT-IR spectroscopy. For this, approximately 2.5 mg of powdered EPS was ground and mixed with 75 mg of KBr in a mortar. The powdered material was pressed to prepare translucent discs of the samples. The respective discs were analysed immediately by a spectrometer adjusted in the range of 1000–4000 cm−1 with a resolution of 5 cm−1 while subtracting the effect of atmospheric H2O and CO2. The samples were visualized under FT-IR spectrometer (Thermo Nicolet, Nexus 670) and characteristic spectral peaks corresponding to each metal were recorded.

NMR analysis of EPS

To further ascertain the structural composition of EPS, solid state 1H nuclear magnetic resonance (NMR) was employed. For spectral analysis, D2O was used as the solvent wherein the dried but crude EPS powder was deuterated with 99% D2O and NMR spectrum of the sample was recorded at 400.3 MHz for 1H NMR using Bruker Avance II-500 spectrometer. The chemical shifts in the EPS sample were measured in ppm using tetramethylsilane (TMS) as an internal standard.

Influence of heavy metals on metallothioneins (MTs) secretion by strain CAZ3

The impact of heavy metals on MTs production was assayed by the method of Murthy et al. (2011) with some modifications. The bacterial strain was grown in NB amended with 50 µg Cd mL−1 and 100 µg mL−1 each of Cr and Ni for 48 h at 28 ± 2 °C on a shaking incubator. A control without heavy metal was also run for comparison. After 48 h, the cultures were centrifuged at 10,000 rpm for 20 min. and the resulting pellets were suspended in lysis buffer comprising of 0.5 M sucrose prepared in 20 mM Tris-HCl buffer (pH = 8.6) and containing 0.01% mercaptoethanol. Sonication of the pellets was done for 2 min. (4 times with 30 sec pulse off) using a Branson Digital Sonifier, Branson Ultrasonics Corporation, Danbury, USA to completely disrupt the bacterial cells. The sonicated sample was centrifuged again at 10,000 rpm for 20 min. and the supernatant was separated from pellet. A-1.05 mL of cold absolute ethanol (chilled at −30 °C) and 80 µL of chloroform were added to one ml of supernatant. The samples were centrifuged under cold conditions at 10,000 rpm for 10 min. with the addition of three volumes of cold ethanol to the supernatant. The suspension was chilled at −30 °C for 1 h. Centrifugation of the samples was done at 10,000 rpm for 10 min. and the cell pellets were washed with a mixture of ethanol, chloroform and homogenization buffer in the ratio 87:1:12 µL and then again centrifuged at 10,000 rpm for 10 min. The pellets were dried to complete evaporation and the dried pellets was re-suspended in 300 µL of 5 mM Tris-HCl and 1 mM EDTA (pH = 7). The MTs fraction so obtained was mixed with 4.2 mL of 0.43 mM 5, 5’-dithiobis nitrobenzoic acid (Ellman’s reagent) in 0.2 M phosphate buffer (pH = 8) and left at room temperature for 30 min. The concentration of reduced sulfhydryl in the samples was measured at 412 nm and MTs in each sample was quantified by plotting a standard curve of reduced glutathione (GSH). The concentration of metallothionein in the samples was calculated using the standard curve equation of Ana and Garcia-Vazquez (2006).

Extraction and characterization of melanin released under metal stress

Melanin secreted by strain CAZ3 was extracted following the method of Banerjee et al. (2014). For this, 7 days old cultures were grown in NB treated with (50 µg Cd mL−1and 100 µg mL−1 each of Cr and Ni) or without metals. The bacterial culture was centrifuged at 5000 rpm for 10 min. to obtain cell pellets. The pellets were washed with 5% trichloroacetic acid (TCA) and later with ether-ethanol mixture in a ratio of 1:1 v/v and absolute ether to remove any impurities. The washed pellets were dissolved in 0.05 M sodium carbonate and the mixture was heated in a boiling water bath for 10 min. The material was stored at room temperature for 15 min. The dark brown material so obtained was washed three times with sterile double distilled water and then lyophilized to obtain a dark brown powder which was characterized using SEM, EDX and FTIR techniques.

