Tim Taylor Department of Chemical Engineering, Kansas State University, Manhattan, Kansas 66506, United States.
Department of Civil Engineering, Kansas State University, Manhattan, Kansas 66506, United States.
ACS Appl Bio Mater. 2022 Jan 17;5(1):134-145. doi: 10.1021/acsabm.1c00971. Epub 2021 Dec 17.
Multispecies biofilms are a common limitation in membrane bioreactors, causing membrane clogging, degradation, and failure. There is a poor understanding of biological fouling mechanisms in these systems due to the limited number of experimental techniques useful for probing microbial interactions at the membrane interface. Here, we develop a new experimental method, termed polymer surface dissection (PSD), to investigate multispecies assembly processes over membrane surfaces. The PSD method uses photodegradable polyethylene glycol hydrogels functionalized with bioaffinity ligands to bind and detach microscale, microbial aggregates from the membrane for microscopic observation. Subsequent exposure of the hydrogel to high resolution, patterned UV light allows for controlled release of any selected aggregate of desired size at high purity for DNA extraction. Follow-up 16S community analysis reveals aggregate composition, correlating microscopic images with the bacterial community structure. The optimized approach can isolate aggregates with microscale spatial precision and yields genomic DNA at sufficient quantity and quality for sequencing from aggregates with areas as low as 2000 μm, without the need of culturing for sample enrichment. To demonstrate the value of the approach, PSD was used to reveal the composition of microscale aggregates of different sizes during early-stage biofouling of aerobic wastewater communities over PVDF membranes. Larger aggregates exhibited lower diversity of bacterial communities, and a shift in the community structure was found as aggregate size increased to areas between 25,000 and 45,000 μm, below which aggregates were more enriched in Bacteroidetes and above which aggregates were more enriched with Proteobacteria. The findings demonstrate that community succession can be observed within microscale aggregates and that the PSD method is useful for identification and characterization of early colonizing bacteria that drive biofouling on membrane surfaces.
多物种生物膜是膜生物反应器中常见的限制因素，会导致膜堵塞、降解和失效。由于用于探测膜界面微生物相互作用的实验技术数量有限，因此对这些系统中的生物污垢机制的了解很差。在这里，我们开发了一种新的实验方法，称为聚合物表面剖析（PSD），用于研究膜表面上的多物种组装过程。PSD 方法使用光降解的聚乙二醇水凝胶，其功能化有生物亲和配体，可将微尺度的微生物聚集体从膜上结合并分离出来，用于微观观察。随后将水凝胶暴露于高分辨率、图案化的紫外光下，允许以高纯度释放任何所需尺寸的选定聚集体，用于 DNA 提取。后续的 16S 群落分析揭示了聚集体的组成，将微观图像与细菌群落结构相关联。优化后的方法可以以微尺度的空间精度分离聚集体，并从面积低至 2000μm 的聚集体中获得足够数量和质量的基因组 DNA，用于测序，而无需进行培养以富集样品。为了证明该方法的价值，PSD 用于揭示有氧废水群落在聚偏二氟乙烯（PVDF）膜上早期生物污垢过程中不同大小的微尺度聚集体的组成。较大的聚集体表现出较低的细菌群落多样性，并且随着聚集体尺寸增加到 25000 至 45000μm 之间，群落结构发生了变化，在这以下聚集体中更多地富集了拟杆菌门，而在这以上聚集体中更多地富集了变形菌门。研究结果表明，可以在微尺度聚集体内观察到群落演替，并且 PSD 方法可用于鉴定和表征驱动膜表面生物污垢的早期定植细菌。
