The Isolation And Screening Of Bacillus Strains Producing Organic Acids On Microbial Corn Stalk Silage

As a mature technology in the utilization of straws, silage treatment results in good retention of nutrients thereby providing a source of cheap but high-quality forage for the animal husbandry industry. Currently, Lactic bacteria are the most commonly used microbial additives for the microbiological fermentation of corn straw. However, this group of bacteria is not resistant to high temperatures and is easily inactivated in the process of production and preservation, thereby limiting its usefulness in application. To resolve this issue, Bacillus strains capable of producing organic acids were screened in this study in order to identify potential agents for microbiological fermentation and the storage of fresh corn stalks.The experiment obtained more than 200 strains of Bacillus spp., which were isolated from fresh cattle manure. Initial selection identified 13 organic acid-producing strains, as demonstrated by their ability to turn bromocresol purple plates yellow. These 13 strains were then rescreened using the drilling method. Finally, R42-16, a strain of bacillus with high acid yield was obtained. Further analysis of this strain revealed it is able to produce many different types of organic acids, with acetic acid and lactic acid being higher than other organic acid produced at 220.70μg/m L, 174.25μg/m L respectively. R42-16 was identified as Bacillus amyloliquefaciens through the analysis of colony morphology, bacteria characteristics, physiological and biochemical experiments and 16 S r DNA gene sequencing.The study of R42-16’s biological characterization showed that the strain had a good tolerance to lower p H conditions, ranging from 1.0 to 7.0 and bile salts, ranging from 0.1% to 0.3%. The strain also demonstrated relatively high utilization of sucrose and fructose with strong antagonistic activity to E. coli O157:H7.In order to provide a theoretical reference to the industrial production of the strain R42-16, we conducted the single factor test and orthogonal test to the fermentation medium components and the fermentation conditions. The results showed that the optimum fermentation medium is as follows: corn meal 1.0%, yeast extract 2.0%, Mn SO4·H2O 0.01%, Ca Cl2·H2O 0.01%, and the optimum fermentation conditions are: initial p H6.0, fermentation time 48 h, inoculation amount 4.0m L, loading volume 50 m L/250 m L. Under these conditions, the rate of spore formation reached 93.76% and total viable bacteria reached 3.19×109CFU·m L-1.This paper evaluated the effect of strain R42-16 on straw fermentation by administering the microbial inoculum in amounts of 8.0×108CFU/kg(Test groupⅠ), 1.0 ×109CFU/kg(Test groupⅡ) and 1.2×109CFU/kg(Test groupⅢ), and allowing for a 30 day fermentation period. The comparison of straw nutrition of the test groups is as follows:Compared with the control group, no significant difference(p>0.05) as found in the content of lactic acid and acetic acid in test group I, where as test groupsⅡ and Ⅲ saw significant increases(p<0.05).The content of lactic acid in Test groupⅡand Ⅲ increased by 24.38% and 25.0% with acetic acid increasing by 292.87% and 338.52% respectively. No significant difference(p>0.05) was found between the test groupⅡ and test groupⅢ. Compared with the control group, no significant difference(p>0.05) was found in the content of butyric acid and ammonia nitrogen/total nitrogen in the test group I. Test group Ⅱ and III saw significant reductions(p<0.05), with butyric acid decreasing by 47.34% and 53.36% and ammonia nitrogen/total nitrogen decreasing by 36.89% and 42.33% respectively. No significant difference(p>0.05) was found between the test groupⅡ and test group Ⅲ.Compared with the control group, no significant difference(p>0.05) as found in the content of crude protein in test group I. Test group Ⅱ and III saw significant increases(p<0.05) at 50.26% and 62.39% to reach 8.43% and 9.11% crude protein content respectively. No significant difference(p>0.05) was found between the test groupⅡ and test group Ⅲ.The content of dry matter, crude fiber, neutral detergent fiber, acid detergent fiber, acid detergent lignin content and other indicators were not significantly different(p>0.05).To address the issue of silage being prone to "secondary fermentation" when exposed to air, this paper researched the stability of silage through evaluation of temperature, p H, smell sensory and etc. At a room temperature of 18 oC, both the three test groups and the control group were close to room temperature from day 1 to 8. The temperature of the control group began to rise on day 9 and was 1.57℃ higher than room temperature, at this time silage was beginning to show signs of instability. The control group was 3.09℃ higher than room temperature by day 14 and presented "secondary fermentation" symptoms, while samples with straw strain R42-16 additives were only about 1.1℃ higher than the room temperature.The p H values of the control group and test groups had small difference between days 1 to 8. However, the p H values of the control group rose quickly between days 9 to 12, to reach 5.59, signaling secondary fermentation. Test groups I, II and III remained relatively low at 4.23, 4.03 and 4.03 respectively on day 15, thereby demonstrating the effectiveness of R42-16 in maintaining the stability of silage.In conclusion, strain R42-16 can be used as an additive in the microbiologically fermentation of corn stalks.