The synergistic cooperation of microbial cells and their extracellular polymeric substances (EPS) in biofilms is critical for the biofilm's resistance to heavy metals and the migration and transformation of heavy metals. However, the effects of different components of biofilms have not been fully understood. In this study, the spatial distribution and speciation of copper in the colloidal EPS, capsular EPS, cell walls and membranes, and intracellular fraction of unsaturated Pseudomonas putida (P. putida) CZ1 biofilms were fully determined at the subcellular level. It was found that 60-67% of copper was located in the extracellular fraction of biofilms, with 44.7-42.3% in the capsular EPS. In addition, there was 15.5-20.1% and 17.2-21.2% of copper found in the cell walls and membranes or the intracellular fraction, respectively. Moreover, an X-ray absorption fine structure spectra analysis revealed that copper was primarily bound by carboxyl-, phosphate-, and hydrosulfide-like ligands within the extracellular polymeric matrix, cell walls and membranes, and intracellular fraction, respectively. In addition, macromolecule quantification, fourier-transform infrared spectroscopy spectra and sulfur K-edge x-ray absorption near edge structure analysis further showed the carboxyl-rich acidic polysaccharides in EPS, phospholipids in cell walls and cell membranes, and thiol-rich intracellular proteins were involved in binding of copper in the different components of biofilm. The full understanding of the distribution and chemical species of heavy metals in biofilms not only promotes a deep understanding of the interaction mechanisms between biofilms and heavy metals, but also contributes to the development of effective biofilm-based heavy metal pollution remediation technologies.

中文翻译：

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对铜形态的亚细胞水​​平研究揭示了细菌细胞与铜结合中涉及的微生物细胞和EPS的协同机制。

微生物细胞及其在生物膜中的细胞外聚合物质（EPS）的协同合作对于生物膜对重金属的抗性以及重金属的迁移和转化至关重要。但是，尚未完全了解生物膜不同成分的作用。在这项研究中，铜在胶态EPS，荚膜EPS，细胞壁和膜以及不饱和恶臭假单胞菌（P.putida）CZ1生物膜的细胞内组分中铜的空间分布和形态在亚细胞水平上得到了充分的确定。发现60-67％的铜位于生物膜的细胞外部分中，其中44.7-42.3％位于荚膜EPS中。另外，在细胞壁和细胞膜或细胞内部分中分别发现有15.5-20.1％和17.2-21.2％的铜。此外，X射线吸收精细结构光谱分析显示，铜主要分别被胞外聚合物基质，细胞壁和膜以及胞内部分中的羧基，磷酸盐和氢硫化物样配体结合。此外，大分子定量，傅立叶变换红外光谱和硫K边缘x射线吸收法在边缘结构附近的分析进一步显示出EPS中富含羧基的酸性多糖，细胞壁和细胞膜中的磷脂以及富含硫醇的细胞内蛋白参与了铜在生物膜不同成分中的结合。全面了解生物膜中重金属的分布和化学种类不仅可以增进对生物膜与重金属之间相互作用机制的深入了解，