MSc, Faculty of Science, University of the Witwatersrand, 2011 / A growing interest is on the biological remediation of pollutants with the added benefit of generating electricity in microbial fuel cells (MFCs). Therefore, the analyses of suitability and potential of full-strength paper mill effluent and cheese whey were separately investigated in such devices. The most promising effluent was selected for biofilm optimization studies. In the biofilm buildup studies, anodes were enriched with microorganisms inherent to whey for a period between one and three months before their application in reactors. Independently, pre-incubated electrodes which were two-month-old were used serially in four MFCs of seven days each. In the preliminary study, the maximum power densities were 24 ± 3 mW/m2 (0.02 % coulombic efficiency − εcb) and 16.7 ± 1.8 W/m2 (εcb = 3.7 %) in paper mill effluent and whey, respectively. Following a three-month acclimation of whey anodophilic microbes, the power increased to 1 800 W/m2 (εcb = 80.9 %) and 92.8 % total chemical oxygen demand (tCOD) removal after a single batch cycle in MFCs. In anode recycling experiments, the operation was characterised by power of 390 ± 21 W/m2 (εcb = 0.25 %) in the third anode reuse; whilst the second reactor cycle had the highest tCOD removal (44.6 %). The anodophilic microbial species identified in cheese whey were from the Lactobacillus genus. This study concluded that wastes can supply fuel for power generation with simultaneous remediation; whey had greater potential than paper mill effluent; and both continual acclimation of inherent waste microbes and anode recycling improved the performance of MFCs.

Fundamental studies for the improvement of photo-microbial fuel cells (pMFCs) within this work comprised investigations into ceramic electrodes, toxicity of metal-organic frameworks (MOFs) and hot-pressing of air-cathode materials. A novel type of macroporous electrode was fabricated from the conductive ceramic Ti2AlC. Reticulated electrode shapes were achieved by employing the replica ceramic processing method on polyurethane foam templates. Cyclic voltammetry of these ceramics indicated that the application of potentials larger than 0.5 V with regard to a Ag/AgCl reference electrode results in the surface passivation of the electrode. Ti2AlC remained conductive and sensitive to redox processes even after electrochemical maximisation of the surface passivation, which was shown electrochemically and with four terminal sensing. Application of macroporous Ti2AlC ceramic electrodes in pMFCs with green algae and cyanobacteria resulted in higher power densities than achieved with conventional pMFC electrode materials, despite the larger surface area of the Ti2AlC ceramic. The effect of electrode surface roughness and hydrophobicity on pMFC power generation and on cell adhesion was examined using atomic force and confocal microscopy, contact angle measurements and long-term pMFC experiments. The high surface roughness and fractured structure of Ti2AlC ceramic was beneficial for cell adhesion and resulted in higher pMFC power densities than achieved with materials such as reticulated vitrified carbon foam, fluorine doped tin oxide coated glass or indium tin oxide coated plastic. Toxicity of the MOF MIL101 and its amine-modified version MIL-101(Cr)-NH2 on green algae and cyanobacteria was assessed on the basis of both growth in liquid culture and by exclusion zones of agar colonies around MOF pellets. MOF MIL101 was found harmless in concentrations up to 480 mg L-1 and MIL-101(Cr)-NH2 did not exhibit toxic effects at a concentration of 167 mg L-1. Air-cathodes were produced from a range of carbon materials and ion-exchange membranes. Hot-pressing of Zorflex Activated Carbon Cloth FM10 with the proton-selective Nafion® 115 membrane provided the best bonding quality and pMFC performance.

