Quantification of Exopolysaccharide Produced by Bacillus subtilis and the Effect of Different Factors on its Production

Abstract

The present study included the extraction and characterization of EPS production by a local soil isolate Bacillus subtilis and examine the effect of different factors pH, inoculums size, incubation periods, and carbon, nitrogen and phosphate sources on EPS production. The exopolysaccharide was recovered from the culture supernatant by using a cold ethanol precipitation. The total carbohydrate content was determined by phenol sulfuric acid method at 488 nm, and major structural groups were detected by Fourier transform infrared (FTIR) spectroscopy at frequency range of 400 to 4000 cm-1. Basal salt solution (BSS) showed higher efficacy in supporting the bacteria to produce EPS (0.986 mg/ml) as dry weight. The study showed that glucose and fructose were gave maximum EPS production as a carbon source along with ammonium chloride as a nitrogen source. The optimal medium conditions were pH 7.0, inoculum size 5-6%, after four days of incubation to promote the maximum EPS production for the bacteria under study. ‫ﺍﻟﻤﻨ‬ ‫ﺍﻟﺨﺎﺭﺠﻲ‬ ‫ﺍﻟﺴﻜﺭﻴﺎﺕ‬ ‫ﻟﻤﺘﻌﺩﺩ‬ ‫ﺍﻟﻜﻤﻲ‬ ‫ﺍﻟﺘﻘﺩﻴﺭ‬ ‫ﺒﻭﺍﺴﻁﺔ‬ ‫ﺘﺞ‬ Bacillus subtilis ‫ﻭ‬ ‫ﺘﺄﺜﻴﺭ‬ ‫ﺇﻨﺘﺎﺠﻪ‬ ‫ﻓﻲ‬ ‫ﻤﺨﺘﻠﻔﺔ‬ ‫ﻋﻭﺍﻤل‬ ‫ﺍﻟﻤﻠﺨﺹ‬ ‫ﺘﻀﻤﻨﺕ‬ ‫ﺍﺴﺘﺨﻼﺹ‬ ‫ﺍﻟﺩﺭﺍﺴﺔ‬ ‫ﺇﻨﺘ‬ ‫ﻭﺘﻭﺼﻴﻑ‬ ‫ﺍﻟﺨﺎﺭﺠ‬ ‫ﺍﻟﺴﻜﺭﻴﺎﺕ‬ ‫ﻤﺘﻌﺩﺩ‬ ‫ﺎﺝ‬ ‫ﻟﺒﻜﺘﺭﻴﺎ‬ ‫ﻲ‬ Bacillus subtilis ‫ﺍﻟﻤﻌﺯﻭﻟ‬ ‫ﺍﻟﺘﺭﺒﺔ‬ ‫ﻤﻥ‬ ‫ﺔ‬ ‫ﻭﺩﺭﺍﺴﺔ‬ ‫ﻤﺨﺘﻠﻔﺔ‬ ‫ﻋﻭﺍﻤل‬ ‫ﺘﺄﺜﻴﺭ‬ ‫ﺍﻻﺱ‬ ‫ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ‬ ‫ﻭ‬ ‫ﺍﻟﻠﻘﺎﺡ‬ ‫ﺤﺠﻡ‬ ‫ﻭ‬ ‫ﺍﻟﺘﺤﻀﻴﻥ‬ ‫ﻓﺘﺭﺓ‬ ‫ﻭ‬ ‫ﻤﻥ‬ ‫ﻤﺨﺘﻠﻔﺔ‬ ‫ﻤﺼﺎﺩﺭ‬ ‫ﺍﻟﻜﺭﺒﻭﻥ‬ ‫ﻭ‬ ‫ﺍﻟﻨﺘﺭﻭﺠﻴﻥ‬ ‫ﻭ‬ ‫ﺍﻟﻔﺴﻔﻭﺭ‬ ‫ﻓﻲ‬ ‫ﺇﻨﺘﺎﺠ‬ ‫ﻪ‬. ‫ﻤﺘﻌﺩﺩ‬ ‫ﺘﺭﺴﻴﺏ‬ ‫ﺘﻡ‬ ‫ﺍﻟﺴﻜﺭﻴﺎﺕ‬ ‫ﺍﻟﺨﺎﺭﺠﻲ‬ ‫ﺍﻻﻴﺜﺎﻨﻭل‬ ‫ﺒﺎﺴﺘﺨﺩﺍﻡ‬ ‫ﺍﻟﻤﺒﺭﺩ‬ ‫ﻭ‬ ‫ﻤﺤﺘﻭﻯ‬ ‫ﻗﺩﺭ‬ ‫ﺍﻟﻔﻴﻨﻭل‬ ‫ﺒﻁﺭﻴﻘﺔ‬ ‫ﺍﻟﻜﻠﻲ‬ ‫ﺍﻟﻜﺎﺭﺒﻭﻫﻴﺩﺭﺍﺕ‬-‫ﺍﻟﻤﻭﺠﻲ‬ ‫ﺍﻟﻁﻭل‬ ‫ﻋﻨﺩ‬ ‫ﺍﻟﻜﺒﺭﻴﺘﻴﻙ‬ ‫ﺤﺎﻤﺽ‬ 488 ‫ﻨﺎﻨﻭﻤﻴﺘﺭ‬ ، ‫ﺍﻟﺘﺭﻜﻴﺒﻴ‬ ‫ﺍﻟﻤﺠﺎﻤﻴﻊ‬ ‫ﺘﺤﺩﻴﺩ‬ ‫ﻭﺘﻡ‬ ‫ﺔ‬ ‫ﺒﺎﺴﺘﺨﺩﺍﻡ‬ ‫ﻁﻴﻑ‬ ‫ﺍ‬ ‫ﻷﺸﻌﺔ‬ ‫ﺘﺤﺕ‬ ‫ﻋﻨﺩ‬ ‫ﺍﻟﺤﻤﺭﺍﺀ‬ ‫ﺍﻟ‬ ‫ﺘﺭﺩﺩ‬ 400-4000 ‫ﺴﻡ‬-1. ‫ﺍﻟﻤﻠﺤﻲ‬ ‫ﺍﻟﻤﺤﻠﻭل‬ ‫ﻭﺴﻁ‬ ‫ﺍﻅﻬﺭ‬) BSS (‫ﻹﻨﺘﺎﺝ‬ ‫ﺍﻟﺒﻜﺘﺭﻴﺎ‬ ‫ﻟﺩﻋﻡ‬ ‫ﻋﺎﻟﻴﺔ‬ ‫ﻜﻔﺎﺀﺓ‬ 0.986 ‫ﻤﻠﻐﻡ‬ / ‫ﻤل‬ ‫ﹰ‬ ‫ﺠﺎﻓﺎ‬ ‫ﹰ‬ ‫ﻭﺯﻨﺎ‬ ‫ﻤﻥ‬ ‫ﺍﻟﺨﺎﺭﺠﻲ‬ ‫ﺍﻟﺴﻜﺭﻴﺎﺕ‬ ‫ﻤﺘﻌﺩﺩ‬. ‫ﺍﻥ‬ ‫ﺍﻟﺩﺭﺍﺴﺔ‬ ‫ﻤﻥ‬ ‫ﻟﻭﺤﻅ‬ ‫ﻭ‬ ‫ﺍﻟﻜﻠﻭﻜﻭﺯ‬ ‫ﺍﻅﻬﺭ‬ ‫ﺍﻟﻔﺭﻜﺘﻭﺯ‬ ‫ﺍ‬ ‫ﺃﻋﻠﻰ‬ ‫ﺇﻨﺘﺎﺠﻴﺔ‬ ‫ﺒﻭﺼﻔﻬﻤﺎ‬ ‫ﹰ‬ ‫ﻤﺼﺩﺭﺍ‬ ‫ﻟﻠﻜﺭﺒﻭﻥ‬ ‫ﻜﻠﻭﺭﻴﺩ‬ ‫ﻤﻊ‬ ‫ﺍﻻﻤﻭﻨﻴﻭﻡ‬ ‫ﹰ‬ ‫ﻤﺼﺩﺭﺍ‬ ‫ﺒﻭﺼﻔﻪ‬ ‫ﻟﻠﻨﺘﺭﻭﺠﻴ‬ ‫ﻥ‬ ، ‫ﻭ‬ ‫ﻜﺎﻨﺕ‬ ‫ﻅﺭﻭﻑ‬ ‫ﺍﻹﻨﺘﺎﺝ‬ ‫ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻨﻲ‬ ‫ﺍﻻﺱ‬ ‫ﻋﻨﺩ‬ ‫ﺍﻟﻤﺜﻠﻰ‬ 7.0 ‫ﺍﻟﻠﻘﺎﺡ‬ ‫ﺤﺠﻡ‬ ، 5-6 % ‫ﻭ‬ ‫ﺒﻌ‬ ‫ﺩ‬ ‫ﺘﺤﻀﻴﻥ‬ ‫ﻓﺘﺭﺓ‬ ‫ﺃﺭﺒﻌﺔ‬ ‫ﺃﻴﺎﻡ‬ ‫ﻟﺩﻋﻡ‬ ‫ﺇﻨﺘﺎﺠﻴﺔ‬ ‫ﺃﻋﻠﻰ‬ ‫ﻟﻤﺘﻌﺩﺩ‬ ‫ﺍﻟﺴﻜﺭﻴﺎﺕ‬ ‫ﺍﻟﺨﺎﺭﺠﻲ‬ ‫ﻗﻴﺩ‬ ‫ﻟﻠﺒﻜﺘﺭﻴﺎ‬ ‫ﺍﻟﺩﺭﺍﺴﺔ‬ .