接种对堆肥进程的影响及堆肥产物功能化研究
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摘要
采用室外堆肥的方法，初步研究了一株嗜热球杆菌(Sphaerobacterium thermophilus sp.) 对猪粪堆肥进程的影响。结果表明：该菌株能有效加快堆肥的腐熟进程，促进堆肥初期温度的上升，处理组最高温度比对照组高6.9℃，到达最高温度的时间比对照组提前15天。试验结束时处理组的全N、全P相对含量均较对照组高，显示了接种还能提高堆肥产品的品质。两组堆肥pH值较堆制前期均有所降低，电导率升高，但处理组的pH值和电导率均比对照组变化快。发芽指数处理组比对照组到达50%的时间提前了22天。接种嗜热球杆菌还影响了堆肥过程中过氧化氢酶、脲酶和纤维素酶活性。处理组过氧化氢酶活性在第12天到达最低点，比对照组提前了7天，此后都略有回升，26天以后，过氧化氢酶活性基本上都维持在800—1300ml左右。两组脲酶在堆制初期活性都很高，进入高温期后，酶活性显著下降，到堆肥后期，脲酶活性基本上维持在3-15mg左右。处理组纤维素酶活性在第6天达到最高，酶活性为90mg左右，到堆肥结束，降到30-50mg左右。而且处理组在第12天以前，酶活性均比对照组高，12天以后比对照组低。说明接种影响了堆肥中微生物活性的强度，从而使酶活性大小发生了变化。
以本实验室自主筛选的一株具对蕃茄青枯病病原菌有拮抗作用、能促进植物生长的肺炎克雷伯氏菌（Klebsiella pneumoniae(Schroeter)Trevisan）（实验室代号101菌株）为材料，对该菌株进行了绿色荧光蛋白（green fluorescent protein ，GFP）标记，研究在不同接种方式下，靶的菌株在堆肥中的消长情况，旨在获得含有较高数量肺炎克雷伯氏菌的堆肥产品，实现堆肥产品的多功能，增加堆肥产品的附加值。
荧光检测和琼脂糖凝胶电泳显示：带GFP基因的质粒pMTC在受体菌中成功表达；在无选择压力的条件下,培养56h后质粒的稳定率仍能达到87.08%，生长曲线和生存能力比较试验结果均表明GFP对靶的菌株的生长速度和生存能力没有明显影响。
在牛粪堆肥腐熟阶段、风干前后等三个不同时段接种肺炎克雷伯氏菌，定殖试验结果表明：以堆肥风干后接种，靶的菌株数量最多；风干后接种，35d后，堆肥中细菌总数为：4.5-5.1×107CFU/g，而靶的菌株达到1.7-2.0×107CFU/g，占一定优势。接种与强化靶的菌株后的堆肥产品，实际应用效果还有待进一步研究。
A field experiment was carried out to study the effect of inoculation with Sphaerobacterium thermophilus sp. on composting pig manure. The results showed that: Temperature in pile inoculated rose faster and reached to 55℃ 15d earlier than the compost uninoculated. Relatively content of total N and P were both higher than that in compost uninoculated, which indicated that inoculating could improve the quality of compost. The pH decreased and EC values increased with the composting and changed faster in the treatment with inoculating compared with uninoculating. The time to reach to 50% seed germination index was also shortened 22days. Peroxidase, Urease and Cellulase activity between compost inoculated and uninoculated were different, which resulted from the Sphaerobacterium thermophilus sp.
Klebsiella pneumoniae(Schroeter)Trevisan(bacterial 101)was isolated and marked with green fluorescent protein (GFP) to study its survival in cattle manure. It was demonstrated that plasmid pMTC was transformed into bacteria 101 by detection of fluorescence and agarose electrophoresis analysis. The transformants was named 101 TC. GFP has many good characteristics, such as simple operation, emitting green fluorescence without requiring any exogenous substrates and cofactors under excitation of long UV light or blue light, reliable and stable property, and so on. Under nonselective condition, the stability of recombinant plasmid was 87.08%. The length of gfp was short, so it has no obvious effect on growing speed and viability of target bacteria. These characteristics showed that GFP might be of application potential in location as a new reporter gene.
