Research On Biodegradation Of Poultry Feather By Bacillus Licheniformis And Its Keratinase Fermentation

There are plenty of resources on feather keratin in our Country. Livestock andpoultry feather accumulate an abundance of potential protein and amino acids, whichcould be used for the source of excellent feedstuff protein. It is the key to find anappropriate way to degrade keratin in this study, as the keratin is very difficult to bedecomposed due to the steady configuration. Different from conventional methodssuch as physical or chemical degradation, biodegradation holds outstanding effect andno defects like low efficiency, high expend and excessive contamination, which notonly makes use of the protein source but also helps to protect environment. As theapplications of keratin and keratinase to all kinds of industry emerged more, those areconsidered to be provided with more values of economic and social, and with brilliantprospect.This study is mainly focused on the utilization of keratin, starting from isolateoptimal strain. The fermentation process and technic have been researched; thecharacterization of crude enzyme and the cultivation dynamics models have beendiscussed. The results completed are reported as follows:1. Two strains degrading feather keratin were identified that could grow on themedium with feather as the solo carbon,nitrogen and energy sources. Between twostrains, Bacillus licheniformis ZJUQH was preferred, which can make the featherbreak down in culture and decompose after 4d～5d fermention in 250ml-flask. Thephenomenon of feather-degrading by Bacillus licheniforrnis ZJUQH was screened byscanning electron microscope during 5d.2. Degrading feather by Bacillus licheniformis ZJUQH cells and adopting theorthogonal design to optimiz the main factors that affect feather decompose, theexperimental results indicated that the best scheme to degrade keratin was as follows:capacity 25ml/250ml, initial culture pH 7.5, cells mass 0.5g, when the cells weregrowed at 40℃, 120r/min in 48h. Then pH7.5 was the best scheme to degrade featherin the same instance above. The former was significant and validated by verifyingexperiments showing that the activity of keratinase was 55.7U, better than theuntreated.3. Culture conditions for keratinase production and feather degradation wereoptimized with using statistical design. Digestion could be improved beforefermentation as feather was treated with Ca(OH)₂ for 3h. Glucose added was a bettercardon source at low concentration to promote synthesizing enzyme and disintegrating feather, while there were no effects as added nitrogen source. Cornsteep flour would make the bacteria grow better and promote keratinase production.Ca²⁺, Mg²⁺ and Tween-80 could promote enzyme production, but the effects werenon-significant. The culture medium was optimized with response surfacemethodology. The optimized culture medium was composed of 2ï¼…treated featherpowder, 1.45ï¼…glucose, 0.735ï¼…corn steep flour, 0.195ï¼…K₂HPO₄, initial medium pH7.5, and with the optimized environment condition at 37℃, 180 r/min. The highestactivity of keratinase would reach 85.8U after 72h, and the content of soluble proteinwould reach 1.13mg/ml after 96h, respectively. The percentage of feather degradationwas 84.06ï¼…at the end of 5d.4. Experiment in 5-L auto fermentor with optimal culture medium. Under the batchcultivation conditions of stirring rate 200r/min, 1L/min aeration rate, 37℃temperature and ratio of liquid content 0.6, the highest keratinase activity producedwas 61U at 72h, the peak content of soluble protein was 0.8874mg/ml at 108h, andthe final percentage of degradation was 64.1ï¼…. It would make a base of scale-upresearch deeply in the future though the result was not as good as in flask experiment.5. The dynamic models of keratinase production were established. One was used forestimate the growth kinetics as keratinase production, and modelwas: X=46.4356/(1+e^{3.8026-0.111125t}). The other was an sudstrate consumption kinetics as featherdegradation, and the model constructed was:X(t)=(1131253.4475-8329.8338t)^0.329965. Their significances were discussed andevaluated, and the parameters of kinetics parameters were computed and analyzed.6. The amino acids from fermentation broth by 5-L fermentor were assayed. Some ofamino acids such as lysine, histidine and methionine were increased greatly than thatof before experiment. The digestion and the nutrition compositions had beenimproved after the biodegradation. So it was viable to produce the amino acid of highquality in nutritional ingredients by Bacillus licheniformis ZJUQH in fermentationprocess. And the scale could be enlarged if the conditions were improved in thefurther research.7. The crude keratinase had been extracted and analyzed from the fermented liquid.The optimum temperature for the activity of keratinase was 37℃, and the keratinsewas stable between 35℃～45℃. The activity would decline above 45℃and losemost of activity when treated for 90 min as temperature above 50℃. The optimum pHwas 8.0, and was stable between 7.5～8.5. The activity would drop out of the range,when the keratinsae treated for 90 rain above pH 10.0 or low pH 6.0. PMSF, EDTA, Cu²⁺ and Hg⁺ were the inhibitors, while the Ca²⁺ and the mercaptoethanol were theactivatora for keratinase activation.