Functional amyloids keep quorum-sensing molecules in check.

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.

Membrane permeabilization underlies the enhancement of extracellular bioactivity in Shewanella oneidensis by a membrane-spanning conjugated oligoelectrolyte.

A stilbene-based membrane spanning conjugated oligoelectrolyte 4,4'-bis(4'-N,N-bis(6"-(N,N,N-trimethyl ammonium) hexyl) amino)-styryl) stilbene tetraiodide (DSSN+) has been reported to be able to interact with bacterial cells and enhance their bioelectricity generation in bioelectrochemical devices, although the mechanism remains elusive. The goal of this study was to elucidate the impacts of DSSN+ on extracellular bioactivity and the underlying mechanism. Specifically, extracellular ferrihydrite reduction by Shewanella oneidensis was used to evaluate the influence of cell-DSSN+ interaction. Our results show that DSSN+ enhanced ferrihydrite reduction by S. oneidensis in a growth-dependent manner. The incorporation of DSSN+ into S. oneidensis cell membrane increased the extracellular concentration of redox shuttles, i.e., flavins, and extracellular enzyme activities without significantly decreasing cell viability. The findings suggested that membrane permeabilization is the dominant mechanism for the enhancement of extracellular bioactivity in S. oneidensis by DSSN+. We further demonstrated that the interaction between DSSN+ and S. oneidensis cells enhanced biofilm formation and stability without compromising the overall biofilm activity. Taken together, our results suggest that membrane spanning conjugated oligoelectrolytes, of which DSSN+ is one of many possible molecular structures, may be applied to enhance extracellular bioactivity in bacteria toward more efficient biofilm-based biocatalysis.

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.

Improving charge collection in Escherichia coli-carbon electrode devices with conjugated oligoelectrolytes.

It is important to tailor biotic-abiotic interfaces in order to maximize the utility of bioelectronic devices such as microbial fuel cells (MFCs), electrochemical sensors and bioelectrosynthetic systems. The efficiency of electron-equivalent extraction (or injection) across such biotic-abiotic interfaces is dependent on the choice of the microbe and the conductive electrode material. In this contribution, we show that spontaneous intercalation of a conjugated oligoelectrolyte, namely 4,4'-bis(4'-(N,N-bis(6''-(N,N,N-trimethylammonium)hexyl)amino)-styryl)stilbene tetraiodide (DSSN+), into the membranes of Escherichia coli leads to an increase in current generation in MFCs containing carbon-based electrodes. A combination of scanning electron microscopy (SEM) and confocal microscopy was employed to confirm the incorporation of DSSN+ into the cell membrane and biofilm formation atop carbon felt electrodes. Current collection was enhanced by more than 300% with addition of this conjugated oligoelectrolyte. The effect of DSSN+ concentration on electrical output was also investigated. Higher concentrations, up to 25 µM, lead to an overall increase in the number of charge equivalents transferred to the charge-collecting electrode, providing evidence in support of the central role of the synthetic system in improving device performance.

Enhancement in hydrogen evolution using Au-TiO2 hollow spheres with microbial devices modified with conjugated oligoelectrolytes.

