Synergistic Microbial Consortium for Bioenergy Generation from Complex Natural Energy Sources

Microbial species have evolved diverse mechanisms for utilization of complex carbon sources. Proper combination of targeted species can affect bioenergy production from natural waste products. Here, we established a stable microbial consortium with Escherichia coli and Shewanella oneidensis in microbial fuel cells (MFCs) to produce bioenergy from an abundant natural energy source, in the form of the sarcocarp harvested from coconuts. This component is mostly discarded as waste. However, through its usage as a feedstock for MFCs to produce useful energy in this study, the sarcocarp can be utilized meaningfully. The monospecies S. oneidensis system was able to generate bioenergy in a short experimental time frame while the monospecies E. coli system generated significantly less bioenergy. A combination of E. coli and S. oneidensis in the ratio of 1 : 9 (v : v) significantly enhanced the experimental time frame and magnitude of bioenergy generation. The synergistic effect is suggested to arise from E. coli and S. oneidensis utilizing different nutrients as electron donors and effect of flavins secreted by S. oneidensis. Confocal images confirmed the presence of biofilms and point towards their importance in generating bioenergy in MFCs

Investigation of charge transfer mechanisms for electroconductivity and bioremediation in bacterial biofilms

This thesis examines extracellular electron transfer (EET) mechanisms and flavins/metabolite exchange in chemically modified biofilms and natural systems to demonstrate syntrophy for energy and bioremediation applications. Recently, chemical modification of bacteria to exhibit enhancement in microbial fuel cells (MFCs), bioremediation of waste products and electrode-driven biosynthesis of by-products has been demonstrated through insertion and self-assembly of unique organic molecules in cellular membranes. The mechanism for enhancement had been attributed to improved charge transfer through the inserted conjugated molecules. Using the novel transmembrane electron transport molecule (TETM), namely, 4, 4’-bis (4’-(N, N-bis (6’’-(N, N, N trimethylammonium)hexyl)amino)-styryl)stilbene tetraiodide, (DSSN+) to chemically modify Escherichia coli, the role of the TETM as a potential charge transfer pathway at the microbe-electrode (biotic-abiotic) interface is studied using MFCs. Significant EET enhancement is observed in this platform and confocal microscopy techniques confirms the incorporation of DSSN+ into the cell membranes of E. coli biofilms formed at the electrodes. To uncover the dominant EET mechanism in DSSN+ incorporated E. coli systems, the bio-reduction process forming gold nanoparticles is monitored. The prevalent mechanism is revealed to be membrane perturbation with the concomitant release of intracellular redox-active components and not enhanced charge transfer as originally proposed. DSSN+ is also unable to restore charge transfer capabilities in the mutant Shewanella oneidensis strain with deleted EET genes, pointing to the absence of charge transfer through the conjugated molecules. In natural systems, EET and flavins/metabolite exchange are investigated by coupling fermentative E. coli and electrochemically active S. oneidensis in a syntrophic community, which significantly affects bioelectricity generation. Notably, the naturally established syntrophic relationship drove preferential colonization in the planktonic/biofilm modes (on the electrode surface), based on the functions of each strain and the metabolite of exchange. In the syntrophic system, EET mechanisms are especially dominated by S. oneidensis. Further exploitation of another simple syntrophy between Pseudomonas putida and S. oneidensis drove concurrent bioelectricity generation and increased rate of bioremediation. In summary, investigation of novel charge transfer mechanisms and flavins/metabolite exchange for electroconductivity and bioremediation in bacterial biofilms was carried out by studying both chemically modified bacteria and natural interactions between cooperative bacterial species.DOCTOR OF PHILOSOPY (MSE

Metabolite-enabled mutualistic interaction between Shewanella oneidensis and Escherichia coli in a co-culture using an electrode as electron acceptor

Mutualistic interactions in planktonic microbial communities have been extensively studied. However, our understanding on mutualistic communities consisting of co-existing planktonic cells and biofilms is limited. Here, we report a planktonic cells-biofilm mutualistic system established by the fermentative bacterium Escherichia coli and the dissimilatory metal-reducing bacterium Shewanella oneidensis in a bioelectrochemical device, where planktonic cells in the anode media interact with the biofilms on the electrode. Our results show that the transfer of formate is the key mechanism in this mutualistic system. More importantly, we demonstrate that the relative distribution of E. coli and S. oneidensis in the liquid media and biofilm is likely driven by their metabolic functions towards an optimum communal metabolism in the bioelectrochemical device. RNA sequencing-based transcriptomic analyses of the interacting organisms in the mutualistic system potentially reveal differential expression of genes involved in extracellular electron transfer pathways in both species in the planktonic cultures and biofilms.Published versio

