Title: Identification of Protease-producing Bacterial Isolates from Soil and Evaluation of their Eggshell and Eggshell Membrane Degrading Capacities

Abstract

Eggshells and eggshell membranes are non-edible wastes largely disposed of, but these are the reserves of many bioactive compounds. Eggshells are one of the most widely used food processing and manufacturing plant by-products. The disposal of eggshell and underlying membrane waste contributes to abrasiveness, odor, and pollution. Most of the eggshell waste is currently cumulated on-site without any pre-treatment. Proteases are enzymes used in industries such as the production and processing of detergents, food, leather, cosmetics, and waste management. The objective of this study was to isolate and identify protease-producing bacterial isolates from soil and evaluate their eggshell-degrading capacities. Soil samples were collected separately from both the cake bakery waste accumulation site and arable land sampling area. Samples weighing 1 g were serially diluted in double-distilled water and spread onto skim milk agar. A total of 14 protease-producing bacterial isolates were isolated from the study samples. Three bacterial isolates showed high proteolytic activity and were selected for enzymatic study based on their zone of proteolysis. The isolates were identified based on morphological tests, biochemical tests, MALDI TOF MAS (Matrix Assisted Laser Desorption ionization time of flight mass spectrometry), and 16S rRNA sequencing. The sequencing results indicated that the isolated bacteria were Lysinibaccillus sphericus (99.69%) and two other isolates that were not sequenced. The PPBI (protease producing bacterial isolates) showed promising results for application in degrading whole eggshells and its membrane. The result of the optimization indicated that all three isolates preferred the alkaline condition since their PH was 8. They preferred yeast extract as a nitrogen source, temperature of 37oC for isolate CB1 and 30oC for the left two isolates (AL1 and CB2), and 48 h for the two isolates and 72 h for CB1 of incubation time. Soils from cake bakery waste accumulation sites and arable land in the study area could be good sources from which to isolate whole eggshell and eggshell membrane degrading alkaline protease-producing bacteria. The alkaline protease from PPBI has to be advanced to various sectors of industries, like the leather industries, cosmetics industries, pharmaceutical industries, and waste management systems. Further purification stages and studies should be done for the wide application of the enzyme producing isolates.