Photosynthetic Microbial Fuel Cell (p-MFC) research aims to develop devices containing photosynthetic micro-organisms to produce electricity. Micro-organisms within the device photosynthesise carbohydrates under illumination, and produce reductive equivalents (excess electrons) from both carbohydrate production and the subsequent carbohydrate break down. Redox mediators are utilised to shuttle electrons between the organism and the electrode. The mediator is reduced by the micro-organism and subsequently re-oxidised at the electrode. However this technology is in its early stages and extensive research is required for p-MFC devices to become economically viable. A basic p-MFC device containing a potassium ferricyanide mediator and the algae Chlorella vulgaris was assembled and tested. From these initial experiments it was realised that much more work was required to characterise cell and redox mediator activities occurring within the device. There is very little p-MFC literature dealing with cellular interaction with redox mediators, but without this knowledge the output of complete p-MFC devices can not be fully understood. This thesis presents research into the reduction of redox mediators by the micro-organisms, including rates of mediator reduction and factors affecting the rate. Both electrochemical and non-electrochemical techniques are used and results compared. Additionally, cellular effects relating to the presence of the mediator are studied; crucial to provide limits within which p-MFCs must be used. After basic characterisation, this thesis presents work into the optimisation of the basic p-MFC. Different redox mediators, photosynthetic species and anodic materials are investigated. Importantly, it is only through fundamental characterization to improve understanding that p-MFCs can be optimised.

Bioelectrochemical systems couple the oxidation of an electron donor at the anode with the reduction of an electron acceptor at the cathode, using microorganisms to catalyse one or both reactions. When the overall reaction is exergonic, a power output is generated and the system is referred to as microbial fuel cell (MFC); when power is added to the system and hydrogen is produced at the cathode through electrolysis of water, the system is referred to as microbial electrolysis cell (MEC). This PhD thesis is principally focused on the microbial fuel cells technology. Microbial fuel cells are regarded as a sustainable technology for electric energy generation from the oxidation of organic substrates contained in wastewater. The rising need for renewable energy sources and sanitation has encouraged intense research in this novel technology. Nevertheless, up untill now the interest has been primarily focused on the anodic oxidation of organic matter contained in wastewater. However, in addition to organics, wastewater also contains other pollutants, such as soluble nitrogen compounds, for which specific treatment is required. In conventional wastewater treatment systems, the organics available in the wastewater are typically used as electron donor during denitrification. However, a considerable fraction (>50%) of the chemical oxygen demand (COD) is still oxidized aerobically due to the large recirculation flows from the nitrification to the denitrification stages required in anoxic/aerobic configurations to allow for low nitrate levels in the final effluent. This increased COD demand is normally fulfilled by supplementary COD addition, with consequent increase of treatment costs. Alternatively, microorganisms can use inorganic carbon substrates and inorganic electron donors such as hydrogen for denitrification. However, the use of compressed hydrogen is hampered by its low solubility. As a solution, electrochemical hydrogen production permits in situ delivery of the electron donor and is advantaged by simplified control and dissolution of H2. The energy requirements to provide reducing power for denitrification can be decreased if bacteria use the electrode directly as electron donor without intermediate hydrogen production in bioelectrochemical systems. However, fundamental knowledge on bioelectrochemical denitrification is still lacking, therefore, this PhD thesis aims to fill some of these knowledge gaps and to solve some of the bottlenecks of the use of biocathodes. In particular, the goals of this work are: (i) to produce a suitable microbial community able to use the cathode as the sole electron donor during denitrification; (ii) to engineer a bioelectrochemical system able to couple the cathodic denitrification with the oxidation of organics at the anode; (iii) to characterize and quantify the electron losses during anodic and cathodic processes; (iv) to develop a bioelectrochemical system that maximises the nitrogen removal by integrating the nitrification stage into the cathode; finally, (v) to provide an insight into the structural properties of the biofilm performing nitrogen removal at the cathode. The results reveal that microbes can effectively utilize the electrode as electron donor for nitrate reduction to gaseous nitrogen at a redox potential that excludes intermediate production of hydrogen. Measurements revealed that acetoclastic methanogenesis and bacterial growth were responsible for causing the major electron losses at the anode. Adjusting the anodic potential did not achieve a significant overall reduction of the electron losses. At the cathode, the charge transfer efficiencies were instead very high, with the losses only due to the generation of nitrous oxide. Moreover, adjustments of the cathode potential resulted in higher efficiency. High carbon and nitrogen removal was obtained with a COD demand for denitrification as low as 2.4 g per g nitrogen denitrified, which is much lower than typically observed in heterotrophic–based nitrogen removal technologies (>7 g g 1). Nitrogen was removed at rates up to 0.256 kg N m-3 d-1, which is comparable to other autotrophic denitrification processes. Simultaneous nitrification and denitrification was observed in a combined system with cathodic aeration, at bulk dissolved oxygen (DO) levels up to 5 mg L-1, which is considerably higher than normally considered feasible for the process. Confocal laser scanning microscope analysis revealed the existence of a structured biofilm where putative nitrifying organisms occupied the outer layers in contact with the aerated bulk liquid, and putative denitrifying organisms occupy the layers closer to the electrode. These findings are significant in the field of bioelectrochemical systems as they help to unravel some of the complex questions relating to biocathodes. Additionally, the system provides an attractive option to achieve a very high level of nitrogen removal from wastewater with low COD/N ratios due to the selective utilisation of the COD for the denitrification reaction via the electrical transfer of reducing equivalents from the anode to the cathode. However, this research creates new questions, particularly regarding the mechanisms of electron transfer at the cathode. Also a number of practical design and optimisation challenges need to be overcome before wider applications can be considered.

