| Side effects caused by overuse of chemical fertilizer on soil environment and crop quality has already drawn wide attention.It's now a consensus that organic fertilizer and microbial agent could enhance soil fertility and realize sustainable use of soil.Cellulose-decomposing and nitrogen-fixing bacillus can grow by using cellulose in crop residues,straw turnovers and organic fertilizers as carbon source and is more adaptive to soil environment.Compared to non-nitrogen-fixing bacillus,nitrogen-fixing bacillus is more stress-resistant and the agent has a longer storage life.Screening bacillus strains with strong cellulose degradation capacity and high nitro-fixation amount is significant in both theoretical and practical aspect.This article mainly discussed the relationship between nitrogen-fixing bacillus AX32 and its associated strain AX322.By using UV and NTG mutation and different screening strategies,a mutant strain with high cellulase activity,nitrogen-fixation amount and biomass was obtained.The article also made some explorations on screening drug resistant mutant,ammonia-secreting mutant and ammonia resistant mutant.The results are as follow:1.Cellulose-decomposing and nitrogen-fixing bacillus was screened in the rhizosphere soil sampled from Shaanxi,Shanxi and Hubei Province.Meanwhile,it was found that associated strains always grow with the bacillus.Based on establishing purification system between bacillus and its associated strains,the strains were identified,and the relationship between them was also discussed.The results showed that cellulose-decomposing and nitrogen-fixing bacillus AX32 was Paenibacillus castaneae while its associated strain was Agrobacterium tumefaciens.Compared to solo cultivation,living cell counts in mixed cultivation was low,which indicated a competitive relationship between the two strains.But when culturing together,both cellulase activity and nitrogen-fixation amount increased.2.Cellulase characteristics and its producing conditions were discussed.The optimum reaction pH was 7.0 and the optimum reaction temperature was 30?.Cellulase activity was relatively stable at pH6.8?7.4,22?45?.Fennenting under the optimized conditions,the crude enzyme activity of AX32 broth was 5.385×10-9mol/s,which increased 15.61%in contrast to initial activity 4.658 X 10-9mol/s.3.UV and NTG mutation was conducted to AX32 in exponential growth phase.Different mutant strains was obtained through different strategies.Cellulase activity of mutant strain NTG-50E was 6.593 × 10-9mol/s and increased to 32.10%compared to its initial strain AX32.Nitrogen-fixation efficient mutant strain NTG-40-2NF could reach a nitrogen-fixation amount of 0.0996mg/mL and was 54.6%higher than its initial strain AX32.High increment mutant strain UV-25-1G could reach a increment of 3.46×108 and was 45.4%higher than its initial strain AX32.Microcrystalline cellulose activity of penicillin resistant mutant strain Pr-1 was 5.189×10-9mol/s and was 7.7%higher than its initial strain AX32.The nitrogen-fixation amount was 0.0649mg/mL and was 5.02%higher than its initial strain AX32.The increment was 2.508×108 and was 13.1%higher than its initial strain AX32.For streptomycin resistant mutant strain Sr-1,the microcrystalline cellulose activity was 5.2×10-9mol/s and was 9.7%higher than its initial strain AX32.The nitrogen-fixation amount was 0.0678mg/mL and was 9.7%higher than its initial strain AX32.The increment was 2.42×108 and was 9.2%higher than its initial strain AX32.For rifampicin resistant mutant strain Rr-1,the microcrystalline cellulose activity was 5.432×10-9mol/s and was 12.7%higher than its initial strain AX32.The nitrogen-fixation amount was 0.0669mg/mL and was 8.3%higher than its initial strain AX32.The increment was 2.5×108 and was 12.8%higher than its initial strain AX32.The passage test suggested that all mutant strains were genetically stable.4.AX32 was treated with UV and NTG.By designing different mutation strategies,ammonia-resistant mutant strain and ammonia-secreting mutant strain were obtained.For ammonia-resistant mutant strain NTG-60-NR,the nitrogen-fixation amount was 0.0953mg/mL and was 53%higher than its initial strain AX32(0.0623mg/mL).For ammonia-secreting mutant strain UV-25-NS,the nitrogen-fixation amount was 0.1076mg/mL and was 75%higher than its initial strain AX32(0.0615mg/mL).The passage test showed a genetic stability fir both mutant strains.The conclusions of the article are as follow:(1)Cellulose-decomposing and nitrogen-fixing bacillus AX32 isolated from soil samples taken from different provinces were Paenibacillus castaneae,AX322,the associated strain,was Agrobacterium tumefaciens,there was a competitive relationship between these two strains.(2)The optimum reaction pH of microcrystalline cellulose was 7.0 and the optimum reaction temperature was 30?.The enzyme was stable between pH6.8?7.4 and 22?45 ?.Under the optimized cultivating condition,the crude enzyme activity of AX32 was 5.385 × 10-9mol/s,compared to the initial enzyme activity 4.658 × 10-9mol/s,the activity increased by 15.61%.(3)By proceeding UV and NTG mutation,mutant strain with high cellulase activity,nitrogen-fixation amount and increment,drug resistant mutant strain,ammonia-resistant mutant strain and ammonia-secreting mutant strain were obtained.Passage test suggested they were genetically stable.The innovative points of the article are:(1)Cellulose-decomposing and nitrogen-fixing bacillus Paenibacillus castaneae and its associated strain Agrobacterium tumefaciens were isolated and they showed a competitive relationship.(2)By proceeding UV and NTG mutation,mutant strain with high cellulase activity,nitrogen-fixation amount and increment,drug resistant mutant strain,ammonia-resistant mutant strain and ammonia-secreting mutant strain were obtained and they were genetically stable.Due to the limited time,this research only gained different mutant strains,if multiple-parent progressive protoplast fusion had been conducted,fusion strains with high enzyme activity,growth rate and nitrogen-fixing capacity might have been obtained. |
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