Departamento de Microbiología e Instituto del Agua, Universidad de Granada, 18071, Granada, Spain.
Environ Sci Pollut Res Int. 2010 Mar;17(3):779-90. doi: 10.1007/s11356-009-0180-y. Epub 2009 May 30.
BACKGROUND, AIM, AND SCOPE: Linear alkylbenzene sulfonate (LAS) is the most used anionic surfactant in a worldwide scale and is considered a high-priority pollutant. LAS is regarded as a readily biodegradable product under aerobic conditions in aqueous media and is mostly removed in wastewater treatment plants, but an important fraction (20-25%) is immobilized in sewage sludge and persists under anoxic conditions. Due to the application of the sludge as a fertilizer, LAS reaches agricultural soil, and therefore, microbial toxicity tests have been widely used to evaluate the influence of LAS on soil microbial ecology. However, molecular-based community-level analyses have been seldom applied in studies regarding the effects of LAS on natural or engineered systems, and, to our knowledge, there are no reports of their use for such appraisals in agricultural soil. In this study, a microcosm system is used to evaluate the effects of a commercial mixture of LAS on the community structure of Alphaproteobacteria, Actinobacteria, and Acidobacteria in an agricultural soil.
The microcosms consisted of agricultural soil columns (800 g) fed with sterile water (8 ml h(-1)) added of different concentration of LAS (10 or 50 mg l(-1)) for periods of time up to 21 days. Sterile water was added to control columns for comparison. The structures of Alphaproteobacteria, Actinobacteria, and Acidobacteria communities were analyzed by a cultivation independent method (temperature gradient gel electrophoresis (TGGE) separation of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes). Relevant populations were identified by subsequent reamplification, DNA sequencing, and database comparisons.
Cluster analysis of the TGGE fingerprints taking into consideration both the number of bands and their relative intensities revealed that the structure of the Alphaproteobacteria community was significantly changed in the presence of LAS, at both concentrations tested. The average number of bands was significantly lower in the microcosms receiving 50 mg l(-1) LAS and in the lower portion of soil cores. The clear differentiation of the samples of the upper portion of the soil columns amended with LAS was specifically related to the presence and intensity of a distinctive major band (named band class 7). There was a statistically significant positive correlation between the concentrations of LAS detected in soil portions taken from LAS 10 mg l(-1) and LAS 50 mg l(-1) microcosms and the relative intensity of band class 7 in the corresponding TGGE profiles. Prevalent Alphaproteobacteria populations in the soil microcosms had close similarity (>99%) to cultivated species affiliated to genera of the Rhizobiaceae, Methylocystaceae, Hyphomicrobiaceae, Rhodospirillaceae, Brucellaceae, Bradyrhizobiaceae, and Caulobacteraceae families. The population represented by band class 7 was found closely related to the genus Phenylobacterium (Caulobacteraceae). According to cluster analysis of TGGE profiles, the structure of both Actinobacteria and Acidobacteria communities in the soil microcosms was remarkably stable in the presence of LAS at the two concentrations tested, as most bands were universally present in all samples and displayed fairly similar relative intensities.
Previous studies by others authors, based on biological and chemical tests, concluded that LAS toxicity was not an important microbial selection factor in sludge amended soil, while work based on the use of molecular fingerprinting to evaluate the impact of LAS in aqueous media and marine sediments showed that concentrations as low as 1 mg l(-1) significantly influence the development of the bacterial community structure. Although TGGE is not a strictly quantitative method due to the bias introduced by the PCR reaction, changes of band intensity through experiments are a consequence of a change in the relative abundance of the corresponding populations in the community and can be used as a semiquantitative measure of bacterial diversity. Our results evidence that the Phenylobacterium population represented by band class 7 was favored by the presence of increasing concentrations of LAS in the soil and turned into a dominant population, suggesting its possible ability to use LAS in soil as a source of nutrients. As studies with pure cultures are required to confirm the ability of this population to degrade LAS, isolation strategies are currently under development in our laboratory. The weak effect of LAS on the structure of Actinobacteria and Acidobacteria communities is particularly interesting, as to our knowledge, there are no previous reports regarding the effects of LAS on these bacterial groups in soil. CONCLUSIONS, RECOMMENDATIONS, AND PERSPECTIVES: The Phenylobacterium-related alphaproteobacterial population identified in this work was selectively enriched in LAS polluted soil and is a plausible candidate to play a relevant role in the biotransformation of the surfactant under the conditions tested. The surfactant had no remarkable effects on the Actinobacteria and Acidobacteria fingerprints in soil, even when present at concentrations widely exceeding those reached in soil immediately after sludge application. TGGE fingerprinting provides a reliable and low time-consuming method for the monitoring of the bacterial community structure and dynamics, and we recommend its integration with the biological and chemical analyses usually applied in risk assessment of LAS in the environment.
