Abstract
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.
Highlights
Discharge of untreated wastewater into the rivers, cannels, lakes, and ponds is one of the major causes of water pollution
Biofilms consist mainly of cells embedded in a self-produced extracellular polymeric substances [EPS (Costerton et al, 1999; Donlan and Costerton, 2002; Flemming and Wingender, 2010)]
11 (27.5%) isolates, such as ENSD101, ENSD102, ENSH201, ENSH202, ENSG301, ENSG302, ENSG303, ENSG304, ENSG305, ENSW401, and ENST501 were found to produce fragile to rigid AL biofilms at the air–liquid interface in the glass test tubes containing 5 mL of SOBG (Figure 1A) after 72 h incubation at 28◦C but not in yeast extract peptone (YP), Luria Bertani (LB), King’s B (KB), yeast peptone dextrose adenine (YPDA), and M63 glycerol minimal media
Summary
Discharge of untreated wastewater into the rivers, cannels, lakes, and ponds is one of the major causes of water pollution. Numerous physicochemical methods (e.g., chemical precipitation, oxidation, reduction, activated carbon, ion-exchange, reverse osmosis, membrane filtration, and evaporation) are being practiced to treat the wastewater. Most of these methods are expensive, ineffective, and required high energy and produced large amount of sludge with hazardous by-products (Ahluwalia and Goyal, 2007; Dixit et al, 2015). Biofilms consist mainly of cells embedded in a self-produced extracellular polymeric substances [EPS (Costerton et al, 1999; Donlan and Costerton, 2002; Flemming and Wingender, 2010)]. Composition of the EPS vary between biofilms, species, surface on which biofilms are formed and environmental conditions, including availability of the nutrients, temperature, and oxygen tension (Prouty and Gunn, 2003; Haque et al, 2012; Koechler et al, 2015)
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