OBJECTIVE: Although photoelectrochemical (PEC) water splitting heralds the emergence of the hydrogen economy, the need for external bias and low efficiency stymies the widespread application of this technology. By coupling water splitting (in a PEC cell) to a microbial fuel cell (MFC) using Escherichia coli as the biocatalyst, this work aims to successfully demonstrate a sustainable hybrid PEC-MFC platform functioning solely by biocatalysis and solar energy, at zero bias. Through further chemical modification of the photo-anode (in the PEC cell) and biofilm (in the MFC), the performance of the hybrid system is expected to improve in terms of the photocurrent generated and hydrogen evolved. METHODS: The hybrid system constitutes the interconnected PEC cell with the MFC. Both PEC cell and MFC are typical two-chambered systems housing the anode and cathode. Au-TiO2 hollow spheres and conjugated oligoelectrolytes were synthesised chemically and introduced to the PEC cell and MFC, respectively. Hydrogen evolution measurements were performed in triplicates. RESULTS: The hybrid PEC-MFC platform generated a photocurrent density of 0.35 mA/cm2 (~70× enhancement) as compared with the stand-alone P25 standard PEC cell (0.005 mA/cm2) under one-sun illumination (100 mW/cm2) at zero bias (0 V vs. Pt). This increase in photocurrent density was accompanied by continuous H2 production. No H2 was observed in the P25 standard PEC cell whereas H2 evolution rate was ~3.4 µmol/h in the hybrid system. The remarkable performance is attributed to the chemical modification of E. coli through the incorporation of novel conjugated oligoelectrolytes in the MFC as well as the lower recombination rate and higher photoabsorption capabilities in the Au-TiO2 hollow spheres electrode. CONCLUSIONS: The combined strategy of photo-anode modification in PEC cells and chemically modified MFCs shows great promise for future exploitation of such synergistic effects between MFCs and semiconductor-based PEC water splitting.

Chemically Functionalized Conjugated Oligoelectrolyte Nanoparticles for Enhancement of Current Generation in Microbial Fuel Cells.

Water-soluble conjugated oligoelectrolyte nanoparticles (COE NPs), consisting of a cage-like polyhedral oligomeric silsesquioxanes (POSS) core equipped at each end with pendant groups (oligo(p-phenylenevinylene) electrolyte, OPVE), have been designed and demonstrated as an efficient strategy in increasing the current generation in Escherichia coli microbial fuel cells (MFCs). The as-prepared COE NPs take advantage of the structure of POSS and the optical properties of the pendant groups, OPVE. Confocal laser scanning microscopy showed strong photoluminescence of the stained cells, indicating spontaneous accumulation of COE NPs within cell membranes. Moreover, the electrochemical performance of the COE NPs is superior to that of an established membrane intercommunicating COE, DSSN+ in increasing current generation, suggesting that these COE NPs thus hold great potential to boost the performance of MFCs.

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.

Anditalea andensis ANESC-ST--An Alkaliphilic Halotolerant Bacterium Capable of Electricity Generation under Alkaline-Saline Conditions.

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, Anditalea andensis ANESC-ST (=CICC10485T=NCCB 100412T), which is capable of generating bioelectricity in alkaline-saline conditions. A. andensis ANESC-ST 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 Escherichia coli or Pseudomonas aeruginosa yielded enhanced electrical output. The ability of A. andensis ANESC-ST to generate energy under alkaline-saline conditions points towards a solution for bioelectricity recovery from alkaline-saline wastewater. This is the first report of A.andensis ANESC-ST producing bioelectricity at high salt concentration and pH.

Uncovering alternate charge transfer mechanisms in Escherichia coli chemically functionalized with conjugated oligoelectrolytes.

Conjugated oligoelectrolytes integrated in Escherichia coli have been proposed to act as 'molecular wires', inducing extracellular electron transfer. This is monitored through reduction of HAuCl4 to form gold nanoparticles. In contrast to direct electron transfer, the reduction mechanism was traced to the release of electroactive cytosolic components during oligoelectrolyte incorporation.

A stable synergistic microbial consortium for simultaneous azo dye removal and bioelectricity generation.

Microbial species coexist in natural or engineered settings, where they encounter extensive competition and cooperation. Interactions occurring through metabolite exchange or direct contact might be important in establishment of functional biodegradation consortium. Understanding these interactions can facilitate manipulation of selected communities and exploitation of their capacity for specific industrial applications. Here, a simple dual-species consortium (Pseudomonas putida and Shewanella oneidensis) was established for examining simultaneous Congo red bioremediation in planktonic culture and power generation in anode biofilms. Compared to mono-species cultures, co-cultures generated higher current densities and could concurrently degrade Congo red over 72h. Disabling the large secreted adhesion protein, LapA, of P. putida greatly enhanced S. oneidensis biofilm formation on the anode, which increased power generation in co-cultures. This demonstrates simultaneous control of specific planktonic and biofilm communities could be effective in manipulating microbial communities for targeted applications.

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.