MOLECULAR CHARACTERIZATION OF SOME TROPICAL RHIZOBIA AND THEIR FRUIT-PEEL-DEGRADING ABILITY

Abstract

This study was carried out to isolate and identify rhizobia from some tropical legumes (lima beans (Phaseolus lunatus), groundnut 1 (Arachis hypogaea var. “Akure”), groundnut 2 (Arachis hypogaea var. “Samaru”), soya beans (Glycine max), cowpea var. 178ID-994, cowpea var. IT90K-277-2, cowpea Ife brown (Vigna uguiculata L. Walp), cowpea Modupe (Vigna uguiculata var. Modupe) and cowpea Oloyin (Vigna uguiculata var. Oloyin) and screen them for fruit-peel-degrading ability. Rhizobia was isolated using yeast extract mannitol agar and identified using cultural, morphological, biochemical and molecular methods of characterization. The potential of the isolates to digest the fruit peels of some Nigerian fruits (orange, water melon, plantain, banana, pineapple, and pawpaw) was investigated by growing the organisms on the peels separately with pectin (as control) after which dinitrosalicylic acid and Biuret reagent methods were used to quantify the amount of glucose and protein released into the medium followed by the calculation of the fruit-peel-degrading (FPD) enzyme activity. Effects of physicochemical factors: temperature and incubation time on the activity of the FPD enzyme and pH changes during the growth of the rhizobia on the peels were determined by various standard analytical methods. The names of the rhizobia were identified through standard molecular methods as Rhizobium leguminosarum, Rhizobium leguminosarum C, Bradyrhizobium oligotrophicum, Nitrobacter winogradiskyi, Bradyrhizobium sp., Nirobacterium hamburgensis, Mesorhizobium opportunistum, Sinorhizobium meliloti and Rhizobium phaseoli. The results showed that the rhizobia were able to degrade all the peels with the highest (0.34 mg/mL) and the lowest (0.094 mg/mL) glucose concentrations expressed by Bradyrhizobium sp. in orange peel and Nitrobacter winogradskyi in pawpaw peel respectively. The FPD enzyme activity ranged from 1.37 Unit/mL in Rhizobium phaseoli to 6.38 Unit/mL in Bradyrhizobium sp. Effect of temperature and incubation periods on the FPD enzyme activity showed that the lowest (0.45 Unit/mL) FPD enzyme

activity occurred at 20oC as revealed by Rhizobium leguminosarium in banana peel and highest (4.7 Unit/mL) FPD enzyme activity at 25oC was expressed by Rhizobium phaseoli. Incubation period showed similar trend of enzyme activity reaching a peak on day 2 (5.2 Unit/mL) by Rhizobium leguminosarium C in banana peel and lowest activity (0.002 Unit/mL) after 7 days with Rhizobium phaseoli. The test showed a general decrease in the pH of each degrading peel (from pH 6.62 to 3.55) during incubation for 7 days. The highest extracellular FPD activity was 0.306 mg/mL as revealed in Rhizobium leguminosarium in banana peel while the lowest was 0.094 mg/mL as expressed by Nitrobacter winogradskyi in pawpaw peel. The highest degradation ability (0.85 mg/mL) was observed in the medium containing Bradyrhizobium sp. and Rhizobium phaseoli in plantain peel while the lowest (0.098 mg/mL) was observed in Bradyrhizobium oligotrphicum in pawpaw peel. The investigation showed that the isolates were able to digest the peels of the fruits as the enzymes synthesized enabled them to breakdown the substrates. This implies that they have potential for use in the peeling of fruits.