Characterization of Biofilm Structure Developed under Varying Concentrations of Fe(III) on Different Support Materials

Abstract

Biofilms are described as consortia of microorganisms adherent to biotic or abiotic surfaces embedded in a self-produced extracellular matrix containing polysaccharides, proteins and DNA. Biofilms can be harmful or useful depending on their area of existence. Biofilm formation is a nuisance, of particular relevance to human health, when found in drinking water reservoirs and distribution systems as these become a source of contamination and hinder the efficient operation of these systems. In addition, they may also pose a health risk to public by providing a habitat for pathogenic microorganisms. Conversely, however, biofilms are positively exploited in processes as diverse as biofilteraion, waste water treatment, production of fine chemicals and biofuel production. The overall objective of the study conducted here was to investigate the coupled effects of exogenous supplementation of different concentrations of Fe(III) and different support materials on microbial community structure and architecture of biofilms. The complexity of multispecies biofilm development and implications of microbial interactions on biofilm performance and their three dimensional structure were also underlined in this study. Four laboratory scale aerobic batch mode biofilm reactors (ABBR) (four liters each) were run under test and control modes at varying conditions of ferric iron 0, 2.5, 6.5 and 8.5 (mg/l) for biofilm development on Stainless steel (SS), Polyethylene based plastic (PE), Polyvinyl chloride (PVC), Iron (Fe) and Tire rubber (TR) by taking activated sludge as an inoculum. After 90 days of incubation results revealed that parametric variation i.e. concentration of Fe3+ and attachment surface properties exerted a significant effect on bacterial density in biofilms (p<0.05). Polyethylene accumulated more bacterial density with increasing concentration of Fe3+ being highest at 8.5 mg/l (1.77131E+11 CFU/ml cm2) whereas Fe and PVC, on the other hand showed a significant decrease in bacterial count with an increase in concentration of Fe3+ across the specified range (Fe = 2.241E+08 CFU/ml cm2, PVC = 8.84E+08 CFU/ml cm2). Biochemical identification of isolated bacteria from biofilms and activated sludge showed a wide diversity of bacteria which were mostly Gram negative and dominant species in biofilms included Pseudomonas sp., Vibrio sp., Shewanella sp., Providencia sp., Serratia sp., Klebsiella sp., Bacillus sp. and Staphylococcus sp. Sludge digestion indicated 38 percent more reduction in COD and BOD5 in the reactor fed with 8.5 mg/l of Fe(III) than untreated reactor representing the undergoing physiological activities of microorganisms. Molecular characterization of autotrophic nitrifiers and denitrifiers based on phylogenetic sequencing analysis indicated the presence of genera Nitrobacter, Bradyrhizobium and Rhodopseudomonas in biofilms on TR in reactor treated at different concentrations of Fe(III), however, more diversity of species was observed at 8.5 mg/l. Likewise, PE at Fe3+-8.5mg/l showed greater species of nitrifiers and denitrifiers related to the genera Bradyrhizobium, Nitrobacter and Rhodopseudomonas in addition to the genera Nitrosomoas and Nitrospira at Fe3+-6.5 mg/l. Fluorescent in situ hybridization (FISH) coupled with high resolution quantitative technique, confocal laser scanning microscopy (CLSM) of biofilm developed on TR revealed a significant increase in density of eubacteria from 3.00E+01 cells/cm2 at 2.5 mg/ to 1.05E+02 cells/cm2 at 8.5 mg/l and beta proteobacteria from 8.10E+01 cells/cm2 at 2.5 mg/l to 1.42E+02 cells/cm2 at 8.5 mg/l as the concentration of Fe(III) increased. Whereas, gamma proteobacteria demonstrated an inverse relationship between their cell density and ferric under different iron treatments (7.30E+01 cells/cm2 at 2.5 mg/ to 3.40E+01 cells/cm2 at 8.5 mg/l). Scanning Electron Microscopy (SEM) of biofilm on PVC showed greater diversity of microorganisms, EPS content and different cell sizes in biofilm developed on TR at 8.5 mg/l than at 0 mg/l. Similarly, effect of Fe3+ and different support materials were noted on biofilm and EPS development through FTIR that varied specifically in the region of 3270 cm- -3384.2 cm- in case of PVC, Fe and PE depicting carboxylic acids. Predominance of amines (1644.38 cm-, 1636.49 cm-) was observed in biofilms on SS and TR at lower concentrations of Fe(III) whereas significant abundance of carboxylic acids (3271.47 cm-) and ethers and esters (1035.03 cm-)in SS and TR, respectively, at its increased concentration. Further studies to understand how different support materials and Fe(III) concentrations mechanistically affect biofilm microbial community structures and functional performance are needed to devise strategies to control biofilms and enable the rational design of new generations of substrata for the improvement of biofilm based biological treatment processes.