Measurement of functional group moieties of metal treated and untreated EPS and melanin by FTIR

The destructive impact of heavy metals and stretching vibrations in various functional groups present in EPS and melanin secreted by strain CAZ3 was determined by FT-IR spectrometer (Thermo Nicolet, Nexus 670). For this, dried powder of EPS and melanin (2.5 mg approx.) was blended and ground with KBr (75 mg) in an agate mortar. The translucent discs were prepared by pressing the ground material with the help of 8 tonnes of pressure bench press. The respective discs were immediately analysed with a spectrophotometer in the range of 1000–4000 cm−1 with a resolution of 5 cm−1. The influence of atmospheric H2O and CO2 was always subtracted.

Statistical analysis

The results were statistically analyzed by Duncan’s multiple range test (DMRT) and the least significant difference (LSD) was calculated using Minitab 17 statistical software at P ≤ 0.05.

Results and discussion

Heavy metal tolerance and bacterial strain identification

Of the total 20 bacterial strains tested for their ability to survive under harsh metal environment, strain CAZ3 exhibited variable growth on NA medium amended with different concentrations of heavy metals. Strain CAZ3 grew well on NA medium treated with 100 µg mL−1 of Cd, 1000 µg mL−1 of Cr and 1200 µg mL−1 of Ni whereas it tolerated 50 µg mL−1 of Cd, 100 µg mL−1 each of Cr and Ni when cultured in liquid medium (Table 1). In general, A. chroococcum CAZ3 exhibited greater tolerance to heavy metals when grown on nutrient agar plates as compared to nutrient broth possibly due to the complex/polymeric nature of agar which renders the metals unavailable for uptake by the bacteria as also reported by other workers (Moghannem et al. 2015). In contrast to this, the metals become soluble and hence, are easily available to bacteria when present in liquid medium, thereby resulting in higher levels of toxicity. Similar variation in survivability and metal tolerance ability among bacterial strains for example, B. cereus and B. amyloliquefaciens has been reported (De Guzman et al. 2016). In this experiment, both bacterial strains tolerated Pb up to a level of 2000 µg mL−1 where as P. aeruginosa, Chryseobacterium sp., and B. subtilis could tolerate 1200 µg mL−1 each of Cd and Ni. In yet another study, Singh and Lal (2015) Pseudomonas sp. (P-3) and Bacillus sp. (B-2) grew well in liquid medium treated with 350 µg mL−1 of chromium. However, to avert the lethal impact of heavy metals and consequently to survive and multiply under heavy metal pressure, the metal tolerant microbes have evolved many interesting mechanisms such as-(a) complexation of metals within the cell (Ianeva 2009) (b) pumping out of metal ions to the exterior of the cell via efflux pump system (Ma et al. 2016) (c) enzymatic degradation (Marzan et al. 2017) and solubilization of heavy metals (Ayangbenro and Babalola 2017) and (d) transformation of metals from toxic to non toxic state (Chaturvedi et al. 2015). However, whatever mechanisms they adopt to circumvent metal toxicity, this feature of strain CAZ3 to survive and proliferate in under metal stressed environment is indeed a unique property which could be exploited for various reasons. For instance, with this ability, the bacterial strain will be able to ramify in soils polluted with single or multiple metals and once established, will express their physiological activities which could be beneficial for crops growing in stressed environment. Since this strain was found environmentally interesting, it was further characterized biochemically and molecularly up to species level. Strain CAZ3 displayed Gram negative reaction, and was rod shaped and exhibited dark brown pigmentation (melanin) on maturation. The bacterial strain tested positive for starch and lipid hydrolysis and also displayed a positive reaction for indole, oxidase, and nitrate reduction (Table 1). On the other hand, strain CAZ3 exhibited a negative reaction towards catalase and urease.

Table 1 Morphological, biochemical and heavy metal tolerance profiling of A. chroococcum strain CAZ3
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On the basis of morphological and biochemical properties, strain CAZ3 was presumed as Azotobacter which was identified molecularly by 16S rRNA gene sequence analysis as Azotobacter chroococcum (GenBank accession number MG252731) based on percent similarity with other A. chroococcum strains already present in the NCBI database (Fig. 1).

