Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA
Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA
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DOI：
10.1111/j.1574-6941.2001.tb00805.x
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发表时间：
2001-04-01
影响因子：
4.2
通讯作者：
Ghiorse, WC
中科院分区：
文献类型：
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作者：
Dees, PM;Ghiorse, WC
高温（大于或等于60 degreesC）的合成食物垃圾堆肥进行了检查，培养依赖和独立的方法，以确定占主导地位的微生物种群。在74 ℃冷冻干燥的堆肥样品中，荧光直接计数总计为6.4（+/- 2.5）× 10（10）个细胞gdw（-1），而嗜热异养需氧菌的平板计数平均为2.6（+/-1.0 × 10（8）CFU gdw（-1）。采用裂解前细胞分级分离法提取细菌群落DNA，利用165和18 S rDNA引物进行PCR扩增，检测细菌、霉菌和真菌的存在。细菌16 S rDNA，包括一个特定的1500 bp的片段和一个特定的放线菌300 bp的片段，通过PCR扩增从所有堆肥样品测试。所有样品中均未扩增出叶绿体rDNA。真菌18 S rDNA仅从达到50摄氏度峰值温度的单独的牛粪堆肥中扩增。利用扩增rDNA限制性内切酶分析（ARP-RFLP）筛选分离的嗜热菌，并构建了全长rDNA片段的克隆文库。在63个分离株中，ARP 19筛选出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个独特的模式，表明高温堆肥中未培养细菌的多样性较高。系统发育分析表明，两个最丰富的菌株属于属Aneurinibacillus和Brevibacillus，这是不常见的与热堆肥。除了一个乳酸杆菌型序列。该克隆文库仅含有聚簇在芽孢杆菌属内的序列。没有分离物或克隆的序列可以被分配到专性嗜热芽孢杆菌属的组。由B表示。嗜热脂肪菌通常被认为在高温堆肥中占主导地位。跨越V3可变区的来自Arctinobacteria的扩增的部分片段（Neefs等（1990）Nucleic Acids Res.18，2237-2242）包括与单孢菌属、戈登氏菌属、红球菌属和棒状杆菌属相关的序列，尽管在分离物或全长克隆的rDNA序列中没有检测到这些生物体。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。(C)2001年，欧洲微生物学会联合会。由Elsevier Science B. V.出版，版权所有。
High-temperature (greater than or equal to 60 degreesC) 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)x 10(10) cells gdw(-1) in a freeze-dried 74 degreesC 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 165 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 degreesC. 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 Arctinobacteria, spanning the V3 variable region (Neefs ct 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. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.