病死猪堆肥高温降解菌的筛选、鉴定及堆肥效果

Isolation and identification of thermophilic bacteria for efficient dead-pig composting

【目的】为分离得到高温高效降解菌,加快病死猪的降解。【方法】本研究通过稀释平板法和选择培养基初筛及酶活性复筛的方法,从锯末和病死猪(粉碎)好氧堆肥样品中筛选获得两株能分别高效降解蛋白质和脂肪的高温菌株N-3和Y-3。通过16S r DNA对两菌株进行鉴定,并采用L9(34)正交设计对菌株培养条件进行优化。再利用10 L全自动发酵罐按优化后的培养条件对两菌进行发酵生产(菌数达到108CFU/m L)并等体积混合制备成液体菌剂,分别按发酵物料湿重的0%、0.3%、0.6%、0.9%接种至锯末+病死猪(粉碎)堆肥中进行堆肥效果验证。【结果】共分离得到两株高效降解菌,N-3为芽孢杆菌(Bacillus aestuarii),可高效降解蛋白质,其最适生长温度55℃,p H为7.2,转速200 r/min,通气量4 L/min;Y-3为嗜热脱氮芽孢杆菌(Geobacillus thermodenitrificans),能高效降解脂肪,其最适生长温度60℃,p H为7.2,转速300 r/min,通气量3 L/min。堆肥过程中对照组和接菌各组(0.3%、0.6%、0.9%)最高温度分别为58.3、69.0、68.9、66.3℃,各接菌组间无显著差异(P>0.05),但均极显著高于对照组(P<0.01),且各接菌组堆肥温度达到60℃以上天数分别为8、10、9 d,极显著高于对照组的0 d(P<0.01)。至堆肥结束时,对照组和接菌各组的病死猪降解率分别为71.2%、75.7%、96.7%、97.1%。接菌各组(0.3%接菌组除外)病死猪降解率均极显著高于对照组(P<0.01),0.3%接菌组与对照组间无显著差异(P>0.05)。【结论】筛选获得的高温腐熟菌N-3和Y-3为能高效降解蛋白质和脂肪的高温菌株,可以用于病死猪腐熟堆肥,且两菌等体积混合后按0.6%添加量接种至病死猪堆肥中,能提高堆肥温度,维持高温时间,加快病死猪的降解,从而有效杀灭病原微生物,达到无害化要求。

[ Objective] To isolate thermophilic bacteria to degrade organic substances of dead-pig. [ Methods] Primary screening was done by using diluted plate count and selective medium, and then enzyme activity was measured for secondary screening. Two thermophilic bacterial strains N-3 and Y-3 were isolated, and could degrade protein and lipids. To test their effect, the isolates were mixed （ V： V = 1 ： 1, the number of bacteria was l0s CFU/mL） and inoculated in dead-pigs and sawdust composting with different doses （0% , 0.3% , 0.6% and 0.9% of the wet weight of fermentation materials）. [ Results ] Strain N-3 was identified as Bacillus aestuarii and Y-3 as Geobacillus thermodenitrificans, based on their 16S rDNA gene sequences. The composting temperature of the 0.3% , 0.6% and 0.9% inoculation group could reach 60 % and maintain at the high temperature for about 10 d, which is higher than control （P〈 0. 01 ）. At the end of composting, the dead-pig degradation rate of the （0%, 0.3%, 0.6% and 0.9% inoculation groups were 71.2%, 75.7% , 96.7% and 97.1% , respectively. The groups of 0.6% and 0.9% were significantly higher than the control （P 〈 0. 01）. [ Conclusion ] Sufficient amount inoculation of thermophilic bacteria （ 〉 0.6% ） could effectively increase composting temperature, maintain thermophilic stage for longer time, and accelerate degradation of dead-pig by composting.