| To promote progress of cotton stalks as roughage and alleviate the shortage of forage resources in southern Xinjiang, we screened lactic acid bacteria, silage microbial flora (SMF), and cellulose-degrading microbial flora (CDMF) to improve unilization rate of cotton stalk. Lactic acid bacteria were isolated from the silage samples and forage. We screened and purified bacteria by the traditional method, conventional physiological & biochemical method, and 16S rDNA method. The stability and diversity of silage microbial flora (SMF), and cellulose-degrading microbial flora (CDMF) were analyzed by DGGE method. The CDMF was determinated enzyme activity, and. We screened the best method of pretreatment for cotton stalk by determining degradation rate of cellulose and lignin. Our aim was to improve the efficiency of lignocellulose degradation in cotton straw using microbial synergy. CDMF, white rot fungi(Phanerochaete chrysosporium, PC) and SMF were incubated with cotton stalks.In the experiment of pretreatment and enzymatic hydrolysis cotton stalk, the highest rate of weight loss was 18.9% by cellulase from Trichoderma viride and alkali or microwave pretreatmnt. The rate of enzyme hydrolysis was the highest to 31.84% by adding cellulase from Trichoderma viride while the cotton stalk was pretreated by alkali and microwave. The rate of saccharification was the highest to 18.3% by sulfuric acid pretreatment. The pretreatment was smaller to 8.85% by the double oxygen water. The rate of glucose yield was the maximum to 1.01% by adding Trichoderma viride while the cotton stalk was pretreated by alkali and microwave.32 strains were isolated from forage and corn silage, through the normal physiological and biochemical experiment and acid yield rate.12 strains of bacteria were chosed for further experiment. The bacteria were idtified by biolog technology and 16 S rDNA technologies. The results were bacillus licheniformis, lactobacillus crustorum, lactobacillus rhamnosus, lactobacillus casei, pediococcus acidilactici, enterococcus, staphylococcus, and bacillus. SMF became to stabilize after 20 generations continuous cultivation. We analyzed diversity and stability by PCR-DGGE technology.The SMF was made from four kinds of microbial silage bacillus lichenniformis, lactobacillus crustorum, lactobacillus rhamnosus, and Lactobacillus casei.We screened five strains for decomposing cellulose from cotton feild soil. After cultured for 20 days, T4 strain the degradation rate of cellulose and lignin were 20.91%,13.71% respectively, T3 strain was 9.46%,21.43%, T2 strain was 22.19%,11.76%, M4 strain was 16.74%,11.45%, M3 strain was 19.74%,11.16%. The five strains of cellulolytic bacteria and PC were mixed culture, and fermentation period was 20 days. We found a mixed strains of cellulose and lignin degradation rate were XT2+PC 34.26%,25.44%; XT3+PC 29.29%,28.69%,35.09%, XT4+PC 27.61%, XM4+PC 27.67%, XM3+PC 32.4%; 44.11%,19.5% in cotton stalk. T2, M3, T4 strains for claodosporium, aspergillus, and penicillium. T3 strain was bacillus subtilis. M4 strain was bacillus megaterium.The experiment was designed to optimize the fermentation parameters. The results showed that the microbial flora degradation of acid pretreatment cotton stalks, at the 5 th day of the fermentation, CMCase, FPase and -G gained the highest value, so 4-day as the fermentation period. Nitrogen source, original pH value and inoculation quantity set in process of acid pretreatment cotton stalks degradation showed very significant effects on CMCase, FPase, B-G and glycosylated ratio (P<0.01). Effects of temperature on FPase and cellulose-decomposing ratio reached very significant levels (P<0.01). The temperature affected glycosylated ratio significantly (P<0.05), while other factors had no significant affect. Considering the effects of all the four factors on cellulase activity, glycosylation and cellulose-degradation of pretreatment cotton stalks, the optimized conditions were (NH4)2SO4 as nitrogen source, temperature at 42℃, original pH value at 6.8, inoculation quantity at 1.0%.The cellulose-degrading microbial flora(CDMF) were created using artificial manure (mixture with ox dung, pig manure, chicken manure). The CDMF degraded natural cellulosic materials and had a high degree of stability.Cellulose-degrading microbial flora, whitelulose degradation in cotton straw rot fungi and silage microbial flora using were incubated with cotton stalks. The results revealed that the β-G, manganese-dependent peroxidase (MnP) and laccase (Lac) were improved by 23.34%,85.85% and 150%, respectively, with treatment D. With treatment E, CMCase and β-G were improved by 41.98% and 48.22%, respectively. With treatment F, Lac, LiP, CMCase and β-G were improved by 50%,41.18%,29.75% and 84.85%, respectively. The glucose conversion coefficient peaked at 26.86% with treatment C.The weight loss of the cotton stalk ranged from 24.27% to 52.87%, indicating that fermentation microbes were able to contribute to degradation of cotton stalks (P<0.01). With treatment F,42.69% of hemicellulose,44.30% of cellulose and 30.45% of lignin were degraded. The CP content was improved by 5.05-9.37%. As measured by an in vitro method, the DM degradation rate was at its highest with treatment F pH and NH3-N concentration did not differ significantly between treatment groups (P>0.05),with the exception of treatment F obviously low in NH3-N concentration (P<0.05). Treatments D, E and F resulted in higher TVTA, acetic acid, propionic acid, butyric acid and CHQ levels compared with treatments B, C and A. In nylon bags, fermentation of cotton stalks had more of a positive effect on DM degradation. The DM degradation rate ranged from 3.90% to 13.84% for F and E (both P<0.01) and D, C and B (all P<0.05). The CP degradation rates with treatments F, D and E were 7.04%,6.65% and 6.46%, respectively (P<0.05). With treatment F, the lignin degradation rate was at its highest at 27.39% (P<0.01), and rates for the other treatments were all higher than with treatment A (P>0.05).Overall, fermentation can greatly improve the degradation of cotton straw in vitro and in nylon bags using microbial synergy in lignocellulose biodegradation. |
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