Screening And Application Of High-efficiency Straw Cellulolytic Bacteria Based On High-throughput Technology

Cellulose biomass resources are one of the most abundant renewable resources in nature.The development and utilization of cellulose resources is of great significance in solving the current global problems such as environmental pollution,energy crisis and food shortage.In order to further increase the decomposition efficiency of cellulosic biomass and obtain more cellulose degrading bacteria resources,the screening of cellulose-degrading bacteria was carried out.This study first established a screening method for high-throughput cellulose-degrading bacteria.Based on the traditional screening principle of cellulose-degrading bacteria,24-well plates and 96-well plates were used as carriers to reduce the system and increase the screening throughput,combined with the identification of Congo red medium,and the ability of the microplate reader to collect data in batches.A set of trace,sensitive,and rapid high-throughput screening methods were established,and the culture system and detection system of the method were optimized.The results showed that when the 24-well plate was filled with 2.5mL of liquid and the incubation time was 48 h,the growth of microorganisms was the best.When the detection wavelength of the microplate reader was 500 nm and the amount of DNS in the 96-well plate was 120?L,the detection system had the best accuracy.Based on the high-throughput screening method established in this study,nine strains of cellulose-degrading bacteria were obtained from the initial screening of cow dung.After the 24-well plate was rescreened,the cellulase-producing strain C-7 was selected for further study.Strain C-7 is Gram-positive Brevibacterium and it has a size of about 1.5 to 2?m×0.5?m,Initially identified as Aneurinibacillus sp.strain N8.The enzyme production conditions were optimized.At the initial pH of 9,shake flask culture at 55°C for 120 h,the strains showed the highest activity of enzyme production.At this time,CMCase activity and FPase activity were 1.68 U/mL and0.91 U/mL,respectively.Using soil,cow dung,and cow rumen fluid as screening sources,under the anaerobic conditions,long-term enrichment and domestication culture,the complex strain H1 with highly efficient cellulose degradation function was obtained.The cellulose degradation rate of filter paper could reach 89%within 5 days.The degradation of filter paper cellulose could be maintained at different cultures,different inoculations and different concentrations of substrates.And the degradation rate of natural cellulose such as corn stalk and rice straw reached 61.5%and 56.4%respectively.The main products of fermentation were butyric acid and acetic acid,and a certain amount of H₂ could be produced during the fermentation.Using 0.5%filter paper,corn stover and rice straw as substrates,fermented for 7 days each,the production of acetic acid was approximately 1566 mg/L,923 mg/L,and 740 mg/L,respectively.Butyric acid production was approximately 2600mg/L,1323mg/L and986mg/L,respectively.The accumulation of H₂ was about 32ml,9.6mL and 5mL respectively,accounting for about 23%,15%and 12%of the total gas production.In the course of continuous enrichment and cultivation,the composition and proportion of micro-organisms in the complex strain H1 tend to be stable.The high-throughput sequencing of the complex strains revealed that Clostridium was the dominant dominant species,with the main function being cellulose degradation and hydrogen production,followed by a portion of rod-shaped bacteria,the main effect being acid production.