Leguminous plants collection:

Different leguminous plants, as follows: Cowpea, Clover, Fenugreek, Leucaena, Alfalfa, Green bean, Puncturevine, and Broad were collected from other farms and areas in Nineveh Governorate/Iraq. After collecting, the leguminous plant species were immediately transported to the laboratory of  Microbial Molecular Biology for analysis, isolation, and characterization of the strains.  The procedure of Vincent [12] was followed to isolate different bacterial isolates from root nodules of collected plants.

Nodules sampling and rhizobia isolation:

Nodules of leguminous plants were carefully detached from the root system plants and washed gently several times under tap water to remove sterile distilled water to remove soil particles. The nodule surface was sterilized by immersion in 3% NaOCl solution for a period of 2 minutes, followed by 70% ethanol for 5 minutes, and six consecutive washes in sterilized distilled water. Each sterilized surface nodule from different leguminous plants was aseptically crushed with a sterile glass rod in a test tube that contained 1 ml sterile normal saline (0.85% NaCl). One loopful of the nodule suspension was streaked on Petri dishes that contained yeast extract mannitol (YEM) agar medium with the following components (gm/mL): mannitol, 10; K₂HPO₄, 0.5; NaCl, 0.1; MgSO4 · 7H2O, 0.2; and agar, 16. Incubation was done for 3-4 days at 30°C. A single colony from each culture was selected and recultured on fresh YEMA plates [13]. The purified cultures were maintained on YEM agar slants and stored at 4°C in a refrigerator for further experiments.

Cultural conditions for exopolysaccharide production:

Rhizobial inoculum preparation:

The inoculum of rhizobial strains was prepared by transferring a loopful of rhizobial bacteria from tryptone yeast solid plates to a 100 ml conical flask containing 20 ml of YEM liquid medium with 6.8 pH. Incubation was done in a shaker incubator with irrigation at 150 rpm with a temperature of 28±2 ºC for 24 hrs. 

Preparation for exopolysaccharide production:

Preparation of inoculates of rhizobial isolates was done by transferring a loopful of rhizobial culture growing on tryptone yeast medium to a conical flask of 100 ml with 10 ml of yeast extract mannitol broth medium. Incubation was done at 150 rpm/minute and 30°C for two days. A transfer of 2% of the inoculum to the 20 mL yeast extract mannitol broth medium was done. Conical flasks containing the inoculated medium were transferred to a shaker incubator with a temperature of 30°C and 150 rpm/minute for 2 days [14].

Determination of final pH, biomass, and exopolysaccharide, biomass:

Final pH valuation of fermented broth culture using the pH meter model PW 9421 Philips. Biomass determinations were done according to Duta et al. [15], i.e., by centrifugation of fermented rhizobial cultures by centrifuge at 6000 rpm/minute for 20 minutes. The pellet was collected and dried in an oven at 80°C for one day. Whereas for exopolysaccharide determination, the addition of 10 mL of cold ethanol to 5 mL of supernatant was done. Appropriately mixed, and the mixture was put in centrifuge tubes for centrifugation at 6000 rpm/minute for 30 minutes. Supernatant was discarded, and pellets (exopolysaccharide) were collected. Wet exopolysaccharides were dried by transfer to an oven at 80°C for 1 day. Triplicates were done for each rhizobial isolate. Biomass determinations were done by [15].   

Tolerance of heavy metals:

An experiment on the tolerance of heavy metals by isolated rhizobial strains was conducted according to Teresa et al. [16]. In this procedure, wells were done on tryptone yeast extract (TY) solid medium containing the following components (g/L): tryptone, 5; yeast extract, 3; CaCl2, 0.87; and agar, 15. Petri dishes were incubated at 28±2 °C for 24 -48 hours. The tested heavy metals were as follows: Pb(CH3COO)₂, ZnSO₄, CuSO₄, and NiCl2 with concentrations: 100, 500, 1000, and 5000 µ/mL. The diameter of the inhibition zone (clear zone) was measured in millimeters (mm) after 2 days of incubation. Isolated stains were considered resistant when no inhibition zones were observed around wells.

Screening of rhizobial isolates for exopolysaccharide production:

According to Sridevi and Mallaiah [17], the rhizobial extreme producer isolates were selected preliminarily by naked eye according to cultural appearance, i.e., mucoid culture with sticky texture.

Statistical analysis:

The values obtained in this research work were statistically analyzed for standard deviation (SD)  using a computer program [18].

Results

Discussion

Ali and Orf [10] from Egypt isolated rhizobial isolates that produced exopolysaccharide [17]. The results revealed that the ability to isolate 16 strains of Rhizobium from root nodules of Sesbania sesban grown in different areas in Indian farms, and they found that the strain SS5 was the best among the isolates in exopolysaccharide production. [19] found that the maximum exopolysaccharide production was 6.31 gm/ L by the isolate R. leguminosarum bv. Viciae WS18 after two days of incubation, when mannitol was used as the sole carbon source. Other studies also revealed that the mannitol supported a maximum exopolysaccharide production [20, 21]. Ten strains of Rhizobium were isolated from Vigna mungo L. According to the research, production of exopolysaccharides is highly dependent on microorganisms, under specific conditions, media composition, as well as the genus and species of bacteria [10, 22]. It was revealed that the decrease in fermented cultures was due to the production of acidic intermediates through incomplete oxidation of sugars in nutrient media. Shen T et al. [23] isolated twenty rhizobial strains that showed different levels of tolerance to copper, nickel, and zinc [24]. The results indicated novel evidence for further exploring more functional microbes from the nickel mine soil. [25] analyzed the diversity of 50 strains isolated from Trifolium sp. Nodules growing on Pb-Zn mine soil. Mechanisms mediating resistance to Cobalt and Nickel have been identified in many metal-resistant rhizobia through the identification of orthologs of metal resistance genes characterized in C. metallidurans CH34 [26]. Microorganisms tolerant of metal contaminants are known to have two mechanisms by which they protect themselves from the toxic effects of heavy metals. Exclusion, as mediated by the exopolysaccharide capsule around the cell, has been shown to prevent cells uptake of metals [27].

Acknowledgement

The author is grateful to the Department of Biology/ College of Education for Pure Sciences/ University of Mosul/ Mosul-Iraq, which helped to improve the quality of this work. 

Author Contributions

Mohammed Jasim is a Researcher was achieved this research work under  guidance  of Dr. Raad Hassani Sultan.

Conflict of Interest

The authors declare no conflict of interest.

References