Sanseverino J, Werner C, Fleming J, Applegate B, King J M, Sayler G S
Center for Environmental Biotechnology, University of Tennessee, Knoxville 37932.
Biodegradation. 1993;4(4):303-21. doi: 10.1007/BF00695976.
Traditional methods for quantifying specific catabolic bacterial populations underestimate the true population count due to the limitations of the necessary laboratory cultivation methods. Likewise, in situ activity is also difficult to assess in the laboratory without altering the sample environment. To circumvent these problems and achieve a true in situ bacterial population count and activity measurement, new methods based on molecular biological analysis of bacterial nucleic acids were applied to soils heavily contaminated with polycyclic aromatic hydrocarbons (PAH). In addition, a naphthalene-lux reporter system was used to determine bioavailability of naphthalene within these soils. DNA extracted from seven PAH-contaminated soils and hybridized with the nahA gene probe indicated that the naphthalene degradative genes were present in all samples in the range of 0.06 to 0.95 ng/100 microliters DNA extract which was calculated to represent 3.2 x 10(6) to 1.1 x 10(10) cells/g soil (assuming one copy of these genes per cell). 14C-naphthalene mineralization was observed in all contaminated soils with 14CO2 mineralization rates ranging from 3.2 x 10(-5) to 304,920.0 x 10(-5) micrograms g soil-1 h-1. Phenanthrene, anthracene, and benzo(a)pyrene were mineralized also in several soils. Messenger RNA transcripts of nahA were isolated and quantified from 4 soils. Only one soil tested, soil B, was inducible with salicylate above the in situ nahA gene transcript level. Two of the soils, C and G, were already fully induced in situ. The naphthalene mineralization rate correlated positively with the amount of nahA gene transcripts present (r = 0.99). Naphthalene was bioavailable in soils A, D, E, G, and N as determined by a bioluminescent response from the naphthalene-lux reporter system. Taken together, these data provided information on what the naphthalene-degrading bacterial population was experiencing in situ and what approaches would be necessary to increase activity.
由于所需实验室培养方法的局限性，传统的定量特定分解代谢细菌种群的方法会低估真实的种群数量。同样，在不改变样品环境的情况下，原位活性在实验室中也很难评估。为了规避这些问题并实现真实的原位细菌种群数量计数和活性测量，基于细菌核酸分子生物学分析的新方法被应用于多环芳烃（PAH）严重污染的土壤中。此外，一个萘 - 荧光素酶报告系统被用于测定这些土壤中萘的生物可利用性。从七种PAH污染土壤中提取的DNA与nahA基因探针杂交表明，萘降解基因存在于所有样品中，含量范围为0.06至0.95 ng/100微升DNA提取物，经计算这相当于3.2×10⁶至1.1×10¹⁰个细胞/克土壤（假设每个细胞有一份这些基因）。在所有污染土壤中均观察到¹⁴C - 萘矿化，¹⁴CO₂矿化速率范围为3.2×10⁻⁵至304,920.0×10⁻⁵微克克土壤⁻¹小时⁻¹。菲、蒽和苯并(a)芘在几种土壤中也发生了矿化。从4种土壤中分离并定量了nahA的信使RNA转录本。所测试的土壤中，只有土壤B用水杨酸盐诱导后高于原位nahA基因转录本水平。其中两种土壤，土壤C和土壤G，在原位已经完全被诱导。萘矿化速率与存在的nahA基因转录本数量呈正相关（r = 0.99）。根据萘 - 荧光素酶报告系统的生物发光反应确定，萘在土壤A、D、E、G和N中具有生物可利用性。综上所述，这些数据提供了关于萘降解细菌种群在原位的情况以及提高活性需要采取何种方法的信息。
