| The northeast is the largest main production area of maize in China,and the amount of straw produced is huge.In northeast China,the cold time is up to six months a year,which greatly limits the decay rate and quality of straw returning to field,and seriously affects the production of the next year,so it is of economic and environmental significance to carry out the research of low temperature humus agent of corn straw.The cellulose degradation bacteria which can adapt to the low temperature environment in northeast China are selected from the soil,which provides theoretical support and strain resources for the final solution of the problems of corn straw in northeast China which is not easy to decompose and low utilization rate under low temperature environment.In this study,16 samples of different bacteria from low temperature environment were cultured by enrichment culture method,and the diluted plate method with different concentration gradients was used to isolate and purify the sodium carboxymethyl cellulose medium.The strains were then cultured in a liquid fermentation medium,and cellulase activity was used to re-screen the strains which could effectively degrade cellulose and have high straw degradation rate.Finally,the morphological characteristics were observed and the strains were analyzed by 16 Sr DNA gene sequence.The nucleic acid sequence of the test gene was compared with the sequence in the Gen Bank,and the phylogenetic tree was established.Results two strains of cellulose degradation bacteria,4# and 11#,which could degrade corn straw efficiently at low temperature,were isolated from soil.4# and 11# can grow stably on 16℃ carboxymethyl cellulose Medium,and have obvious transparent ring,and the ratio of transparent ring diameter to microbial colony diameter can reach 0.4-0.6.The cellulase activity of crude enzyme solution measured by 4# strain in enzyme culture medium under 16℃ was larger,the activity of filter paper enzyme was 18.24U/m L,excision enzyme was 11.69U/m L,β-glucose glycosides was 5.37U/m L,and the strain was cultured at 30 d under 16℃.The degradation rate of corn straw segment was as high as 27.6%.The 11# of crude enzyme liquid cellulase,which was measured after culture in 16℃ enzyme culture Medium,was the most significant among the 11 strains screened,the filter paper enzyme was 22.59U/m L,the enzyme activity was 11.07U/m L,and excision enzyme activity was 12.39U/m L,β-glucose glycosides 7.16U/m L The degradation rate of corn straw segment was 29.1% after the strain was cultured under 16℃.Strain 4# and 11# were analyzed by homologous ratio to 11# strain was the genus Ochrobactrum(Ochrobactrum sp.),and the strain of 4# was the genus Pseudomonas(Pseudomonas sp.).The composition and fermentation conditions of the medium have important effects on the growth,development and enzyme production of the strain,so it is particularly important to construct a liquid fermentation condition suitable for the growth of the strain.In this study,the liquid fermentation conditions of strain 11# were optimized,and it was found that the strain was 0.7%-1.2% carbon source concentration,organic nitrogen source,medium starting p H of 4,inoculation volume was 6%,25℃ under the condition of culture can be better real estate cellulase,of which,carbon source concentration,Nitrogen source concentration and culture temperature were the three most significant factors among these factors.The fermentation conditions of these three factors were optimized by response surface method,and the results were optimized from the original 39.518U/m L to 59.146U/m L,which increased by 49.67% compared with the previous enzyme activity.Although the fermentation conditions of single strain 11# liquid enzyme production were successfully improved,the enzyme activity of cellulase was increased,but in practice,there was a certain gap between the single strain of low temperature cellulose degradation bacteria and other low temperature cellulose degradation strains.In this experiment,two and two strains of the known strains that could degrade corn straw efficiently were combined,and six groups of stable combination strains were obtained,enzyme activity study and straw degradation test were carried out on the combination strains,and the results showed that the 11#-A2 combined bacteria had the best degradation effect in low temperature environment,the degradation rate of straw could reach more than 41%,The degradation rate of straw increased by 41.38% in 29%.And the combination fungus 11#-A2 in different low temperature environment four cellulase activity than other combination of the enzyme activity is high,so the combination bacterium 11#-A2 is the dominant combination of bacteria.Using the response surface method to optimize the 11#-A2 of the combination fungus,the optimum conditions for 11#-A2 enzyme production in the fungus system were cultured 8.93 d,the starting p H was 4.93,the culture temperature was 22.89℃,and the inoculation amount was 6.15%,the maximum value of endonuclease enzyme activity was 72.631U/m L,And the degradation rate of straw reached 41%. |