Microbial electrochemical systems (MESs) have attracted much research attention in recent years due to their promising applications in renewable energy generation, bioremediation, and wastewater treatment. In a MES, microorganisms interact with electrodes via electrons, catalyzing oxidation and reduction reactions at the anode and the cathode. The bacterial community of a high power mixed consortium MESs (maximum power density is 6.5W/m��) was analyzed by using denature gradient gel electrophoresis (DGGE) and 16S DNA clone library methods. The bacterial DGGE profiles were relatively complex (more than 10 bands) but only three brightly dominant bands in DGGE results. These results indicated there are three dominant bacterial species in mixed consortium MFCs. The 16S DNA clone library method results revealed that the predominant bacterial species in mixed culture is Geobacter sp (66%), Arcobacter sp and Citrobacter sp. These three bacterial species reached to 88% of total bacterial species. This result is consistent with the DGGE result which showed that three bright bands represented three dominant bacterial species. Exoelectrogenic bacterial strain SX-1 was isolated from a mediator-less microbial fuel cell by conventional plating techniques with ferric citrate as electron acceptor under anaerobic conditions. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy-48 being the most closely related species. The bacterial strain SX-1 produced electricity from citrate, acetate, glucose, sucrose, glycerol, and lactose in MFCs with the highest current density of 205 mA/m�� generated from citrate. Cyclic voltammetry analysis indicated that membrane associated proteins may play an important role in facilitating electron transfer from the bacteria to the electrode. This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors. Citrobacter strain SX-1 is capable of generating electricity from a wide range of substrates in MFCs. This finding increases the known diversity of power generating exoelectrogens and provids a new strain to explore the mechanisms of extracellular electron transfer from bacteria to electrode. The wide range of substrate utilization by SX-1 increases the application potential of MFCs in renewable energy generation and waste treatment. Anode properties are critical for the performance of microbial electrolysis cells (MECs). Inexpensive Fe nanoparticle modified graphite disks were used as anodes to preliminarily investigate the effects of nanoparticles on the performance of Shewanella oneidensis MR-1 in MECs. Results demonstrated that average current densities produced with Fe nanoparticle decorated anodes were up to 5.9-fold higher than plain graphite anodes. Whole genome microarray analysis of the gene expression showed that genes encoding biofilm formation were significantly up-regulated as a response to nanoparticle decorated anodes. Increased expression of genes related to nanowires, flavins and c-type cytochromes indicate that enhanced mechanisms of electron transfer to the anode may also have contributed to the observed increases in current density. The majority of the remaining differentially expressed genes were associated with electron transport and anaerobic metabolism demonstrating a systemic response to increased power loads. The carbon nanotube (CNT) is another form of nano materials. Carbon nanotube (CNT) modified graphite disks were used as anodes to investigate the effects of nanostructures on the performance S. oneidensis MR-1 in microbial electrolysis cells (MECs). The current densities produced with CNT decorated anodes were up to 5.6-fold higher than plain graphite anodes. Global transcriptome analysis showed that cytochrome c genes associated with extracellular electron transfer are up-expressed by CNT decorated anodes, which is the leading factor to contribute current increase in CNT decorated anode MECs. The up regulated genes encoded to flavin also contribute to current enhancement in CNT decorated anode MECs. / Graduation date: 2013

A study of how mediators interact with the catabolic pathways of microbes was undertaken with a view towards improving the performance of microbial fuel cells. The use of mediators is known to improve the power density in microbial fuel cells, but this work suggests that no single mediator is ideally suited to the task. Instead, a carefully selected mixture of two targeted mediators (Methylene Blue and Neutral Red) might be optimal. To test this hypothesis, a yeast-catalyzed microbial fuel cell was built and empirically evaluated under different mediation conditions while keeping all other parameters constant. The results clearly show that an appropriate mix of the two mediators mentioned could indeed achieve significantly superior performance, in terms of power-density, than when either mediator is used singly. All tests were carried out using the same overall mediator concentration.

