【作者】 耿丽平；

【导师】 刘文菊； 陆秀君；

【作者基本信息】 河北农业大学 ， 土壤学， 2012， 硕士

【摘要】 中国是农业大国,年产各种农作物秸秆约8.0亿t,占世界秸秆总产量的20%～30%,其中绝大部分作为废弃物被丢弃焚烧,不仅浪费了资源,也造成了严重的环境问题。秸秆还田是当今世界上普遍关注的一项培肥地力和增产增效的农艺措施,但还田玉米秸秆存在腐解难的问题,如何加速还田玉米秸秆的腐解是目前研究的热点。本研究采用纤维素-刚果红染色法从河北省山前平原小麦-玉米轮作区且长期秸秆还田的小麦季农田土壤中分离筛选出高效分解纤维素的菌株,在优化其产酶条件、发酵条件和CMC酶活力的基础上,进行了该菌株对玉米秸秆的腐解能力、土壤中固定态磷的释放和小麦生长及其磷营养状况的影响等方面研究。主要研究结果如下:1.采用纤维素-刚果红染色法,经形态鉴定初步筛选出具有纤维素分解菌真菌8株,细菌20株;进一步采用水解圈测定,酶活力比较分析,得到高效纤维素分解真菌1株,编号为HB1,经18S rDNA基因序列分析,确定为草酸青霉(Penicillium oxalicum),命名为草酸青霉HB1(P. oxalicum HB1),该菌株现保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏号为CGMCC NO. 4842;得到高效纤维素分解细菌3株,编号为B1,B2和B3,其中B1、B2为芽孢杆菌,B3为无芽孢杆菌;室内摇瓶试验研究了草酸青霉HB1和3株细菌的生长曲线,并对内切葡聚糖酶(CMC酶)和滤纸酶(FPA酶)活力进行了测定。结果表明,利用CMC-Na培养基,按1%接种量,30℃,180rpm摇瓶培养72h,草酸青霉HB1菌株的CMC酶和FPA酶活力分别达672.8U和774.6U。培养12h时,B1和B3菌CMC酶活力近峰值,分别为31.2U和24.4U;培养18h时,B2菌CMC酶活力达最高,为36.3U。培养12h时,B1菌FPA酶活力达高峰,为34.1U;培养8h时,B2、B3菌FPA酶活力最大,分别为39.2U和163.6U。综合比较发现草酸青霉HB1菌株的CMC酶和FPA酶活力最高,因此对该菌株展开如下系统的研究。2.草酸青霉HB1菌株发酵条件的优化首先选择氮源、接种量、培养温度、发酵时间、培养基初始pH进行单因子试验,对草酸青霉产酶条件的影响,结果表明该菌最佳产酶条件为3%的牛肉膏蛋白胨为氮源,接种量为5%,培养温度为28℃～35℃,pH=4.0～7.0,培养48h～72h。进一步采用正交设计研究了pH、温度、固液比和培养时间四因素对草酸青霉HB1菌株发酵的影响,综合考虑CMC酶、FPA酶、纤维素酶、半纤维素酶四种酶活力,该菌株最优发酵条件为固液比为1:10,培养时间为48h,培养温度为30℃,pH为6.5。在草酸青霉HB1菌株作为纤维素分解菌,以CMC-Na液体培养作基质,研究CMC酶活力的结果表明,该酶反应的最适pH为5.0左右,其pH的稳定性在4.0～5.0范围内,酶反应的最适温度为50℃,其热稳定性在40℃～50℃之间。3.草酸青霉HB1对玉米秸秆腐解能力评价采用室内摇瓶试验、土样培养试验及小麦盆栽试验对其进行了系统研究。利用玉米秸秆替代CMC-Na的培养基中,按1%接种量,30℃,180rpm摇瓶培养72h,CMC酶和FPA酶活力则分别为282.9U,618.3U,纤维素酶活力为376.1U;利用3%的牛肉膏蛋白胨为氮源,接种量为5%,培养温度为30℃,pH=7.0,培养72h,其CMC酶活力高达888.8U。从腐解秸秆的形态特征发现,草酸青霉HB1处理的秸秆与对照相比形态发生明显改变,在培养10d后,秸秆腐解率达87.3%,为对照的1.90倍;初步说明该菌株具有较强的玉米秸秆腐解能力。在土壤培养试验中,培养30d时,接种草酸青霉HB1的土壤中玉米秸秆的腐解率达83.5%,是对照的1.62倍;在小麦盆栽试验中,培养50d时,接种草酸青霉HB1的土壤中还田秸秆腐解率达70.8%,高于对照15.1%。4.土壤中接种草酸青霉HB1的解磷作用及其生物效应研究土壤培养试验以及小麦盆栽试验研究还表明,接种草酸青霉HB1有利于土壤固定态磷的释放和提高植物对磷的吸收利用。在土壤培养30d时,处理组和对照组中土壤速效磷含量分别达98.6mg/kg和77.1mg/kg,高出对照27.9%;在培养50d时,小麦植株地上部和地下部,处理组和对照组全磷含量分别为12.6g/kg、7.62g/kg和25.9g/kg、21.1g/kg,分别高出对照65.3%和22.7%,差异显著性分析表明,各处理组与其对照组差异显著;同时有利于植物的生长,在培养30d时,小麦植株生物量增加16.8%。本试验所获得的草酸青霉HB1对玉米秸秆腐解的同时在土壤中解磷的特性还未见其他作者报道,具有进一步利用研究的价值。更多还原