Fig. 1
figure 1

Phylogenetic analysis based on 16S rRNA gene comparison showing relationship between Azotobacter chroococcum CAZ3 and other closely related bacteria. The tree was constructed using Clustal W sequence alignment tool in MEGA 6.0 software

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Cellular damage to A. chroococcum cells revealed under SEM, EDX and CLSM studies

A. chroococcum CAZ3 cells when grown in NB amended with 50 µg mL−1 Cd (Fig. 2a) and 100 µg mL−1 each of Cr (Fig. 2b) and Ni (Fig. 2c) had distorted cellular morphology when observed under SEM as compared to untreated control cells (Fig. 2d). The metal treated cells displayed shrinkage and elongation and other surface distortions as compared to the untreated bacterial cells whose morphology and size remained unaltered/normal. The average size of cells (1.6 µm) was reduced even though marginally by 6.25% in the presence of 50 µg mL−1 Cd, compared to un-treated cells (1.7 µm) yet the distortions were visible. However, among metals, maximum reduction in cell size was observed for Ni which reduced it by 24% (1.3 µm) as compared to control. In contrast, the cells grown in the presence of Cr had elongated cell and the average length of cells was recorded as 1.8 µm. Similar reduction in size of bacterial cells grown with Cd and Ni (Bhagat et al. 2016) and Pb (Girisha 2014) while increase in cell length of bacteria for instance, Ochrobactrum tritici 5bvl1 grown under Cr (VI) stress has been reported (Francisco et al. 2010). In yet other study, a significant elongation in the size of Cr treated E. coli and B. subtilis cells have been revealed under SEM (Samuel et al. 2013). The elongation in bacterial cells following exposure to Cr could possibly be an adaptive mechanism employed by bacteria to protect itself from the toxic impact of metal. Despite the damage caused by heavy metals, it was interesting to note that the metals were found localized within the cells of A. chroococcum CAZ3, as presented in the EDX spectra prepared from bacterial cells after growing them with Cd (Fig. 2e), Cr (Fig. 2f) and Ni (Fig. 2g).

Fig. 2
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SEM micrographs of A. chroococcum CAZ3 grown in the presence of a 50 µg mL−1 Cd b 100 µg mL−1 Cr c 100 µg mL−1 Ni d Control. e–g represent the EDX spectra of A. chroococcum CAZ3 grown with 50 µg mL−1 Cd, 100 µg mL−1 Cr and 100 µg mL−1 Ni, respectively. h represents the EDX spectra of untreated control

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Also, the toxicity of heavy metals to A. chroococcum CAZ3 expressed as number of dead cells was clearly visible which however increased progressively with consequent increase in metal concentrations (Fig. 3). In the microimage, the PI stained cells exhibiting red fluorescence indicate the dead cells. Generally, the number of dead cells increased with a progressive increase in metal concentration as presented in Fig. 3a. Conventionally, the untreated control cells did not show any red fluorescence. Mechanistically, the PI dye (a DNA intercalating dye) permeates only through the membranes of dead cells and hence the cells become red in colour. Due to this, the PI dye is considered as an excellent indicator for assessment of metal toxicity. Prior to this study, a similar increase in the number of dead cells of Burkholderia cepacia strain PSBB1 with the progressive increase in the concentration of glyphosate was reported by Shahid and Khan (2018). Additionally, the toxic impact of Se causing oxidative stress and DNA damage in Oreochromis mossambicus has previously been reported (Gobi et al. 2018). Conclusively, even though, A. chroococcum CAZ3 tolerated higher concentrations of Cd, Cr and Ni, yet the bacterial membrane was damaged by heavy metals in a dose dependent manner.

Fig. 3
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Propidium iodide (PI) staining of metal treated and untreated cells of A. chroococcum CAZ3: Number of dead cells increased with increase in metal concentrations. a Graphical representation depicting the effect of progressively increasing concentrations of Cd, Cr and Ni on the number of PI stained cells of A. chroococcum CAZ3. b Growth kinetics of A. chroococcum CAZ3 under varying doses of Cd, Cr and Ni compared with untreated control; Error bar shows the mean ± S.D. of three independent replicates

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After evaluating the growth pattern under CLSM, the inhibitory impact of varying concentrations of Cd, Cr and Ni on A. chroococcum CAZ3 were further tested under in vitro conditions. Here also, a variable growth pattern was observed when measured at 620 nm (Fig. 3b). Among metals, Cd in general was found most toxic and inhibited the bacterial growth substantially at 100 ug/ml compared to other metals after 24 h of incubation. The inhibition of bacterial growth exposed to Ni, Cd, and Cr observed under in vitro conditions corroborated the findings recorded by CLSM.