This thesis presents studies that examine microbial extracellular electron transfer that an emphasis characterizing how environmental conditions influence electron flux between microbes and a solid-phase electron donor or acceptor. I used bioelectrochemical systems (BESs), fluorescence and electron microscopy, chemical measurements, 16S rRNA analysis, and qRT-PCR to study these relationships among chemical, physical and biological parameters and processes. / Engineering and Applied Sciences

Η μικροβιακή κυψελίδα καυσίμου (ΜΚΚ) είναι ένας βιοαντιδραστήρας ο οποίος μετατρέπει απευθείας τη χημική ενέργεια ποικίλων υποστρωμάτων σε ηλεκτρική ενέργεια μέσω μικροβιακών καταλυτικών αντιδράσεων, σε αναερόβιες συνθήκες. Η διττή υπόστασή της τεχνολογίας να επεξεργάζεται λύματα με ταυτόχρονη παραγωγή ηλεκτρικής ενέργειας, έχει κερδίσει τα τελευταία χρόνια το ενδιαφέρον της επιστημονικής κοινότητας. Η παρούσα διδακτορική διατριβή προτείνει μια πρωτότυπη ΜΚΚ ενός θαλάμου ιδιαίτερης αρχιτεκτονικής, η οποία συνδυάζει πληθώρα πλεονεκτημάτων. Τα πειράματα που διεξήχθησαν είχαν ως απώτερο στόχο τη βελτιστοποίηση τόσο των σχεδιαστικών όσο και των λειτουργικών παραμέτρων της κυψελίδας, η οποία μελετήθηκε κάτω από το πρίσμα της εφαρμογής της σε μονάδες βιολογικού καθαρισμού αστικών λυμάτων. Η λογική που εργάστηκα βασίστηκε στη λειτουργία της συσκευής με πλήρη αντικατάσταση των ακριβών υλικών από φθηνότερα, ενώ ταυτόχρονα προσπάθησα σταδιακά να βελτιώσω την απόδοσή της, ακόμα και σε λειτουργίες μακράς διαρκείας. Η καινοτόμος κυψελίδα λειτούργησε σε συνθήκες διαλείποντος και συνεχούς έργου. Παράλληλα, με τη λειτουργία της καινοτόμου διάταξης, μελετήθηκε η επίδραση διαφορετικών παραμέτρων στην απόδοση ΜΚΚ δύο θαλάμων (τύπου H). Η εμπειρία που αποκτήθηκε από την προκειμένη λειτουργία, καθώς και τα αποτελέσματα των πειραμάτων αυτών, είναι πρωταρχικής σημασίας, καθώς αποτέλεσαν τον οδηγό για την καινοτόμο κατασκευή και τη λειτουργία της ΜΚΚ ενός θαλάμου. Έτσι λοιπόν, στα πλαίσια της παρούσας έρευνας μελετήθηκαν τόσο συνθετικά (γλυκόζη, πεπτόνη από χωνευμένο με τρυψίνη κρέας και αραβοσιτέλαιο) όσο και πραγματικά απόβλητα (ορρός τυρογάλακτος, αστικό λύμα). Ειδικότερα, μελετήθηκαν οι παράμετροι της ιοντικής ισχύος, του pH, του είδους του αποδέκτη ηλεκτρονίων, της θερμοκρασίας, της αρχικής συγκέντρωση του υποστρώματος, του υδραυλικού χρόνου παραμονής (HRT), της επιφάνειας του ανοδικού ηλεκτροδίου αλλά και της ποσότητας του καταλύτη της καθόδου. Επιπρόσθετα, προκειμένου να επιτευχθεί πλήρης ηλεκτροχημικός χαρακτηρισμός των κυψελίδων, διεξήχθηκαν πειράματα Φασματοσκοπίας Ηλεκτροχημικής Εμπέδησης (Electrochemical Impedance Spectroscopy, EIS) ενώ παράλληλα ελήφθησαν ηλεκτρονικές μικρογραφίες των ανοδικών ηλεκτροδίων με ηλεκτρονικό μικροσκόπιο σάρωσης (SEM). Τέλος, στα πλαίσια αξιοποίησης των πειραματικών αποτελεσμάτων της παρούσας διατριβής το μαθηματικό μοντέλο των Zeng et al τροποποιήθηκε κατάλληλα ώστε να καταστεί δυνατή η περιγραφή των αποτελεσμάτων της ΜΚΚ δύο θαλάμων. / A microbial fuel cell (MFC) is a bioreactor that converts the chemical energy of the bonds of organic compounds to electrical energy, through the catalytic reactions of microorganisms under anaerobic conditions. Over the last years the MFC technology has gained increasing interest from the scientific community, because it offers the advantage of simultaneous wastewater treatment and electricity generation. The present thesis proposes an innovative single chamber MFC design of a special architecture, which combines several advantages. The aim of the experiments was to optimise the design and the operational parameters of the proposed MFC, under the view of its practical implementation at wastewater treatment plants. In order to accomplish this goal the cost was kept low, by replacing all the expensive materials with lower-cost ones, while gradually increasing the cell performance even during long term operation. The MFC was operated both in batch and continuous mode. In parallel with single chamber MFC operation, the effects of several parameters on the performance of a dual chamber MFC (H-type) were examined. The findings from these experiments as well as the experience gained are of great significance, because they were used as guides for the construction and operation of the prototype cell. In conclusion, during the present research, synthetic (glucose, peptone, trypsin from meat digested and corn oil) as well as real wastewater (cheese whey, domestic wastewater) were examined. Specifically, the ionic strength, pH, the type of electron acceptor, the temperature, the initial substrate concentration, the Hydraulic Retention Time (HRT), the surface area of the anodic electrode as well as the quantity of the cathode catalyst were tested. Additionally, aiming at a detailed electrochemical characterization of the MFCs, the impedance characteristics were also investigated by performing Electrochemical Impedance Spectroscopy (EIS) experiments, while Scanning Electron Microscopy (SEM), images of the anodic biofilm were collected. Finally, for the valorization of the experimental results of the present thesis, the mathematical model of Zeng et al was appropriately modified in order to describe the experimental results of the dual chamber MFC.

