养猪微生物发酵床芽胞杆菌空间分布多样性

了解微生物发酵床大栏养猪垫料中的芽胞杆菌多样性和空间分布规律,为微生物发酵床管理、芽胞杆菌新资源挖掘及菌剂开发奠定基础。将发酵床划分为32个方格(4行×8列),采用五点取样法获得每个方格的样品。采用可培养法从32份样品中分离芽胞杆菌菌株,利用16S rRNA基因序列初步鉴定所分离获得的芽胞杆菌种类。利用聚集度指标和回归分析法,分析芽胞杆菌的样方空间分布型。通过Shannon-Wiener多样性指数、Simpson优势度指数、Hill指数及丰富度指数分析,揭示微生物发酵床中芽胞杆菌的空间分布多样性。从32份样品中共获得芽胞杆菌452株,16S rRNA基因鉴定结果表明它们分别隶属于芽胞杆菌纲的2个科、8个属、48个种。其中,种类最多的为芽胞杆菌属(Bacillus),30种;赖氨酸芽胞杆菌属(Lysinibacillus),6种;类芽胞杆菌属(Paenibacillus),5种;短芽胞杆菌属(Brevibacillus),3种;鸟氨酸芽胞杆菌属(Ornithinibacillus)、大洋芽胞杆菌属(Oceanibacillus)、少盐芽胞杆菌属(Paucisalibacillus)和纤细芽胞杆菌属(Gracilibacillus)各1个种。芽胞杆菌种类在发酵床空间分布差异很大,根据其空间出现频次,可分为广分布种类,如地衣芽胞杆菌(Bacillus licheniformis);寡分布种类,如根际芽胞杆菌(B.rhizosphaerae);少分布种类,如弯曲芽胞杆菌(B.flexus)。依据其数量,可分为高含量组优势种群,如地衣芽胞杆菌(B.licheniformis);中含量组常见种群,耐盐赖氨酸芽胞杆菌(Lysinibacillus halotolerans);寡含量组寡见种群,如根际芽胞杆菌(B.rhizosphaerae);低含量组偶见种群,如土地芽胞杆菌(B.humi)。空间分布型聚集度和回归分析测定表明,芽胞杆菌在微生物发酵床的分布类型为聚集分布。微生物发酵床垫料中芽胞杆菌种类总含量高达4.41×108个/g,其种类含量范围为0.01—94.1×106个/g(均值为8.96×106个/g),丰富度指数(D)、优势度指数(λ)、Shannon-Wiener指数(H')和均匀度指数(J')分别为0.4928、0.2634、1.3589和0.9803,其中香农指数最大的单个芽胞杆菌种类为地衣芽胞杆菌(B.licheniformis)。根据芽胞杆菌种类多样性指数聚类分析,当欧式距离λ=17时,可分为高丰富度高含量和低丰富度低含量类型。微生物发酵床的芽胞杆菌种类丰富、数量高,是一个天然的菌剂"发酵罐",有望直接作为微生物菌剂,应用于土壤改良、作物病害防控、污染治理等领域。

Spatial distribution pattern and community diversity of the Bacillus-like species in a fermentation bed system for pig rearing

To provide a foundation for the management of microbial fermentation beds, new resource mining of Bacillus-like species, and development of microbial agents, we investigated the spatial distribution pattern and community diversity of Bacillus-like species in the litters of a pig-on-litter system. The microbial fermentation bed was divided into 32 square areas of 4 rows × 8 columns. A sample from each area was obtained using a five-point sampling mode. Bacillus-like bacteria were isolated from the 32 samples using a culture method and preliminarily identified based on 16S rRNA gene sequences. The spatial distribution pattern of the Bacillus-like bacteria was analyzed using a concentration index and regression analysis. Moreover, the Shannon-Wiener diversity index, Simpson dominance index, Hill index, and richness were separately calculated to reveal the community structure and diversity of the Bacillus-like bacteria in the microbial fermentation bed. A total of 452 Bacillus-like isolates were obtained from the 32 litter samples, and identified as 48 species belonging to eight genera and two Families (Bacillaceae and Paenibacillaceae) within the Bacilli by 16S rRNA gene sequences. The species numbers for the genera were as follows: 30 for Bacillus, six for Lysinibacillus, five for Paenibacillus, three for Brevibacillus, and one for each of Ornithinibacillus, Oceanobacillus, Paucisalibacillus, and Gracilibacillus. There was a large difference in the distribution of Bacillus-like species in the fermentation bed. On the basis of their occurrence frequency, the Bacillus-like species could be discriminated as the widespread species, including Bacillus licheniformis; sparsely distributed species, including B. rhizosphaerae; and rare species, including B. flexus. According to the abundance of each species, the Bacillus-like species could be classified into the high-content dominant populations, including B. licheniformis; medium-content common populations, including Lysinibacillus halotolerans; oligocontent oligo populations, including B. rhizosphaerae, and low-content occasional populations, including B. humi. The concentration class and regression analysis results indicated that the spatial distribution pattern of the Bacillus-like species in the microbial fermentation bed was an aggregated distribution. In the litters, the content range, total content, richness, Simpson index, Shannon-Wiener index, and Pielou''s evenness index of the Bacillus-like species were 0.01-94.1 × 10⁶ cfu/g, 4.41 × 10⁸ cfu/g, 0.4928, 0.2634, 1.3589, and 0.9803, respectively. Moreover, B. licheniformis appeared to have the highest Shannon-Wiener index value. On the basis of the diversity index cluster analysis, all the Bacillus-like species could be divided into a high richness high content group and a low richness low content group. The Bacillus-like bacteria can actively inhabit microbial fermentation beds and have rich species and abundant contents. Thus, the microbial fermentation bed can be considered as a natural bacterial "fermentation tank", which is expected to be directly used as a microbial inoculum and applied in the fields for purposes including soil improvement, crop disease prevention and control, and pollution management.