Metal detoxification strategies adopted by A. chroococcum CAZ3

Characterization of metal chelating EPS secreted by A. chroococcum

Exopolysachharides are polymeric substances synthesized by microorganisms as a protective material to overcome many stressful conditions (Gupta and Diwan 2017). Chief among them are the ionic stresses, antibiotics, heavy metals, and desiccation (Shameer 2016). Due to large surface area, EPS has been found to play tremendous roles in biosorption and removal of metals from the contaminated sites (Muthu et al. 2017). Considering this useful and applicable activity of microbes, the production of EPS by A. chroococcum CAZ3 while growing under heavy metal stress was assayed. Interestingly, it was observed that A. chroococcum CAZ3 survived and maintained the synthesis of EPS even in the presence of metal stress. The strain CAZ3 produced 260 µg mL−1 EPS in the presence of 25 µg mL−1 Cd which was increased by 23% (320 µg mL−1) when Cd concentration was raised to 100 µg mL−1 (Fig. 4). Likewise, at 100 µg Cr mL−1, the concentration of EPS was enhanced by 25% relative to those observed at 25 µg Cr mL−1. Although, the secretion of EPS increased with increasing metal concentrations but Ni at 100 µg mL−1 caused a significant drop (77%) in EPS production over control (Fig. 4). In contrast, under conventional growth environment, A. chroococcum CAZ3 secreted maximum amounts (580 µg mL−1) of EPS. In a similar study, Exiguobacterium strains UE1 and UE4 under Cr stress have shown maximum release of EPS (Batool et al. 2017). In a follow up study, Abinaya et al. (2018) have confirmed the antioxidant potential, antibiofilm activity and other important biomedical applications of EPS released by Bacillus licheniformis Dahb1.

Fig. 4
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Quantification of EPS secreted by A. chroococcum CAZ3 under control and metal stressed environments

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After quantifying the EPS, the powder of EPS was analyzed under SEM. The EPS micrographs of metal treated bacterial cells showed certain polymeric structures while the EDX spectra of the EPS extracted from metal treated cells demonstrated the presence of Cd (Fig. 5a), Cr (Fig. 5b) and Ni (Fig. 5c). The presence of these metals in bacterial cells clearly validated the fact that EPS could chelate heavy metals when compared with control (Fig. 5d).

Fig. 5
figure 5

SEM micrographs and EDX spectra of EPS secreted by A. chroococcum CAZ3 in the presence of a 50 µg mL−1 Cd b 100 µg mL−1 Cr c 100 µg mL−1 Ni and d Control. The EDX spectra represent the elemental composition of EPS extracted from metal treated and untreated cells

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The EPS extracted from metal treated and untreated cells of A. chroococcum CAZ3 was further characterized using FT-IR and various functional groups involved in binding the heavy metal ions to bacterial cell surface were determined. The FT-IR data on metal loaded and unloaded EPS of A. chroococcum CAZ3 was recorded in the range of 1000–4000 cm−1. Multiple peaks were obtained in the spectra of metal treated and untreated EPS which indicated diverse functional groups which in turn reflected a complex nature of the extracted EPS. It was interesting to note that some deviations in the peaks corresponding to various functional groups attached to the surface of EPS were recorded when the strain was grown in the presence of 100 µg mL−1 each of Cd, Cr and Ni (Fig. 6a). A broad peak appearing in the range of wave number 3281–3428 cm−1 could be assigned to N-H stretching of proteins and O-H stretching of hydroxyl groups. The changes in functional group moieties of EPS and shifting/disappearance of peaks due to binding of metal ions have been illustrated in Table 2. Interestingly, the FTIR spectrum of the metal loaded EPS revealed certain modifications. Shifting of peaks was observed at few regions of EPS extracted from Cd, Cr and Ni treated cells of A. chroococcum CAZ3 when compared with control. Similar disappearance/shifting of peaks of EPS extracted from numerous bacterial cells grown under metal pressure have been reported (Seneviratne et al. 2016; Batool et al. 2017). The shifting in peaks as compared to control could possibly be due to the change in functional groups present on the surface of metal loaded EPS because of the interactions and binding of metal ions with the bacterial EPS. The production of EPS, thus illustrates a putative mechanism for the elimination of heavy metals from contaminated soils which could eventually serve as a cost effective and eco-friendly biological approach for environmental management.

