Enrichment and Electrochemical Performance of Electricigens from Different Environmental Niches using Microbial Fuel Cells

Abstract

Renewable energy technologies (solar, wind, hydropower, 1st, 2nd, 3rd generation biofuels etc) are under development that may replace conventional approaches which are adding more carbon footprints and causing environmental deterioration. In current scenario, among other renewable solutions, microbial fuel cells (MFCs) has been viewed as promising energy technology, besides it can help in treatment of waste and pollutants in more viable and cost-effective manner. This thesis focused on boosting electrochemical performance of MFCs by investigating key factors like enrichment of electricigens from different environmental niche, the electrogenic properties of electricigens under varying situation of anolytes and fuel cell configurations. Experimental strategy include selection of (1) different environmental samples (petroleum contaminated soil, hot spring water, sludge, wastewater, sewer contaminated soil), (2) electrode materials (carbon cloth, graphite rod, carbon felt, graphite plates), (3) Fabrication of different MFCs [double chamber MFCs (PEM and salt bridge separators), air-cathode MFCs, parallel circuit air-cathode MFCs, membraneless air-cathode MFCs] (4) anolyte substrates (acetate, glucose, starch, bacterial growth medium) (5) catholytes (Bio-cathode (activated sludge), reducing compounds (K2MnO4), Air-cathode) under batch mode of operation. In phase 1, bacterial consortia from two extreme environments i.e., petroleum contaminated soil (MFC-1) and hot spring water (MFC-2) has been investigated as a potential source of electricigens in bio-cathode MFCs. Maximum power density 2.9Wm-2 to 5.5Wm-2 and 7.6Wm-2 to 1.3Wm-2 was recorded during enrichment stage 1 and 2 during operation of MFC-1 and MFC-2. The electrochemical performance of MFCs was significantly improved (1.8fold and 1fold) when applied in successive modes with enriched biofilms during enrichment stage 2 in two respective MFC setups. Additionally, biodegradation of petroleum contaminants and enhanced electrochemical performance of MFC-1 was further confirmed by the presence enriched bacterium Stenotrophomonas maltophilia on anode surface. In phase 2, the succession and enrichment of bacterial communities from activated sludge in double chamber salt bridge MFCs was evaluated. During enrichment stage 1, maximum voltage output of 136.2mV was recorded, that was increased by 3fold (418mV) during enrichment stage 2 with COD removal efficiency of 86.04% at ambient temperature. Molecular based phylogeny confirmed the enrichment of major contributing classes of α-proteobacteria 48.51%, β-proteobacteria 31.48% and γ-proteobacteria 16.16%. Several novel bacterial species i.e. Massilia timonae, Duganella sp. and Paracoccus aestuarii were identified to have bioelectrogeneic activity in MFC technology. Enrichment of anodic biofilms from already operating fuel cells, showed faster start-up and better in performance. In phase 3, biocatalytic activity of sewer contaminated soil bacterial flora on two different anode materials (MFC1 = carbon cloth anode and MFC2 = graphite rod anode) using PEM-MFCs under batch mode with continuous anolyte mixing at 50 rpm at 35±2oC. It has been observed that there is significant difference in power output between carbon cloth anode (27Wm-2 ) and graphite rod (12Wm-2 ). These results were further validated through ultra-micrographs of anodes using LSM and SEM. Biofilm developed on carbon cloth anode was much thicker (enriched) in terms of electricigens as compared to graphite anode. Maximum density of phylum Proteobacteria 99.1% including classes (Class: β-Proteobacteria>γ-Proteobacteria>α-Proteobacteria) was comparatively higher on graphite anode than carbon cloth anode [(94.5%) (Class: γ Proteobacteria>β-Proteobacteria > Opitutae)]. Carbon cloth anode was enriched with Pseudomonas sp. (35.73%) followed by Methyloversatilis universalis (16.237%), Pseudomonas plecoglossicida (7.16%), Pseudoxanthomonas Mexicana (5.589%) etc, while, the graphite anode enriched with Methyloversatilis universalis (55.7) followed by Nitrosomonas europaea (13%), Stenotrophomonas acidaminiphila (11%) etc. In phase 4, parallel circuit air-cathode MFCs were operated under different concentrations (0mlL-1 DMFC-1, 10mlL-1 DMFC-2, 50mlL-1 DMFC-3 and 100mlL-1 DMFC-4) of diesel taking bacteria from diesel oil contaminated soil. Maximum current (Imax = 43.11mA) under applied potential (-1V to 1V) was recorded using DMFC-3. Bioelectrochemical activity (13mA) of Bacillus toyonensis (MN173853) was monitored for the first time in MFC reactors. Bacillus sp. was found to have greatest electrochemical activity (22mA) and biodegradation capability (88%) in MFC. Up scaling of MFCs at bench pilot scale, indicated that maximum current (Imax) of about 795mA in (2L) AC-MFC. Whereas, Imax (1098mA) was about 1.38 folds higher in 8L AC-MFC. In phase 5, membrane less air-cathode MFCs (MLAC MFCs) were fabricated in order to optimize of anode to cathode spacing (between 20mm, 40mm, 60mm, 80mm) and associated biodegradation of simple (acetate, glucose) to complex (starch, wastewater) substrates which previously reported to have an influence on MFC performance was evaluated. Maximum potential output (Imax 1.8mA, PD 113.8±10.6 Wm-2 with COD removal efficiency of 95%) at minimum carbon felt anode distance (20mm) in MLAC-MFCs was recorded. Statistically, significant difference is observed between maximum current density generated (117mAm-2 ± 7.5) with carbon felt and graphite plate anode (94.31mAm-2±5.7). Fermentation rate constant (k=0.1523h-1 ) was much larger than hydrolysis and fermentation rate constant (k = -0.0747h-1 ) which means hydrolysis is rate limiting step in performance of MLAC-MFCs when operated with simple (acetate, glucose) to complex (starch, wastewater) substrates. Enrichment of electricigens from diesel contaminated soil under diesel influence (50mlL-1 ) in parallel circuit air-cathode MFC was done. Efficiently enriched electricigens (Bacillus sp. (22mA) followed by Bacillus lichenformis (16mA), Bacillus toyonensis (13mA) etc) has significantly improved the MFC performance. Simple anolytes (acetate (137mAm-2 ), glucose (135mAm-2 )) has been proved to be better substrates for boosting MFC performance than complex substrates (starch, wastewater), showed faster start-up in less than 24hours and remained sustainable for 120hrs using acetate, 100hrs with glucose. Carbon felt anode (111.62Wm-2 ) was shown to be better in performance and cost-effective material as compared to other electrode materials like graphite plate (106.62Wm-2 ), carbon cloth anode (27Wm-2 ) and graphite rod (12Wm 2 ). The current study deciphered the relationships and profiles of enriched electricigens from different environmental niches on MFC performance that could be helpful guide for future up-scale MFC studies. Through our investigations we opened a plethora of possibilities to use MFC as cost-effective renewable energy technology in Pakistan in near future.