Engineering PQS biosynthesis pathway for enhancement of bioelectricity production in Pseudomonas aeruginosa microbial fuel cells

The biosynthesis of the redox shuttle, phenazines, in Pseudomonas aeruginosa, an ubiquitous microorganism in wastewater microflora, is regulated by the 2-heptyl-3,4-dihydroxyquinoline (PQS) quorum-sensing system. However, PQS inhibits anaerobic growth of P. aeruginosa. We constructed a P. aeruginosa strain that produces higher concentrations of phenazines under anaerobic conditions by over-expressing the PqsE effector in a PQS negative ΔpqsC mutant. The engineered strain exhibited an improved electrical performance in microbial fuel cells (MFCs) and potentiostat-controlled electrochemical cells with an approximate five-fold increase of maximum current density relative to the parent strain. Electrochemical analysis showed that the current increase correlates with an over-synthesis of phenazines. These results therefore demonstrate that targeting microbial cell-to-cell communication by genetic engineering is a suitable technique to improve power output of bioelectrochemical systems

<i>Anditalea andensis</i> ANESC-S^T - An Alkaliphilic Halotolerant Bacterium Capable of Electricity Generation under Alkaline-Saline Conditions

<div><p>A great challenge in wastewater bioremediation is the sustained activity of viable microorganisms, which can contribute to the breakdown of waste contaminants, especially in alkaline pH conditions. Identification of extremophiles with bioremediation capability can improve the efficiency of wastewater treatment. Here, we report the discovery of an electrochemically active alkaliphilic halotolerant bacterium, <i>Anditalea andensis</i> ANESC-S^T (=CICC10485^T=NCCB 100412^T), which is capable of generating bioelectricity in alkaline–saline conditions. <i>A</i>. <i>andensis</i> ANESC-S^T was shown to grow in alkaline conditions between pH 7.0–11.0 and also under high salt condition (up to 4 wt% NaCl). Electrical output was further demonstrated in microbial fuel cells (MFCs) with an average current density of ~0.5 µA/cm², even under the harsh condition of 4 wt% NaCl and pH 9.0. Subsequent introduction of secreted extracellular metabolites into MFCs inoculated with <i>Escherichia coli</i> or <i>Pseudomonas aeruginosa</i> yielded enhanced electrical output. The ability of <i>A</i>. <i>andensis</i> ANESC-S^T to generate energy under alkaline–saline conditions points towards a solution for bioelectricity recovery from alkaline–saline wastewater. This is the first report of <i>A</i>.<i>andensis</i> ANESC-S^T producing bioelectricity at high salt concentration and pH.</p></div

Current density vs. time plots of <i>E</i>. <i>coli</i> DH5α and <i>P</i>. <i>aeruginosa lasIrhlI</i> mutant – based MFCs.

<p>EM stands for extracellular metabolites of <i>A</i>. <i>andensis</i> ANESC-S^T (2.5 mg/mL). Arrows indicate introduction of EM. Data represents an average of triplicates. </p

Some recent advances in automated analysis

<p>(A) Growth curves of <i>A</i>. <i>andensis</i> ANESC-S^T in LB broth with varied NaCl concentrations (Initial OD₆₀₀ ≈ 0.4). (B) <i>A</i>. <i>andensis</i> ANESC-S^T was inoculated and cultured for 14 hours in LB broth with varied NaCl concentrations (Initial OD₆₀₀ ≈ 0.4).</p

CV analysis of <i>A</i>. <i>andensis</i> ANESC-S^T in MFCs.

<p>(A) CV traces of MFCs inoculated with fresh M9 media, <i>A</i>. <i>andensis</i> ANESC-S^T and free cell spent media of <i>A</i>. <i>andensis</i> ANESC-S^T. The working electrode is carbon felt, counter electrode is Pt wire and the reference electrode is Ag/AgCl. Scan rate is 5 mV/s. Voltammograms were acquired in the presence of 10 mM L-Arabinose. (B) Plot of peak current vs. scan rate of the CV traces. All experiments were performed in triplicates.</p

Schematic illustration of two-chambered MFC for bioelectricity generation.

<p>Schematic illustration of two-chambered MFC for bioelectricity generation.</p