Photosynthetic biofilms have much higher cell density than suspended cultures and when grown in a stacked waveguide configuration, can have orders of magnitude higher areal productivity. Evanescent and plasmonic growth of biofilm cultures have been demonstrated, solving issues with light penetration impeding growth, but thus far the technology has been limited to biofuel production applications. In this thesis, plasmonically excited cyanobacterial biofilms are used to produce electrical power in a photosynthetic-plasmonic-voltaic device. This approach uses red lasers to deliver light to cells via an optical waveguide through the generation of surface plasmons at the interface between a metal and dielectric, in this case a glass-gold-air interface. This gold film serves a dual purpose as a current collector for electrons generated at the cell surface. Experiments presented here demonstrate positive power output light response under both direct light and plasmonic excitation and produced equivalent power output of 6 uW/m2 under similar light power intensities.

Photosynthetic biofilms have much higher cell density than suspended cultures and when grown in a stacked waveguide configuration, can have orders of magnitude higher areal productivity. Evanescent and plasmonic growth of biofilm cultures have been demonstrated, solving issues with light penetration impeding growth, but thus far the technology has been limited to biofuel production applications. In this thesis, plasmonically excited cyanobacterial biofilms are used to produce electrical power in a photosynthetic-plasmonic-voltaic device. This approach uses red lasers to deliver light to cells via an optical waveguide through the generation of surface plasmons at the interface between a metal and dielectric, in this case a glass-gold-air interface. This gold film serves a dual purpose as a current collector for electrons generated at the cell surface. Experiments presented here demonstrate positive power output light response under both direct light and plasmonic excitation and produced equivalent power output of 6 uW/m2 under similar light power intensities.