Enhanced Production of Butyric Acid through Solid State Fermentation of Agriculture Waste by Mutant Clostridium tyrobutyricum and its utilization in early Rumen development

Abstract

Due to lessening availability of crude oil, increasing requirement for natural foodstuffs, increasing worries over environment and one of alternative fuel, production of butyric acid through bacterial activity is greatly increased. The present study was designed to improve its production through solid state fermentation technology by microbe using cheaper renewable biomass. For this purpose three agriculture wastes as carbon source were tested for optimum yield applying solid state fermentation technique by wild C.tyrobutyricum. Rice polishings with 2 mL inoculum volume of the growth medium at 37oC, pH 6.0 for 3 days incubation period and 10:32 substrate: water ratio gave the best yield (6.49mg) among them and was selected for further optimization procedure to get higher yield of butyric acid. Various concentrations of different ionic salts such as NaCl, CaCl2, MgSO4.7H2O and KH2PO4.2H2O were optimized and obtained 6.52, 6.89, 8.15 and 8.95 mg butyric acid respectively. The addition of optimized 1:20 nitrogen: carbon ratio of molasses and optimized 0.5 mL concentration of corn steep liquor as an additive gave significantly (P< 0.05) highest yield i.e.11.07 and 18.23 mg butyric acid respectively. To further enhance the yield of butyric acid, bacterial strain was upgraded. For this purpose two chemical mutagens ethidium bromide, nitrous acid and physical mutagen UV irradiation were utilized. The RCM plates containing more than 90% kill rate were selected and mutants were screened on RCM media for optimized yield of butyric acid against wild C.tyrobutyricum. The UV mutant gave the better yield of butyric acid 4.46 mg than wild and other two mutants (nitrous acid and ethidium treated). The optimized three day incubation period gave butyric acid yield (6.86 mg %) which was further increased at10:32 substrate: water ratio (7.47 mg) and by optimizing inoculum volume, better yield (7.69 mg) of butyric acid was achieved with 2.5 mL inoculum volume. Different ionic salts such as NaCl, CaCl2, MgSO4.7H2O and KH2PO4.2H2O raised butyric acid yield 8.8 and 9.6, 10.52, 10.66 mg respectively. The addition of optimized 1:25 nitrogen: carbon ratio and under optimized 0.6 mL concentration of corn steep liquor of the growth medium as an additive gave significantly (P<0.00) the highest yield 11.63 and 26.09 mg of butyric acid respectively. The mutant strain C.T UV showed more butyric acid capability compared to wild C.tyrobutyricum. Detection and estimation of butyric acid was carried out by organic analysis method by G. Deniges (1918). The parameters optimized on small scale by mutant C.T UV were then used on laboratory scale for butyric acid production and was biologically evaluated. It was carried out by conducting 90 days feeding trials on sixteen Lohi sheep kids of age between 25-35 days, almost uniform weight and mixed sex. The animals were randomly grouped into A, B, C, and D containing four sheep each. D group of sheep was served as control group and was fed on control diet. Whereas A, B, C were supplemented with 0.2, 0.4, 0.6 % (w/w) butyric acid along with the same rations fed to control group. All animals were weighed initially and thereafter weighed weekly. While blood sampling for estimation of butyric acid, complete blood count and glucose were done initially and thereafter fortnightly. To visualize the inner surface of walls and papillae development in the rumen the Pentax EPK-100 P endoscope was used for sheep. One fasted sheep from each group was randomly selected for this purpose as well as for slaughtering at the end of feeding trial. From different portions of rumen i.e. dorsal, ventral, caudal dorsal, caudal ventral, dorsal cranial and from ventral cranial sacs of each slaughtered animal samples were taken for histological examination. Slides were observed under light microscope (Labomed USA) for measuring the papilla length, height, width, denseness and surface area of papilla by using the Morphometric program pro ResR 2.1.1 Capture Prog Camera Control software. The results showed non-significant (P > 0.05) increase in CBC and in blood glucose levels in experimental animals as well in control groups. Whereas significant (P<0.05) difference was seen in butyric acid levels in serum among treatments and control. Group C showed significantly higher concentration of butyric acid in serum than others. Among all groups Non significant (P >0.05) effect on body weights was noted. However a significance difference in the papillae length, width, rumen sacs surface areas and denseness was noted, when histological examination of rumen sacs was performed. It is concluded that solid state fermentation technique can be applied on industrial scale to produce massive quantity of butyric acid by mutated strain of C.tyrobutyricum. Butyric acid augmented in the diet improved the rumen growth in sheep.