Dees P M., Ghiorse W C.
Department of Microbiology, Wing Hall, Cornell University, 14853, Ithaca, NY, USA
FEMS Microbiol Ecol. 2001 Apr;35(2):207-216. doi: 10.1111/j.1574-6941.2001.tb00805.x.
High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.
采用依赖培养和不依赖培养的方法对高温（≥60℃）合成食品垃圾堆肥进行检测，以确定主要的微生物种群。在一个冻干的74℃堆肥样品中，荧光直接计数法测得的细胞总数为6.4（±2.5）×10¹⁰个细胞·克干重⁻¹，而嗜热异养需氧菌的平板计数平均值为2.6（±1.0）×10⁸CFU·克干重⁻¹。开发了一种细胞预裂解分级分离方法来获取群落DNA，并使用一套针对16S和18S rDNA的PCR引物来检测细菌、古菌和真菌的存在。通过PCR从所有测试的堆肥样品中扩增出细菌16S rDNA，包括一个1500bp的结构域特异性片段和一个300bp的放线菌特异性片段。在任何样品中均未扩增出古菌rDNA。真菌18S rDNA仅从一个单独的奶牛粪便堆肥中扩增出来，该堆肥的最高温度达到50℃。扩增的rDNA限制性分析（ARDRA）用于筛选分离出的嗜热细菌和全长rDNA片段的克隆文库。ARDRA筛选在63个分离株中揭示了14种独特的模式，其中一种模式占31个分离株。在克隆文库中，70个克隆中检测到52种独特的模式，表明热堆肥中未培养细菌具有高度多样性。系统发育分析表明，两个最丰富的分离株属于Aneurinibacillus属和Brevibacillus属，它们通常与热堆肥无关。除了一个乳酸杆菌型序列外，克隆文库仅包含聚在芽孢杆菌属内的序列。没有一个分离株或克隆序列可归为以嗜热脂肪芽孢杆菌为代表的专性嗜热芽孢杆菌属，通常认为该属在高温堆肥中占主导地位。来自放线菌的扩增部分片段跨越V3可变区（Neefs等人，（1990年）《核酸研究》18，2237 - 2242），包括与糖单孢菌属、戈登氏菌属、红球菌属和棒状杆菌属相关的序列，尽管在分离株或全长克隆rDNA序列中未检测到这些生物。本研究中检测的所有嗜热分离株和测序的rDNA片段均来自革兰氏阳性菌。
