Table_1_Metal-Adapted Bacteria Isolated From Wastewaters Produce Biofilms by Expressing Proteinaceous Curli Fimbriae and Cellulose Nanofibers.DOCX

Bacterial biofilm plays a pivotal role in bioremediation of heavy metals from wastewaters. In this study, we isolated and identified different biofilm producing bacteria from wastewaters. We also characterized the biofilm matrix [i.e., extracellular polymeric substances (EPS)] produced by different bacteria. Out of 40 isolates from different wastewaters, only 11 (27.5%) isolates (static condition at 28°C) and 9 (22.5%) isolates (agitate and static conditions at 28 and 37°C) produced air–liquid (AL) and solid–air–liquid (SAL) biofilms, respectively, only on salt-optimized broth plus 2% glycerol (SOBG) but not in other media tested. Biomass biofilms and bacteria coupled with AL biofilms were significantly (P ≤ 0.001) varied in these isolates. Escherichia coli (isolate ENSD101 and ENST501), Enterobacter asburiae (ENSD102), Enterobacter ludwigii (ENSH201), Pseudomonas fluorescens (ENSH202 and ENSG304), uncultured Vitreoscilla sp. (ENSG301 and ENSG305), Acinetobacter lwoffii (ENSG302), Klebsiella pneumoniae (ENSG303), and Bacillus thuringiensis (ENSW401) were identified based on 16S rRNA gene sequencing. Scanning electron microscope (SEM) images revealed that biofilm matrix produced by E. asburiae ENSD102, uncultured Vitreoscilla sp. ENSG301, A. lwoffii ENSG302, and K. pneumoniae ENSG303 are highly fibrous, compact, and nicely interlinked as compared to the biofilm developed by E. ludwigii ENSH201 and B. thuringiensis ENSW401. X-ray diffraction (XRD) results indicated that biofilm matrix produced by E. asburiae ENSD102, uncultured Vitreoscilla sp. ENSG301, and A. lwoffii ENSG302 are non-crystalline amorphous nature. Fourier transform infrared (FTIR) spectroscopy showed that proteins and polysaccharides are the main components of the biofilms. Congo red binding results suggested that all these bacteria produced proteinaceous curli fimbriae and cellulose-rich polysaccharide. Production of cellulose was also confirmed by Calcofluor binding- and spectrophotometric assays. E. asburiae ENSD102, Vitreoscilla sp. ENSG301, and A. lwoffii ENSG302 were tested for their abilities to form the biofilms exposure to 0 to 2000 mg/L of copper sulfate (for Cu), zinc sulfate (for Zn), lead nitrate (for Pb), nickel chloride (for Ni), and potassium dichromate (for Cr), several concentrations of these metals activated the biofilm formation. The polysaccharides is known to sequester the heavy metals thus, these bacteria might be applied to remove the heavy metals from wastewater.

细菌生物膜（Bacterial biofilm）在废水重金属生物修复中发挥着关键作用。本研究从废水中分离并鉴定了多株产生物膜细菌，同时对不同细菌所产生的生物膜基质（即胞外聚合物（extracellular polymeric substances, EPS））开展了表征分析。从不同废水中分离得到的40株菌株中，仅11株（占比27.5%，28℃静置培养条件）与9株（占比22.5%，28℃、37℃振荡及静置培养条件）可分别在仅添加2%甘油的盐优化肉汤（salt-optimized broth plus 2% glycerol, SOBG）培养基中形成气-液（air-liquid, AL）型与固-气-液（solid-air-liquid, SAL）型生物膜，其余受试培养基均未诱导产生此类生物膜。各菌株的生物膜生物量及气-液型生物膜所耦合的细菌数量均存在极显著差异（P ≤ 0.001）。通过16S rRNA基因测序，共鉴定出以下菌株：大肠杆菌（Escherichia coli，菌株ENSD101、ENST501）、阿氏肠杆菌（Enterobacter asburiae，菌株ENSD102）、路德维希肠杆菌（Enterobacter ludwigii，菌株ENSH201）、荧光假单胞菌（Pseudomonas fluorescens，菌株ENSH202、ENSG304）、未培养透明颤菌属（uncultured Vitreoscilla sp.，菌株ENSG301、ENSG305）、路氏不动杆菌（Acinetobacter lwoffii，菌株ENSG302）、肺炎克雷伯菌（Klebsiella pneumoniae，菌株ENSG303）以及苏云金芽孢杆菌（Bacillus thuringiensis，菌株ENSW401）。扫描电子显微镜（scanning electron microscope, SEM）成像结果显示，相较于路德维希肠杆菌ENSH201与苏云金芽孢杆菌ENSW401形成的生物膜，阿氏肠杆菌ENSD102、未培养透明颤菌属ENSG301、路氏不动杆菌ENSG302以及肺炎克雷伯菌ENSG303所产生的生物膜基质呈现出高度纤维状、结构紧凑且连接紧密的特征。X射线衍射（X-ray diffraction, XRD）分析结果表明，阿氏肠杆菌ENSD102、未培养透明颤菌属ENSG301以及路氏不动杆菌ENSG302所产生的生物膜基质为非晶态无定形结构。傅里叶变换红外（Fourier transform infrared, FTIR）光谱分析显示，蛋白质与多糖是生物膜的主要组成成分。刚果红结合实验结果表明，所有受试菌株均可产生蛋白质类卷曲菌毛纤丝（curli fimbriae）及富含纤维素的多糖。纤维素的产生进一步通过钙荧光白结合法与分光光度法实验得到了验证。选取阿氏肠杆菌ENSD102、透明颤菌属ENSG301以及路氏不动杆菌ENSG302，测试其在0~2000 mg/L的硫酸铜（对应Cu²+）、硫酸锌（对应Zn²+）、硝酸铅（对应Pb²+）、氯化镍（对应Ni²+）及重铬酸钾（对应Cr⁶+）环境中形成生物膜的能力，结果显示不同浓度的上述重金属均可激活生物膜的形成。已知多糖能够螯合重金属，因此此类菌株可用于废水中重金属的去除修复。