Wagner-Döbler I, Bennasar A, Vancanneyt M, Strömpl C, Brümmer I, Eichner C, Grammel I, Moore E R
Department of Microbiology, GBF National Research Institute for Biotechnology, D-38124 Braunschweig, Germany.
Appl Environ Microbiol. 1998 Aug;64(8):3014-22. doi: 10.1128/AEM.64.8.3014-3022.1998.
A microcosm enrichment approach was employed to isolate bacteria which are representative of long-term biphenyl-adapted microbial communities. Growth of microorganisms was stimulated by incubating soil and sediment samples from polluted and nonpolluted sites with biphenyl crystals. After 6 months, stable population densities between 8 x 10(9) and 2 x 10(11) CFU/ml were established in the microcosms, and a large percentage of the organisms were able to grow on biphenyl-containing minimal medium plates. A total of 177 biphenyl-degrading strains were subsequently isolated and characterized by their ability to grow on biphenyl in liquid culture and to accumulate a yellow meta cleavage product when they were sprayed with dihydroxybiphenyl. Isolates were identified by using a polyphasic approach, including fatty acid methyl ester (FAME) analysis, 16S rRNA gene sequence comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, and genomic fingerprinting based on sequence variability in the 16S-23S ribosomal DNA intergenic spacer region. In all of the microcosms, isolates identified as Rhodococcus opacus dominated the cultivable microbial community, comprising a cluster of 137 isolates with very similar FAME profiles (Euclidean distances, <10) and identical 16S rRNA gene sequences. The R. opacus isolates from the different microcosms studied could not be distinguished from each other by any of the fingerprint methods used. In addition, three other FAME clusters were found in one or two of the microcosms analyzed; these clusters could be assigned to Alcaligenes sp., Terrabacter sp., and Bacillus thuringiensis on the basis of their FAME profiles and/or comparisons of the 16S rRNA gene sequences of representatives. Thus, the microcosm enrichments were strongly dominated by gram-positive bacteria, especially the species R. opacus, independent of the pollution history of the original sample. R. opacus, therefore, is a promising candidate for development of effective long-term inocula for polychlorinated biphenyl bioremediation.
采用微观世界富集法分离代表长期适应联苯的微生物群落的细菌。通过将来自污染和未污染地点的土壤和沉积物样本与联苯晶体一起培养来刺激微生物生长。6个月后，在微观世界中建立了8×10⁹至2×10¹¹CFU/ml的稳定种群密度，并且很大比例的生物体能够在含联苯的基本培养基平板上生长。随后共分离出177株联苯降解菌株，并通过它们在液体培养中对联苯的生长能力以及用二羟基联苯喷雾时积累黄色间位裂解产物的能力进行表征。通过多相方法鉴定分离株，包括脂肪酸甲酯（FAME）分析、16S rRNA基因序列比较、全细胞蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及基于16S-23S核糖体DNA基因间隔区序列变异性的基因组指纹图谱。在所有微观世界中，鉴定为不透明红球菌的分离株在可培养微生物群落中占主导地位，包括一组137株分离株，它们具有非常相似的FAME谱（欧氏距离，<10）和相同的16S rRNA基因序列。所研究的不同微观世界中的不透明红球菌分离株无法通过所使用的任何指纹方法相互区分。此外，在分析的一个或两个微观世界中发现了另外三个FAME簇；根据它们的FAME谱和/或代表的16S rRNA基因序列比较，这些簇可归为产碱菌属、地杆菌属和苏云金芽孢杆菌。因此，微观世界富集主要由革兰氏阳性菌主导，尤其是不透明红球菌，与原始样本的污染历史无关。因此，不透明红球菌是开发用于多氯联苯生物修复的有效长期接种物的有前途的候选者。