Fig. 6
figure 6

a FT-IR spectra of metal loaded and unloaded EPS showing characteristic peaks corresponding to various functional groups involved in binding of heavy metal ions. b1H NMR spectrum of EPS extracted from the cells of A. chroococcum CAZ3

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Table 2 FT-IR assignments of metal loaded and unloaded EPS extracted from Azotobacter chroococcum CAZ3
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Since the NMR spectroscopy has widely been used to reveal the structure of EPS (Liang and Wang 2015; Dahech et al. 2013), the EPS extracted from A. chroococcum CAZ3 in this study and characterized employing SEM, EDX and FTIR, was further analyzed by NMR in order to better understand the structure of EPS. The 1H NMR spectrum of EPS obtained from A. chroococcum CAZ3 revealed that the protons appeared at 9.497, 9.369, 9.242 and 4.717 ppm (Fig. 6b). The highest peak signal appearing at 6.438 ppm could represent the peak of solvent D2O. The NMR peaks were however, poorly resolved due to viscosity/impurity of EPS. The specific characteristic functional groups representing the structure of polysachharides were not detected because the EPS obtained from A. chroococcum CAZ3 was not purified in this study. Similar chemical shifts and corresponding functional groups indicating glucan anomeric protons of 1H NMR spectrum of EPS extracted from P. aeruginosa B01 has recently been reported (Benit and Roslin 2018).

Metal induced production of metallothionein by A. chroococcum CAZ3

Metallothioneins (MTs), a group of low molecular weight proteins (with molecular weight generally ranging from 3500–14000 Da) are rich in cysteine. The thiol group of MTs binds to heavy metals and makes it inaccessible to microbes (Enshaei et al. 2010). While doing this, MTs safeguard the bacterial cells from metal toxicity and oxidative stress that could otherwise have deleterious impact on their survival. Considering the importance of MTs in metal removal, A. chroococcum CAZ3 was tested to evaluate its potential to induce MTs production while growing in the presence of Cd (50 µg mL−1), Cr (100 µg mL−1) and Ni (100 µg mL−1). The bacterial cell pellets believed to contain MTs was mixed with Ellman’s reagent. For determination of MTs synthesis, the yellow colour so produced was quantified spectrophotometrically in terms of concentration of reduced sulfhydryl in the samples calculated from the standard curve of reduced glutathione. The amount of MTs in the samples was calculated from the straight line equation (y = mx + c where ‘m’ is the slope and ‘c’ is the intercept) of GSH standard curve, wherein the MT content was further calculated by dividing the x-value by 20 since 1 mole of metallothionein is assumed to contain approx. 20 moles of cysteine residues. While comparing the synthesis of MTs both in the presence and absence of heavy metals, bacterial cells grown in metal treated medium showed maximum production of MTs suggesting the inducible role of metals (Table 3). For instance, the untreated cells showed lowest production of metallothioneins (0.4 µ mol) whereas the Cd, Cr and Ni treated cells released 3.6, 0.7 and 2.6 µ moles of MTs, respectively (Table 3). Among metals, Cd was found as the strongest inducible metal and hence, showed maximum production of MTs by A. chroococcum CAZ3 compared to Ni and Cr. The production of MTs apart from other active biomolecules under metal stress in general, could therefore, be a possible mechanism which aid the bacterial cells to survive even under metal pressure. In agreement to our findings, similar induction in MTs production by Bacillus cereus cells grown in the presence of increasing rates of Pb (Murthy et al. 2011) and P. aeruginosa and P. putida cells exposed to Cu and Cd have been reported (Enshaei et al. 2010).

