| In recent years,the straw produced by agricultural production activities in northern China has increased year by year.The average temperature in winter is low and precipitation is low.The main component of straw is cellulose,which is not easy to decompose in a short time.Waste straw has become an obstacle to agricultural production.Therefore,screening straw degrading microorganisms adapted to low-temperature field environment,establishing high-efficiency cellulose degrading microorganisms and accelerating the decomposition process of straw in-situ returning to the field can provide theoretical basis and technical support for the full utilization of straw resources,soil fertilization and sustainable development of ecological agriculture in China.In this study,low-temperature cellulose-degrading strains were screened from low-temperature surface soil and some humus soil by means of primary screening,secondary screening,DNS colorimetric method,molecular biological identification and single factor test.The response surface optimization of enzyme-producing conditions of the composite strains was studied.The main results were as follows:(1)43 strains of low-temperature cellulose-degrading bacteria were isolated and purified through primary and secondary screening,and could still degrade cellulose at low temperature of 4-20 degrees.However,Congo red staining and enzyme activity test showed that only three strains(X24,X26 and X37)met the conditions of high efficiency and low-temperature tolerance.After colony morphology and molecular biological identification,the strain X24 is Stenotrophomonas maltophilia,strain X26 is Flavobacterium johnsoniae,strain X37 is Pantoea rodasii and all three strains are Gram negative bacteria.There was no antagonistic reaction among three strains,and then four groups of low temperature and high efficiency composite strains were constructed: A(X24 + X26),B(X24 + X37),C(X24 + X26 + X37),D(X26 + X37).(2)The results of cellulase activity determination showed that the enzyme production ability of composite strain C was generally higher than that of other single strains and composite strains.The cellulase activity of strain X37 was the highest among the three strains.The CMC activity of strain X37 was 20.826 U/mL,the filter paper activity was 13.68 U/mL and the activity of beta-glucosidase was 12.03U/mL after incubation at 12 ℃ for 3 days.Among all composite strains,low temperature complex strain C had the most prominent ability to produce cellulase enzymes.The CMC activity was 26.96U/mL,the filter paper enzyme activity was 19.73 U/mL,and the beta-glucosidase activity was 14.69U/mL after incubation at 12 C for 3 days.Therefore,the cellulase activity of low temperature complex strain C is more efficient than that of single strain.(3)45d solid-state and 45 d liquid-state fermentation experiment at low-temperature showed that compared with natural straw degradation rate,straw degradation rate of strain X37 liquid-state fermentation increased by 30.63%,straw degradation rate of solid-state fermentation increased by 22.25%,degradation rate of composite strain C solid-state fermentation increased by 30.1%,degradation rate of liquid-state fermentation increased by 39.86%,far higher than other single strain and composite strains A,B and D.This indicated that compound strain C had strong degradation ability to crop straw.(4)According to preliminary determination of single factor test results,the optimum conditions for cellulase production of the composite strain C were as follows: culture time 96 h,inoculation volume 4%,temperature 12 ℃ and pH 6.0.The results of response surface analysis and optimization by DesignExpert 8.0 software showed that the model was well fitted.The better optimum conditions of CMC enzyme production by composite strain C were predicted as follows: inoculation volume 4.6%,temperature 12.5 ℃,incubation time 98 h and pH 6.4.It was proved that the activity of CMC enzyme produced by the strain was(26.26 +0.15)U/mL,which was very close to the predicted value of the model(26.19 U/mL),1.75 U/mL higher than that before optimization,and the optimum rate was 7.13%.It indicated that the model could test the predicted results.In summary,the components of low temperature efficient cellulose degradation complex strains are Stenotrophomonas maltophilia,Flavobacterium johnsoniae and Pantoea rodasii,which have the characteristics of low temperature resistance and high cellulase production capacity.Therefore,the breeding of the low temperature cellulose degradation complex strain has very broad application prospects and research and development value for improving the comprehensive utilization rate of straw and soil fertility under low temperature environment. |
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