The survival study on Klebsiella pneumoniae(Schroeter)Trevisan marked by GFP showed that: When the compost was inoculated by 10% after air-drying, the quantity of 101TC was 1.7 -2.0×107CFU/g and total bacterial was 4.5-5.1×107CFU/g after air-drying 35d, which indicated that the treatment was the best with most 101TC and least otiose bacterial among the three group of treatments. But the total benefical bacterial was failed to reaching the standard of Chinese agricultural standard NY-227-94, which regulated that the quantity of benefical bacterial should be more than 1-3×108 CFU/g in solid inoculant, so its optimum condition in composting needed further research.
以本实验室自主筛选的一株具对蕃茄青枯病病原菌有拮抗作用、能促进植物生长的肺炎克雷伯氏菌（Klebsiella pneumoniae(Schroeter)Trevisan）（实验室代号101菌株）为材料，对该菌株进行了绿色荧光蛋白（green fluorescent protein ，GFP）标记，研究在不同接种方式下，靶的菌株在堆肥中的消长情况，旨在获得含有较高数量肺炎克雷伯氏菌的堆肥产品，实现堆肥产品的多功能，增加堆肥产品的附加值。
荧光检测和琼脂糖凝胶电泳显示：带GFP基因的质粒pMTC在受体菌中成功表达；在无选择压力的条件下,培养56h后质粒的稳定率仍能达到87.08%，生长曲线和生存能力比较试验结果均表明GFP对靶的菌株的生长速度和生存能力没有明显影响。
在牛粪堆肥腐熟阶段、风干前后等三个不同时段接种肺炎克雷伯氏菌，定殖试验结果表明：以堆肥风干后接种，靶的菌株数量最多；风干后接种，35d后，堆肥中细菌总数为：4.5-5.1×107CFU/g，而靶的菌株达到1.7-2.0×107CFU/g，占一定优势。接种与强化靶的菌株后的堆肥产品，实际应用效果还有待进一步研究。
A field experiment was carried out to study the effect of inoculation with Sphaerobacterium thermophilus sp. on composting pig manure. The results showed that: Temperature in pile inoculated rose faster and reached to 55℃ 15d earlier than the compost uninoculated. Relatively content of total N and P were both higher than that in compost uninoculated, which indicated that inoculating could improve the quality of compost. The pH decreased and EC values increased with the composting and changed faster in the treatment with inoculating compared with uninoculating. The time to reach to 50% seed germination index was also shortened 22days. Peroxidase, Urease and Cellulase activity between compost inoculated and uninoculated were different, which resulted from the Sphaerobacterium thermophilus sp.
Klebsiella pneumoniae(Schroeter)Trevisan(bacterial 101)was isolated and marked with green fluorescent protein (GFP) to study its survival in cattle manure. It was demonstrated that plasmid pMTC was transformed into bacteria 101 by detection of fluorescence and agarose electrophoresis analysis. The transformants was named 101 TC. GFP has many good characteristics, such as simple operation, emitting green fluorescence without requiring any exogenous substrates and cofactors under excitation of long UV light or blue light, reliable and stable property, and so on. Under nonselective condition, the stability of recombinant plasmid was 87.08%. The length of gfp was short, so it has no obvious effect on growing speed and viability of target bacteria. These characteristics showed that GFP might be of application potential in location as a new reporter gene.
The survival study on Klebsiella pneumoniae(Schroeter)Trevisan marked by GFP showed that: When the compost was inoculated by 10% after air-drying, the quantity of 101TC was 1.7 -2.0×107CFU/g and total bacterial was 4.5-5.1×107CFU/g after air-drying 35d, which indicated that the treatment was the best with most 101TC and least otiose bacterial among the three group of treatments. But the total benefical bacterial was failed to reaching the standard of Chinese agricultural standard NY-227-94, which regulated that the quantity of benefical bacterial should be more than 1-3×108 CFU/g in solid inoculant, so its optimum condition in composting needed further research.
引文
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