背景、目的和范围：直链烷基苯磺酸盐（LAS）是全球范围内使用最广泛的阴离子表面活性剂，被认为是高度优先的污染物。在水介质中有氧条件下，LAS 被认为是一种易于生物降解的产品，并且主要在污水处理厂中去除，但重要的一部分（20-25％）被固定在污水污泥中，并在缺氧条件下持续存在。由于将污泥用作肥料，LAS 会到达农业土壤，因此，微生物毒性测试已被广泛用于评估 LAS 对土壤微生物生态的影响。然而，基于分子的群落水平分析在关于 LAS 对自然或工程系统影响的研究中很少应用，并且据我们所知，在农业土壤中没有关于使用它们进行此类评估的报告。在这项研究中，使用微宇宙系统来评估商业 LAS 混合物对农业土壤中α变形菌、放线菌和酸杆菌群落结构的影响。
微宇宙由农业土壤柱（800 克）组成，用无菌水（8 毫升/小时）连续进料，添加不同浓度的 LAS（10 或 50 毫克/升），持续时间长达 21 天。为了进行比较，无菌水被添加到对照柱中。通过培养独立方法（PCR 扩增的 16S rRNA 基因的温度梯度凝胶电泳（TGGE）分离）分析 α变形菌、放线菌和酸杆菌群落的结构。通过随后的再扩增、DNA 测序和数据库比较来鉴定相关种群。
考虑到条带的数量及其相对强度的 TGGE 指纹聚类分析表明，LAS 的存在显著改变了 α变形菌群落的结构，在测试的两种浓度下均如此。在接收 50 毫克/升 LAS 的微宇宙中和在土壤芯的较低部分中，条带的平均数量明显较低。在 LAS 10 毫克/升和 LAS 50 毫克/升微宇宙中添加 LAS 的土壤柱上部样品的明显分化特别与独特的主要条带（命名为条带类 7）的存在和强度有关。在 LAS 10 毫克/升和 LAS 50 毫克/升微宇宙中从土壤部分中检测到的 LAS 浓度与相应 TGGE 图谱中条带类 7 的相对强度之间存在统计学上显著的正相关。土壤微宇宙中普遍存在的 α变形菌种群与根瘤菌科、甲基球菌科、噬氢菌科、红螺菌科、布鲁氏菌科、慢生根瘤菌科和柄杆菌科的培养物种密切相似（>99％）。条带类 7 所代表的种群与苯基杆菌属（柄杆菌科）密切相关。根据 TGGE 图谱的聚类分析，LAS 在两种浓度下对土壤微宇宙中放线菌和酸杆菌群落的结构具有显著的稳定性，因为大多数条带普遍存在于所有样品中，并且相对强度相当相似。
其他作者先前基于生物和化学测试的研究得出结论，LAS 毒性不是污泥添加土壤中微生物选择的重要因素，而基于使用分子指纹图谱评估 LAS 在水介质和海洋沉积物中的影响的工作表明，浓度低至 1 毫克/升就会显著影响细菌群落结构的发展。尽管由于 PCR 反应的偏差，TGGE 不是一种严格的定量方法，但通过实验改变条带强度是群落中相应种群相对丰度变化的结果，并且可以用作细菌多样性的半定量测量。我们的结果表明，由条带类 7 代表的苯基杆菌种群被土壤中 LAS 浓度的增加所青睐，并成为优势种群，表明其可能具有利用 LAS 作为土壤中养分的能力。由于需要进行纯培养研究来确认该种群降解 LAS 的能力，目前正在我们的实验室中开发分离策略。LAS 对放线菌和酸杆菌群落结构的微弱影响特别有趣，因为据我们所知，在土壤中没有关于 LAS 对这些细菌群体的影响的先前报告。结论、建议和展望：在这项工作中鉴定的与苯基杆菌相关的 α变形菌种群在 LAS 污染的土壤中被选择性富集，并且是在测试条件下对表面活性剂进行生物转化的潜在候选物。表面活性剂甚至在污泥施用后立即在土壤中达到的浓度远超过时，对土壤中的放线菌和酸杆菌指纹图没有明显影响。TGGE 指纹图谱为监测细菌群落结构和动态提供了一种可靠且耗时少的方法，我们建议将其与通常在 LAS 环境风险评估中应用的生物和化学分析相结合。