【Abstract】 China is a large agricultural country with the production of crop straw 800 million tons per year, accounting for 20% ~30% of total output in the world. Most of them are burned in the farmland after grain harvest as waste, which not only waste the resources, but also cause the pollution of the environment. Straw returning is a beneficial measure in farmland, which can increase soil fertility and crops production generally.The decomposition of corn stalk returning to soil is very difficult and slow. It has been the hot topic how to accelerate the decomposition of corn stalk in soil around the world. Isolation of cellulose-decomposing bacteria or fungi was conducted using method of cellulose-Congo red stain in soil with straw returning which was collected from wheat-corn rotation farmland in Hebei province, its application of beneficial soil rapidiy available phospprus has a decomposition release and can increase the absorption of plant phosphorus in the soil. On the basis of optimize the fermentation process, the conditions for enzyme production and enzymatic properties. The main results showed as follows:1. Eight strains of fungi or 20 strains of bacteria were isolated using method of cellulose-Congo red stain. One fungus and three strains of bacteria were screened with hydrolysis circle determination and enzyme activity analysis.The 18SrDNA gene sequence showed that the fungus strain isolated was identified as Penicillium oxalicum. The isolated strain was named Penicillium oxalicum HB1 (P.oxalicum HB1), preserved in the China General Microbiological Culture Collection Center (CGMCC), preserved No. of CGMCC NO.4842. Moreover, B1 and B2 are strains with Bacillus and B3 is a strain without Bacillus. The growth curve and CMC and FPA enzyme activities were studied further for HB1 and three strains of bacteria. The results showed that the enzymic activities of CMC and FPA of HB1 were 672.8U and 774.6U on the sodium carboxymethyl cellulose (CMC-Na) medium when inoculum size kept at 1% and grew on the condition of 30℃and shaking at 180rpm for 72h. CMC enzyme activities of B1 and B3 are close to the peak at 12h after cultured, up to 31.2U and 24.4U, respectively. CMC enzyme activities of B2 are the highest and reached 36.3U at 18h after cultured. For another enzyme-FPA, the enzyme activities of B1 strain had reached the peak at 12h after cultured and maximum value is 34.1U. However, the FPA enzyme activities of B2 and B3 strains is the highest at 8h after cultured and reached 39.2U and 63.6U, respectively. Comparison Penicillium oxalicum HB1 to 2 strains of bacteria in enzyme activities and growth conditions, CMC and FPA enzymes activities of Penicillium oxalicum HB1 were highest. Therefore, further study was conducted for Penicillium oxalicum HB1.2. The study of fermentation optimization and CMC enzyme activities of Penicillium oxalicum HB1The results showed that CMC enzyme activities were high when HB1 grew on the medium with 3% N as beef extract peptone, 5% inoculum size, incubation temperature at 28℃～35℃, pH at 4～7 and incubation time for 48h～96h. The further experiment was conducted on the base of this result to explore fermentation optimization.The best combination of pH, temperature, solid-liquid ratio and incubation time was solid-liquid ratio of pH=6.5, 30℃, 1:10 and incubation time of 48h.CMC enzyme activity was higheat when HB1 grew in the medium with pH5 and kept at 50℃. In addition, CMC enzyme activity was stable on the conditions of pH4~5 and 40℃～50℃.3. The ability evaluate of Penicillium oxalicum HB1 decomposing corn strawsThe incubation experiment illustrated that the enzymic activities of CMC and FPA and the total activity of celluase were 282.9U, 618.3U and 376.1U respectively when HB1 grew on the medium withcorn straw, 1% of inoculum size, shaking at 30℃and 180rpm for 72h. When the HB1 strain was cultured in agar medium with powder of corn stalk together for 10 days, the 87.3% of core stalk was decomposed and 1.90 times higher than that in the control; When the HB1 strain were incubated in soil with corn stalk for 30 days, the decomposition rates were 83.5% and 1.62 times higher than that in the control; Finally, the pot experiment with wheat plants was carried out to investigate the effect of HB1 strain on corn stalk decomposition in the soil for 50 days. The result showed that the straw decomposition rate was 70.8% and 15.1% higher than that in the control.4. The functions of HB1 on fixed P release in soil and beneficial effects on plant growthWhen the HB1 strain were incubated in soil with corn stalk for 30 days, the soil available phosphorus (Olsen-P) concentrations were 98.6mg/kg and 77.1mg/kg in HB1 application treatment and control, respectively. When incubation time expanded to 50d, P concentrations in wheat shoots were 12.6g/kg and 7.62g/kg for treatment and control, and P concentration in wheat roots were 25.9g/kg and 21.1g/kg in treatment and control.moreover,There were significant differences in P concentrations between control and HB1 treatment(P<0.05). Meanwhile, the biomass of wheat increased by 16.8% in treatment comparing to the control when plant growth for 30d.Penicillium oxalicum HB1 not only accelerates the decomposition of corn stalk in soil, but also enhances the available P released from soil. So it is necessary to do further research on its application in farmland.更多还原