Centre for Water, Environment, Sustainability and Public Health, Department of Civil and Environmental Engineering, University of Strathclyde, Glasgow G1 1XJ, UK.
Centre for Water, Environment, Sustainability and Public Health, Department of Civil and Environmental Engineering, University of Strathclyde, Glasgow G1 1XJ, UK.
J Microbiol Methods. 2022 Jan;192:106377. doi: 10.1016/j.mimet.2021.106377. Epub 2021 Nov 17.
Toxicological batch assays are essential to assess a compound's acute effect on microorganisms. This methodology is frequently employed to evaluate the effect of contaminants in sensitive microbial communities from wastewater treatment plants (WWTPs), such as autotrophic nitrifying populations. However, despite nitrifying batch assays being commonly mentioned in the literature, their experimental design criteria are rarely reported or overlooked. Here, we found that slight deviations in culture preparations and conditions impacted bacterial community performance and could skew assay results. From pre-experimental trials and experience, we determined how mishandling and treatment of cultures could affect nitrification activity. While media and biomass preparations are needed to establish baseline conditions (e.g., biomass washing), we found extensive centrifugation selectively destabilised nitrification activities. Further, it is paramount that the air supply is adjusted to minimise nitrite build-up in the culture and maintain suitable aeration levels without sparging ammonia. DMSO and acetone up to 0.03% (v/v) were suitable organic solvents with minimal impact on nitrification activity. In the nitrification assays with allylthiourea (ATU), dilute cultures exhibited more significant inhibition than concentrated cultures. So there were biomass-related effects; however, these differences minimally impacted the EC values. Using different nutrient-media compositions had a minimal effect; however, switching mineral media for the toxicity test from the original cultivation media is not recommended because it reduced the original biomass nitrification capacity. Our results demonstrated that these factors substantially impact the performance of the nitrifying inoculum used in acute bioassays, and consequently, affect the response of AOB-NOB populations during the toxicant exposure. These are not highlighted in operation standards, and unfortunately, they can have significant consequential impacts on the determinations of toxicological endpoints. Moreover, the practical procedures tested here could support other authors in developing testing methodologies, adding quality checks in the experimental framework with minimal waste of time and resources.
毒性批量分析对于评估化合物对微生物的急性影响至关重要。这种方法常用于评估来自废水处理厂（WWTP）的敏感微生物群落中污染物的影响，例如自养硝化种群。然而，尽管硝化批量分析在文献中经常被提及，但它们的实验设计标准很少被报道或被忽视。在这里，我们发现培养物准备和条件的微小偏差会影响细菌群落的性能，并可能使测定结果产生偏差。通过预实验和经验，我们确定了培养物的不当处理和处理方式如何影响硝化活性。虽然需要培养基和生物量准备来建立基线条件（例如生物量洗涤），但我们发现广泛的离心会选择性地破坏硝化活性。此外，至关重要的是，应调整供气以减少培养物中亚硝酸盐的积累，并在不吹脱氨的情况下保持适当的曝气水平。DMSO 和丙酮最高达 0.03%（v/v）是合适的有机溶剂，对硝化活性的影响最小。在使用烯丙基硫脲（ATU）的硝化测定中，稀释培养物比浓缩培养物表现出更显著的抑制作用。因此，存在与生物量相关的影响；然而，这些差异对 EC 值的影响最小。使用不同的营养培养基组成的影响最小；然而，不建议将毒性测试的矿物培养基从原始培养物培养基切换，因为这会降低原始生物量的硝化能力。我们的结果表明，这些因素会极大地影响急性生物测定中使用的硝化接种物的性能，从而影响毒物暴露期间 AOB-NOB 种群的反应。这些在操作标准中没有被强调，不幸的是，它们可能对毒理学终点的确定产生重大影响。此外，这里测试的实际程序可以为其他作者提供支持，在实验框架中添加质量检查，以最小化时间和资源的浪费。
