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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.

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