Table 3 Metallothionein content in untreated and metal treated cells of A. chroococcum CAZ3
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The number of free thiol groups located within a MT protein represents the formation of metal-cysteine clusters responsible for bridging thiolates which eventually results in binding of more metal ions to even smaller number of cysteine residues. In particular, Cd2+ ions eventhough have fixed geometry but the bridging ligands generally modify the ratio of bound vs free thiols forming a varied cluster (Rubino 2015; Irvine and Stillman 2017). The metal bound MTs appear as protein chains wrapped around a cluster formed from thiol-metal binding with numerous cysteine residues holding the metal ions via thiolate groups. On the other hand, MTs generally cannot directly bind Cr ions but can eliminate the ROS (developed due to Cr toxicity) by means of cysteine residues, thereby providing protection to the cells from toxic Cr ions (Kimura et al. 2015). This was evident from our findings also where, minimum amounts of MTs were recorded in Cr treated cells as compared to Cd and Ni. Moreover, MT-1 and MT-2 molecules, in some cases, are known to bind chromate ions as well. Interestingly, the apo-MT molecule exhibits higher binding affinity to Cr ions and forms a stable complex once bound to the MT molecule (Dziegiel et al. 2016). In case of Ni, saturation binding of Ni2+ ions occurs at seventh position, resulting in the formation of M-7 MT. The binding is assisted by means of thiol groups via displacement of native Cd or Zn ions. Despite this, the exact stoichiometry of Ni binding to MTs is still not clearly understood. A hypothetical model representing the structure of metal bound MT is presented in Scheme 1 where Cd, Cr and Ni ions have been shown to bind at specific sites on the protein chain, resulting in a cluster held together through thiol groups.

Scheme 1
scheme 1

Hypothetical structure of metal bound metallothionein showing the attachment of various metal ions to the peptide chain a; Chemical reactions involved during binding of metal ions to MTs b; 2D and 3D molecular models of metal bound MTs conjugated through thiol groups c

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Due to their metal binding ability, the MTs could serve efficiently in the abatement of metal toxicity and hence, the bacteria possessing this potential could be exploited for removal of metals (bioremediation) from contaminated environment.

Melanin: importance in metal detoxification

Melanin, a dark brown to black coloured polymeric and amorphous substance, is synthesized by many prokaryotic organisms (Banerjee et al. 2014) including nitrogen fixing organisms (Maru and Gadre 2016). They are known to protect microbial cells from harmful effects of electromagnetic radiations, ROS, high temperatures and heavy metals (Drewnowska et al. 2015) by trapping environmental stressors for example, heavy metals (El-Naggar and El-Ewasy 2017). Taking this into consideration, the melanin synthesized by A. chroococcum CAZ3 was extracted and characterized. The SEM micrographs of melanin extracted from metal treated and untreated cells revealed that the pigment was an amorphous substance with no well defined structures. Moreover, the SEM images showed irregular clumping of the aggregates (Fig. 7a–d). In earlier experiments, Tarangini and Mishra (2013) have also confirmed similar features of melanin extracted from Pseudomonas sp. The EDX analysis of melanin obtained from untreated cells of A. chroococcum CAZ3 revealed the presence of C, O, N and Na (Fig. 7d) as the key elements found in bacterial melanin. Also, the melanin extracted from strain CAZ3 grown with metals displayed the adsorption of Cd (Fig. 7a), Cr (Fig. 7b) and Ni (Fig. 7c) when visualized under EDX spectroscopy. Moreover, it was interesting to note that in addition to C, O, N and Na, the melanin obtained from metal treated A. chroococcum cells, also exhibited a fairly good percentage of Cd, Cr and Ni which clearly indicated the metal chelating/binding property of melanin.

Fig. 7
figure 7

SEM micrographs and EDX spectra of melanin pigment isolated from the cells of A. chroococcum CAZ3 in the presence of a 50 µg mL−1 Cd b 100 µg mL−1 Cr c 100 µg mL−1 Ni and d Control. The EDX spectra displays the key elements present in melanin along with heavy metal ions trapped inside the melanin extracted from A. chroococcum CAZ3

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The relationship between metal ion concentration and melanin production is however, still not clearly understood. Yet in avian species, it has been found that higher levels of metals like Zn, results in greater synthesis of melanin (Chatelain et al. 2014). This study therefore, has tried to establish a link between metal concentration and melanin based colouration in birds. Likewise, the melanin pigment formation within the feathers of birds influenced by accumulation of Cu has been reported (Edwards et al. 2016). In case of bacterial cells, there are few reports available suggesting the synthesis of melanin by bacteria under heavy metal stress as a protective mechanism and its role in metal ion chelation. However, how the increasing concentration of heavy metals enhances the secretion of melanin is yet to be established. Despite all these conflicting reports, the melanin synthesized by A. chroococcum CAZ3 in our study was induced largely by highest amounts of Cd, which was followed by Cr and Ni. The release of melanin under heavy metal influence thus suggests that melanin had higher affinity for Cd2+ ions as compared to Cr and Ni ions. And hence, greater amounts of melanin were released under Cd pressure.

