| Lignocellulose,the main product of carbon dioxide formed by using solar energy and photosynthesis,is the most valuable renewable resource on earth,yet today the disposal of cellulosic waste is still a huge challenge.Currently,most cellulosic materials(e.g.corn stover)are disposed of by in-situ burning,which not only pollutes air quality and damages human health,but also leads to a huge waste of biomass resources.Therefore,developing methods to effectively treat and utilize cellulosic waste as a high-quality carbon source has become a key research goal,and degradation of lignocellulose into biofuel by cellulase is one of the most effective methods to utilize lignocellulose on a large scale.Therefore,in this study,the traditional culture method was used to screen degrading microbial strains from nature that can produce high cellulase,and then select high yielding mutant bacteria by mutagenesis at room temperature under atmospheric pressure(ARTP),optimize the culture process to make them reach the optimal enzyme production state,and analyze the degradation effect of straw under the optimized fermentation conditions to provide an experimental basis and theoretical basis for obtaining efficient cellulase resources.Twenty-four strains with cellulose degradation ability were isolated and purified from humusrich soil by Congo red staining,and the CMC enzyme activity and FPA enzyme activity of the strains were measured,and finally one strain Ax4 with high enzyme activity was obtained,whose CMC enzyme activity was 36.01 U/mL and FPA enzyme activity was 22.70 U/mL,and was identified by molecular biology as Bacillus subtilis,and named Bacillus subtilis Ax4.In order to improve the cellulase producing ability of Bacillus subtilis Ax4,it was mutagenized and bred by atmospheric pressure room temperature plasma mutagenesis,and after Congo red primary screening and enzyme activity re-screening,a mutant strain T-26 with CMC enzyme activity of 71.89 U/mL was obtained,which was 98% higher than the starting bacterium,and its FPA enzyme activity was also increased by 43.15%,and this strain was After 6 consecutive generations of culture,the cellulase activity of its fermentation broth retained more than 85% compared with the first generation strain and was able to be inherited stably.In order to improve the enzyme production ability of mutant strain T-26,the fermentation process was optimized by response surface methodology.The best fermentation conditions were investigated by single-factor experiments with T-26 as the starting bacteria: incubation temperature of 30 ℃,inoculum amount of 6 %,and fermentation time of 96 h.Based on this,PB design was conducted to analyze the significant factors affecting the enzyme production,which showed that corn starch,yeast powder,and initial pH value were the significant influencing factors,and the best enzyme production process of the strain was determined by response surface optimization as corn starch The optimized enzyme activity of T-26 cellulose reached 118.90 U/mL,which was 65.39 %higher than that before optimization.The optimized mutant strain T-26 was used to study the degradation effect of corn stover for30 days.The results showed that the pH value of strain T-26 did not change significantly during the whole fermentation process,and it always maintained a weak acid and neutral environment,indicating that it has a certain self-regulation ability to maintain a stable living environment.The strain was fermented on the 18 th day.At this time,the OD540 value was 0.761,and the CMC enzyme activity and FPA enzyme activity reached the highest at this time,which were 111.09 U/mL and58.69 U/mL,respectively.The results showed that the number of strains had a certain effect on enzyme production.At 30 d,the weight loss rate of the strain’s corn stalk reached 56.31 %,and the corn stalk tissue was damaged to a certain extent,indicating that the mutant strain T-26 had a high ability to degrade the stalk. |
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