Biofilm Dynamics and Role of Electricigens under Stressful Conditions for Wastewater Treatment, Bio-electricity Generation and Bio-monitoring

Abstract

The growing concern in the environmental perspective demands cost-effective treatment systems for wastewater and monitoring before it is discharged into the environment. Biofilms have successfully been used in water and wastewater treatment processes for well over a century. Biological wastewater treatment in suspended or attached growth reactors is an energy intensive process and there is need for improving the nutrient removal efficiency. Bioelectricity production through bioremediation of liquid and solid waste is one of the thrust areas of research in the environmental circles. Among different treatment technologies currently under investigation or being practically used, Microbial fuel cell (MFC) has been proposed quite effective. This technique uses electrochemically active microbes (EAM) for electricity generation using organic and inorganic waste in liquid or solid state. Electrochemically active microbial strains are cost effective biological catalysts in wastewater treatment and bioelectricity (energy) production in MFC. Microbial fuel cell technology also has scope for biomonitoring of different pollutants in the wastewater. In MFC based biosensor, the strength of current corresponds to the amounts of pollutants. The first phase of the study employed a multifaceted strategy, whereby, the microbial consortia of municipal sewage/wastewater was employed in attached growth batch reactors (AGBR) for investigating the treatment efficiency and microbial diversity under the effect of different metals such as ferric iron (Fe3+) and aluminum (Al3+). The results of the studies revealed 38% improved COD removal in the AGBR with 8.5mg/L iron and its supplementation supported more biofilm biomass. Fluorescent in situ hybridization (FISH) with phylogenetic oligonucleotide probes coupled with confocal laser scanning microscopy (CLSM) and digital image analysis showed an increase in the density of eubacteria and beta proteobacteria whereas gamma proteobacteria decreased with an increasing concentration of Fe3+ . Likewise, the COD (mg/l) of the sludge was reduced (28.68%) with Al3+ (2.5 mg/l) but the sludge digestibility decreased at higher concentrations (4.5 mg/l-6.5 mg/l) of Al3+ compared to the control. Conventional (culture based heterotrophic plate count) and molecular based studies (FISH-CLSM, and 16S rDNA sequencing) indicated limiting effect of increasing concentrations of Al3+ (0mg/l-6.5 mg/l), though non-significant, on the bacterial density and diversity. The bacterial density decreased (7.33%) with an increase in Al3+ concentration (0 mg/l-6.5 mg/l). Extracellular polymeric substances (EPS) are biopolymers self-produced by microorganisms, widely studied for their economic importance as the biocoagulants to replace chemical-based coagulants in the sludge flocculation and for applications in food industries.EPS producing bacteria were isolated from biofilms developed on support material such as waste tire rubber in the attached growth batch reactors with and without the incorporation of different concentrations of iron and aluminum. The bacteria were screened, identified and characterized for their EPS production potentials using different assays. An about 66% of the bacterial isolates were capsule forming (Anthony’s capsule staining) whereas 30% of xviii them strong biofilm formers. Dry weight and viscosity measurement revealed higher slime EPS (two fold) and viscosity production in pure cultures of bacteria rather than in consortium. Nevertheless, higher carbohydrates production was higher in consortia compared to pure culture of bacteria. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectra further confirmed variations in EPS production and its components. Highest EPS production was noticed in Enterobacter sp., Citrobacter sp., Clostridium sp. and Candidimonas sp. through 16SrRNA gene sequencing. In the second phase of the study, the conventional approach of treating wastewater in the attached growth reactors was studied in microbial fuel cells (MFCs) where this biocatalysis was basically integrated with the energy production and biomonitoring. MFCs, operated in a fed batch mode with microbial communities from cropland soil and lake sediment showed an about 87.7% and 45% improved voltage output, respectively when supplemented with iron compared to control. Illumina MiSeq sequencing showed predominant enrichment of genera; Pseudomonas, Sedimentibacter, Aminobacterium, Clostridium and Flavobacterium. Alpha rarefaction curves and Shannon index revealed higher enrichment of diverse bacterial community on anodic biofilm from soil than the sediment treated with Fe3+ .MFC inoculated with the sediment sample supported enrichment of delta-proteobacteria (included Shewanella sp. and Geobacter sp.) with iron. The isolated electrochemically active bacteria (EAB) were identified as Staphylococcus sp., Bacillus sp., Streptomyces sp. and Gordonia sp. based on 16SrRNA sequencing. Mediator-less dual chamber microbial fuel cells (MFC) were fabricated and operated to evaluate their role as biosensing devices for BOD measurements using natural (activated sludge) and artificial formulated bacterial (Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus circulans) consortia. The study validated a considerable application of MFC as a biosensor device for effective and continuous monitoring of BOD in water. The MFC biosensors were optimized and calibrated at pH 7, temperature 37 oC using 100 mM phosphate buffer with 100 mM NaCl solution as a catholyte at 10 kΩ external resistance. The power density (14.2 mWcm-2) achieved with MFC-I with the sludge consortium was 5 folds higher than MFC-II with artificial consortium. The relative performance of MFC-I, in terms of stability (55-60 days) and reproducibility (±15.4%), was double than MFC-II biosensor. The co-existence of different electron acceptors such as phosphate, nitrate and nitrite at low concentration in the anolyte had an insignificant effect on the biosensor performance. Molecular based phylogeny revealed the bacterial community structure in the sludge was considerably altered during enrichment in MFC. Gram positive bacteria are generally considered weak electricigens due to the presence of thick non conductive cell wall. An eight genes-based locus, responsible for extracellular electron transfer (EET), has recently been reported in Listeria monocytogenes, a Gram-positive food borne pathogen. So, the role of Ndh1/NDH-2a and Ndh2/NDH-2b in respiration and EET was investigated using electrochemical techniques and by growing wild type (WT), mutant and complementary strains of L. monocytogenes xix EGDe in single chamber bioelectrochemical cells with ultra-smooth template stripped gold (TSG) electrodes modified with self-assembled monolayers (SAMs) of carboxylic acid terminated thiols. The results of the study revealed that Ndh2/NDH-2b is involved in EET and L. monocytogenes was able to respire in the absence of Ndh1/NDH-2a. Overall, the findings of the studies indicate that supplementation of Fe3+ enhanced the sludge digestibility, microbial fuel cell (MFC) performance and significantly impacted the bacteria density and diversity. Natural activated sludge-based consortium in MFC biosensor proved to be more stable for continuous monitoring of BOD. The identified bacteria have high potential to be used for different applications such as wastewater treatment, biopolymer and electricity production and can be considered as the potential candidates for further studies.