Also, the peaks of various functional groups obtained in the FT-IR analysis of melanin extracted from metal treated and untreated (control) cells of A. chroococcum CAZ3 was investigated in the range of 1000–4000 cm−1 (Fig. 8).

Fig. 8
figure 8

FT-IR spectra of melanin pigment derived from metal treated and untreated cells of A. chroococcum CAZ3 exhibiting characteristic peaks which corresponds to vibrations/ disturbances in various functional groups that participate in metal binding

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The IR spectra of melanin recovered from metal treated cells exhibited significant deviations corresponding to several functional groups when compared with the peaks in untreated control (Table 4).

Table 4 FT-IR assignments of melanin extracted from metal treated and untreated cells of Azotobacter chroococcum CAZ3
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In case of melanin obtained from Cd treated cells, a disappearance of peaks at 1399 and 1170 cm−1 was observed in IR spectra. Also, peak disappearance at 1170 cm−1 was also recorded in the presence of Ni. Peak disappearance is indicative of disturbances in the functional groups in the presence of heavy metals. In this regard, a study revealed the stretching vibrations and bending in NH group, C=C stretching and CH2–CH3 bending which are characteristic of melanin (Apte et al. 2013). In our studies, a broad peak in the range of 3291–3431 cm−1 was also observed in the presence of Cr. In another similar study, several peaks were detected in the range of 650–1750 cm−1 in P. aeruginosa cells corresponding to alterations in carbohydrates, lipids and proteins of the bacterial cells. In addition to this, some other peaks representing hydroxyl groups were also recorded in the synchronous and asynchronous spectra (Durve and Chandra 2014). Overall, the metal chelating property of melanin could play an important role in detoxifying heavy metals from metal enriched soils. And hence, the melanin producing A. chroococcum CAZ3 could be a superb choice for removing metals and thus successfully remediating the soils contaminated with various heavy metals.

Mechanistic basis of melanic plumage/coat coloration

The melanin pigment in general, play important roles in protection against UV radiations, chemical stresses, thermoregulation and camouflage (Côte et al. 2018). Melanin synthesis is a complex process which involves a series of reactions catalyzed by various enzyme complexes. Primarily, the process of melanogenesis begins with the oxidation of L-tyrosine catalyzed by tyrosinase (a key regulator of melanin synthesis) which is encoded by the TYR locus and is highly conserved among various species. This enzyme has two main components- (a) tyrosine hydroxylase and (b) dopa oxidase. Both of these components work in unison to synthesize L-dopaquinone. This o-quinone so generated, plays a pivotal role in animal melanogenesis (Solano 2014) resulting in the formation of either eumelanin or pheomelanin. The compound L-dopaquinone is highly reactive and during the synthesis of melanin, reacts with multiple functional groups including amino, thiol and hydroxy groups. A well known pathway for melanogenesis known as the Raper–Mason pathway, involves an intramolecular cyclization of L-dopaquinone which gets converted into L-leukodopachrome (L-cyclodopa) through a nucleophilic reaction where the side chain amino group on position 6 of the aromatic ring is attacked. This reaction depends mainly on pH. Similarly, the synthesis of eumelanin is also influenced by the pH which however, is inhibited under acidic environment (Galván and Solano 2016). Post eumelanogenic pathway, L-leukodopachrome behaves as an unstable indoline exhibiting strong properties of reduction. It then undergoes a spontaneous redox reaction along with the precursor L-dopaquinone. The second most pivotal intermediate formed during melanogenesis, L-dopachrome, plays a crucial role in eumelanogenesis pathway (Nasti and Timares 2015). Conclusively, during melanin synthesis, L-Tyrosine serves as the precursor, L-dopachrome enhances the tyrosinase activity and L-tyrosine induces the synthesis of melanosome. The product, dopa, so released, is known to up regulate melanin synthesis and melanocytes regulate the local and global homeostasis of melanogenic systems (D’Mello et al. 2016). Once secreted endogenously, the melanin pigments provide coat colouration to mammals and birds. For instance, eumelanin imparts black and grey coat colour while pheomelanin imparts brown and red coat colour. For example, coat colour in animals is determined mainly by the ratio of eumelanin and pheomelanin along the hair and feathers and its spatial distribution across the body (Lin et al. 2013a).

