Introduction

Heavy metals (HMs) are metals with relatively high densities, atomic weights, or atomic numbers. HMs are natural assets on Earth, but they are becoming increasingly scarce as a result of fast growth, urbanisation, and modern technology breakthroughs. Natural and anthropogenic sources of HMs include mining, volcanic eruptions, metal processing, ammunition, coal ash, industrial waste, residential and agricultural waste (1). HMs poisoned the land, water, and air of the ecosystem. Since of its connection to food production, agricultural soil is a key source of concern because it may have an impact on the health of living beings (2). In humans, an excess of HMs causes oxidative stress through the generation of free radicals (3). It can lower antioxidant levels in living cells and alter protein and DNA structure (4). The toxicity of HM varies according on the element; for example, As and Cd are extremely toxic; Hg, Pb, and Ni are moderately toxic; while Cu, Zn, and Mn are less harmful in living systems (5). HMs, on the other hand, have severe deleterious effects on human health, including central nervous system disturbance, kidney malfunction, and lung damage (6). Adsorption, filtration, reverse osmosis, precipitation, oxidation-reduction, and other physical and chemical techniques for dealing with HMs contamination have been developed in recent decades. However, because to the large input requirements and the generation of secondary waste, these approaches were proven to be inefficient. Bioremediation of HMs has the potential to remedy this problem since it provides unique approaches for addressing the main issue of HM contamination in soil, while also serving as a platform for new research and progress in bioremediation technology (7). This strategy entails the use of living systems to reduce dangerous toxicants/pollutants from the environment by their accumulation or transformation in an organism's cellular system. Bacteria and fungi are the species capable of this HM elimination metabolic activity (8). The ability of one of these creatures to bioremediate HM varies greatly amongst species, even within the same genus (9). As a result, the selection of a live organism for the HM bioremediation process varies correspondingly and is dependent on a number of criteria, including soil types, physical properties, and the level of HM contamination in the study site (10). The bacterial population in HM-contaminated locations is dominated by Actinobacteria, Firmicutes, and Proteobacteria, all of which belong to the genera Arthrobacter, Bacillus, and Pseudomonas, respectively (11). Fungi belonging to the phyla Ascomycota and Basidiomycota have also been found in HM-contaminated soils (12).

The influence of specific heavy metals was investigated in this study (Cr, Hg, Cu, Zn and Mg) on growth of selected indigenous microflora has been evaluated in terms of minimum inhibitory concentration and OD₆₂₀. Each heavy metal was used individually with three concentrations to check the growth or the severe toxic effects and compared with the type of organism, the nature, as well as the concentration of heavy metals.

II. Materials and Methods

A. Isolation and Identification:

The Indigenous species viz. (Bacteria- Rhizobium and Azotobacter and Fungi- Aspergillus) were isolated from the soils of different areas of Durg, Chhattisgarh for the study. These selected species were grown on suitable growth medium viz. Rhizobium Hi media (Rhizobium selective media) for the isolation of Rhizobium bacteria, Jensen’s media (Azotobacter selective media) for the isolation of Azotobacter bacteria and the Potato Dextrose Agar media was prepared for the isolation of fungus Aspergillus from the collected soil samples. Following serial dilution of obtained soil sample inoculation on respective plates, these inoculated plates were then incubated for 24 hours at 37°C for bacteria and 48-72 hours at 25°C for fungus. Overnight culture was then streaked on respective selective medium at same culture condition to get the pure culture for further analysis. Gram staining and biochemical testing of the following organisms were done for identification.

B. Stock Preparation:

Sterilized stock solution (1000mg/l) of selected heavy metals Potassium dichromate, Mercuric chloride, Copper sulphate, Zinc sulphate and Magnesium sulphate of concentration (100,300 and 500ppm for bacteria and additionally 1000,3000 and 5000ppm for fungi) was prepared in distilled water which was further diluted as per the requirement.

C.  Determination of MIC and OD:

The selected species were spread on suitable growth medium and three wells were cut on each plate. Then the solution of three different concentrations were poured in the respective wells through micropipette (50 µl) and kept for incubation for 24-48hrs at 37°C for bacteria and at 25°C for fungus. Zone of inhibition was observed after incubation. The cultures were then transferred to heavy metal-containing broth for 7 days. Individual monocultures were seen to grow at OD₆₂₀ after 7 days of incubation as compared to the control (13) and data were analysed using appropriate statistical procedures.

III. Result and Discussion

Among the heavy metals believed to be required for regular functioning of living organisms are Hg, Cr, Cu, Zn, and Mg, however an excess or deficiency of these metals can cause severe toxic effects depending on the organism, heavy metal concentration, and environmental conditions (14). The effect of different heavy metals in terms of MIC and OD₆₂₀ on the growth of Rhizobium and Azotobacter is depicted in Table 1 and 3, and that of Aspergillus is depicted in Table 2 and 4 respectively. It was found that Aspergillus sp. was tolerant against all heavy metals of different concentrations from 100-5000ppm except for Cr. Cr was found to be toxic at 1000ppm and above concentration and Hg was found to be toxic up to some extent at 5000ppm concentration (Fig-1, Fig-3 and Fig-4). Whereas, it was found that Rhizobium and Azotobacter were tolerant against Zn and Mg at lower concentration(100ppm) and toxic at higher concentration (500ppm), but for the rest heavy metal it showed complete toxicity at all concentration (Fig-1 and Fig-6). Further the confirmation of tolerance was done in terms of OD and the tolerance capacity was compared with the control (Fig-2, Fig-5, and Fig-7). The decrease in growth was seen in the tubes of metals that were toxic to the following microflora.

Fig 1: Effect of Heavy Metals on Rhizobium sp., Azotobacter sp. and Aspergillus sp. showing Minimum inhibitory concentration (MIC).

Fig 2: Effect of Heavy Metals on Rhizobium sp., Azotobacter sp. and Aspergillus sp. showing Toxicity.

Fig 3: Result obtained using three different concentrations of heavy metal against Aspergillus showing zone of inhibition at 100ppm, 300ppm and 500ppm conc.

Fig 4: Result obtained using three different concentrations of heavy metal against Aspergillus showing zone of inhibition at 1000ppm, 3000ppm and 5000ppm conc.

Fig 5: Result showing confirmation of tolerance at varying concentrations for fungus Aspergillus. First image showing control followed by 1000,3000 and 5000ppm conc.

(a)    Rhizobium plates                                      (b)  Azotobacter plates

Fig 6: Result obtained using three different concentrations of heavy metal against (a)Rhizobium sp. and (b) Azotobacter sp showing zone of inhibition at 100ppm, 300ppm and 500ppm conc.

Fig 7: Results showing confirmation of tolerance and toxicity for Rhizobium and Azotobacter bacteria. First image showing control followed by 100,300 and 500ppm conc.

Table 1: Minimum inhibitory concentration (MIC) at varying concentrations against Rhizobium and Azotobacter.

Table 2: Minimum inhibitory concentration (MIC) at varying concentrations against Aspergillus.

Table 3: Confirmation of tolerance for Rhizobium and Azotobacter at varying concentrations.

Table 4: Confirmation of tolerance for Aspergillus at varying concentrations.