油茶壳堆肥微生物的培养及功能芽孢杆菌的筛选和初步应用

Functional Bacillus Species in Camellia Seed Shell Compost

【目的】从自然状态下的油茶壳堆肥产品中筛选功能芽孢杆菌,为油茶壳堆肥高效生产、饲料发酵等应用提供菌剂。【方法】利用高通量测序技术测定广西某油茶壳堆肥中微生物群落分布,筛选适宜堆肥微生物生长的培养基以获得多样性较丰富的堆肥芽孢杆菌菌群,经平板稀释涂布法分离纯化菌株,使用水解圈法和酶活测定方法进行水解酶功能分析,利用菌株形态特征观察、16S rDNA分子鉴定法明确菌株的种属,并运用分子生物学软件MEGAX构建系统发育树。使用重铬酸钾法测定腐殖酸含量。【结果】芽孢杆菌科(Bacillaceae)是优势菌科,占比达55.58%;分离到15株芽孢杆菌,同时具有淀粉酶、纤维素酶、蛋白酶3种水解酶活性的菌株有6株;同时具有淀粉酶和纤维素酶活性的有1株;仅具有蛋白酶或纤维素酶一种酶活性的有2株。其中菌株Bacillus sp.YX11的蛋白酶活力达到27.07±3.28 U·mL^(-1),淀粉酶活力达123.97±3.19 U·mL^(-1),纤维素酶活力达15.75±0.23 U·mL^(-1)。含有这15株菌的复合菌制剂有利于腐殖酸的生成,提高了堆肥品质。【结论】鉴定出的Bacillus cereus YX02和Bacillus flexus FYF01等菌株具有进一步研究开发的价值,可用于开发油茶壳堆肥微生物菌制剂。

【Objective】Bacillus spp that contribute to the fermentation of Camellia oleifera seed shells were isolated for effective composting of the waste material.【Method】The microbial community in natural camellia seed shell compost found in Guangxi was studied using the high-throughput sequencing technology.Suitable culture medium to foster the growth of richly diverse Bacillus spp from the compost was selected.Flora isolation by dilution with a streaking plate method followed.The hydrolase activities of the isolates were determined by using the hydrolysis circle method and enzyme activity analysis,the species identified by a 16S rDNA analysis,and the phylogeny constructed by MEGAX.The content of humic acid in the compost was measured by a potassium dichromate method.【Result】Bacillaceae was the dominant family in the compost.It accounted for 55.58% of all isolated flora.Among the 15 Bacillus isolates,6 exhibited activities of amylase,cellulase,and protease,one of amylase and cellulase,and 2 of protease or protease.Strain YX11 showed a protease activity of 27.07±3.28 U·mL^(-1),an amylase activity of 123.97±3.19 U·mL^(-1),and a cellulase activity of 15.75±0.23 U·mL^(-1).In the presence of numerous Bacillus spp that secreted varieties of hydrolases,formation of humic acid in the compost was enhanced.【Conclusion】Some of the isolated strains,such as B.cereus YX02 and B.flexus FYF01,might warrant further investigation to develop microbial inoculants for efficient composting camellia seed shells.