The production of a specific type of melanin is regulated by melanocortin-1 receptor (MC1R) which is present within melanocytes and its two ligands- (i) α-melanocyte stimulating hormone (α-MSH) which is secreted from the pituitary glands and (ii) the agouti signalling protein (ASIP) (Chen et al. 2015). The melanocytes produce dark eumelanin when they are activated by α-MSH. The MC1R activity is inhibited when ASIP ligand binds and results in the synthesis of pheomelanin. On the contrary, in many species of birds and mammals, white coat colour is due to the absence of pigment in hair or feathers (Zimova et al. 2018). In case of avian species, the anagen stage of hair/feather growth represents melanogenesis and is restricted within the melanocytes (pigment-producing cells). Melanins get transferred to keratinized cells i.e., keratinocytes of the developing hair or feathers in birds through melanosomes (Chen et al. 2015). Broadly, the entire phenomenon of melanic plumage/coat coloration is genetically controlled involving a series of reactions along with the formation of intermediates and products responsible for melanin synthesis.

Finally, based on the findings observed in this study, the metal-microbes interaction and the roles of EPS, MTs and melanin secreted by metal tolerant A. chroococcum CAZ3 in metal removal/detoxification is summarized in Fig. 9.

Fig. 9
figure 9

The schematic representation elucidating the interaction of bacterial cell with the heavy metal ions and their removal/detoxification by active biomolecules (EPS, MTs and melanin) secreted by A. chroococcum CAZ3 under metal stress

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Conclusion

The deleterious impact of heavy metals on A. chroococcum strain CAZ3 observed under in vitro studies was further confirmed by microscopic observation under SEM and CLSM. Moreover, despite the toxicity shown by heavy metals, some percentage of metals was found inside the bacterial cells as revealed by EDX. This accumulation of heavy metals confirmed that the bacterial strain could thrive well even under harsh metal stress. The EPS secreted by A. chroococcum was found to accumulate significant amounts of Cd, Cr and Ni which was revealed by EDX. The release of EPS by A. chroococcum induced by heavy metals and its ability to chelate metals could be a defense strategy adopted by the bacterial strain to protect itself from metal toxicity and consequently to survive, proliferate and perform normal physiological activities in stressed environments. This novel and fascinating trait of A. chroococcum is likely to provide new insight in this research area of inexpensive bioremediation technology. The secretion of MTs by viable cells under the influence of heavy metals was yet another interesting feature of A. chroococcum CAZ3 which after binding, greatly diminished the metal toxicity. This finding thus, established the protective role of MTs secreted by A. chroococcum CAZ3 while growing under metal stressed environment. Among all the test metals, Cd was found as the most efficient inducer of MTs in A. chroococcum cells. Also, A. chroococcum synthesized a brown pigment melanin which very effectively trapped the heavy metals and hence, reduced its availability and toxicity. The entrapment of heavy metal ions by melanin was visible under EDX which further strengthened the fact that bacterial melanin can play significant role in heavy metal removal/detoxification. This melanin related finding is likely to help readers to better understand the metal removal strategy (bioremediation) and to apply melanin producing bacterial strains under contaminated soils. Overall, the microbial management confirmed the potential of nitrogen fixing A. chroococcum CAZ3 to detoxify the contaminated environment through secretion of EPS, MTs and melanin. These metal detoxifying properties together with conventional nitrogen fixing ability makes A. chroococcum CAZ3 an exciting and most appropriate candidate for heavy metal removal from contaminated soils. Broadly, the application of A. chroococcum could be a cost effective and environmentally viable approach in metal cleanup program vis-a-vis, the management of polluted environment.