Singapore Delft Water Alliance (SDWA), National University of Singapore, 2 Engineering Drive 2, Engineering Workshop 1, #02-05 Singapore 117577, Singapore.
Singapore Synchrotron Light Source (SSLS), National University of Singapore, 5 Research Link, Singapore 117603, Singapore.
Water Res. 2014 Nov 1;64:123-133. doi: 10.1016/j.watres.2014.06.039. Epub 2014 Jul 9.
Understanding the spatial heterogeneity within environmental biofilms can provide an insight into compartmentalization of different functions in biofilm communities. We used a non-destructive and label-free method by combining Synchrotron Radiation-based Fourier Transform Infrared Microspectroscopy (SR-FTIR) with Confocal Laser Scanning Microscopy (CLSM) to distinguish the spatial chemical changes within multispecies biofilms grown from natural storm waters in flow cells. Among the different surfaces tested for biofilm growth and optimal imaging, mylar membranes were most suited and it enabled successful spatial infrared imaging of natural biofilms for obtaining reliable and interpretable FTIR spectra. Time series analysis of biofilm growth showed that influx of water during biofilm growth, results in significant changes in biofilm formation. Early biofilms showed active nutrient acquisition and desiccation tolerance mechanisms corresponding with accumulation of secreted proteins. Statistical approach used for the evaluation of chemical spectra allowed for clustering and classification of various regions of the biofilm. Microheterogeneity was observed in the polymeric components of the biofilm matrix, including cellulose, glycocalyx and dextran-like molecules. Fructan and glycan-rich regions were distinguishable and glycocalyx was abundant in the strongly adhering peripheral regions of biofilms. Inner core showed coexistence of oxygen dimers and ferrihydrite that will likely support growth of Fe (II)-oxidising bacteria. The combined SR-FTIR microspectroscopy and CSLM approach for complex natural biofilms described here will be useful both in understanding heterogeneity of matrix components and in correlating functions of juxtaposed microbial species in complex natural biofilms with physicochemical microenvironment to which they are exposed.
了解环境生物膜内的空间异质性可以深入了解生物膜群落中不同功能的分区。我们使用了一种非破坏性且无标记的方法，将同步辐射傅里叶变换红外显微镜（SR-FTIR）与共焦激光扫描显微镜（CLSM）相结合，来区分在流动池中从天然雨水生长的多物种生物膜内的空间化学变化。在测试的不同表面中，用于生物膜生长和最佳成像的聚对苯二甲酸乙二醇酯（mylar）膜最合适，它使成功对天然生物膜进行空间红外成像成为可能，从而获得可靠且可解释的 FTIR 光谱。生物膜生长的时间序列分析表明，生物膜生长过程中进水会导致生物膜形成发生显著变化。早期生物膜表现出活跃的营养获取和干燥耐受性机制，对应于分泌蛋白的积累。用于评估化学光谱的统计方法允许对生物膜的各个区域进行聚类和分类。生物膜基质的聚合物成分存在微异质性，包括纤维素、糖萼和葡聚糖样分子。可区分出果聚糖和聚糖丰富的区域，并且糖萼在生物膜的强附着外围区域丰富。核心内部显示出氧二聚体和水铁矿的共存，这可能支持 Fe（II）氧化细菌的生长。这里描述的用于复杂天然生物膜的组合 SR-FTIR 显微镜和 CSLM 方法将有助于理解基质成分的异质性，并将毗邻微生物物种的功能与它们暴露的物理化学微环境相关联。
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