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Utilizing photosynthetic entities in electrochemical systems enables converting solar energy into electrical energy, obtaining bio-hybrid photo-electrochemical systems. Such systems have been recently proposed for micro power generation, bioelectrosynthesis, and biosensing for in-situ water quality monitoring.[1] However, due to the photosynthetic apparatus in bacteria (i.e., purple bacteria and cyanobacteria) being physically separated from the electrode surface by the presence of various membrane layers, artificial approaches to divert the photoexcited electrons are required. As a result, research efforts have been focused on developing bio-compatible approaches to facilitate the transfer of photoexcited electrons from these bacteria to the electrodes (and vice versa).[2] Herein, a sustainable biophotoanode based on intact purple bacteria is utilized for the monitoring of phenol-class contaminants that affects photocurrent generation. Specifically, we focused on nitro-phenols and other phenols that might be released in water when food/agricultural wastes are disposed into the environment. All the investigated compounds are known to be toxic, affecting both humans and animals’ health. Furthermore, to follow the green chemistry and bio-circular economy principles, approaches for the possible removal/recovery of these phenols and their degradation products were also investigated, with their possible re-use as active compounds in biomedical applications.[3] Challenges and future research directions for the application of these systems in the field will be discussed. References: [1] M. Grattieri, Purple bacteria photo-bioelectrochemistry: enthralling challenges and opportunities, Photochem. Photobiol. Sci., 19 (2020) 424-435. [2] M. Grattieri, K. Beaver, E.M. Gaffney, F. Dong, S.D. Minteer, Advancing the fundamental understanding and practical applications of photo-bioelectrocatalysis, Chem. Commun., 56 (2020) 8553-8568. [3] J. Gubitosa, V. Rizzi, A. Lopedota, P. Fini, A. Laurenzana, G. Fibbi, F. Fanelli, A. Petrella, Laquintana, N. Denora, R. Comparelli, P. Cosma, One pot environmental friendly synthesis of Gold Nanoparticles using Punica Granatum Juice: a novel antioxidant agent for future dermatological and cosmetic applications, J. Colloid Interface Sci. 521 (2018) 50–61.

Photobioelectrocatalysis (PBEC) adopts the sophistication of photosynthetic units, their abundant availability, easy processing and sustainability, to convert solar energy into electricity. Specifically, the use of chloroplasts as photobioelectrocatalysts is desirable due to their metabolic independence and photoprotection mechanisms. However, the protein-based outer membranes of chloroplasts impede efficient charge transfer onto electrode surfaces during PBEC. Typical bio-inspired redox polymers used to ameliorate this charge transfer are branched, and contain redox pendants peripherally attached to a backbone. Pendants extract, and propagate electrons via collision-based electron tunneling. Conversely, we investigate the ability of an unbranched hybrid conductive-redox polymer, polydihydroxy aniline (PDHA), which has its redox moiety embedded in a conductive backbone. The redox moiety of PDHA is a quinone derivative, with redox potentials higher than the quinone-based redox active sites of chloroplasts. The biohybrid chloroplast-PDHA electrode encapsulates chloroplasts in a conductive matrix of PDHA, improving charge transfer. Holistically, PDHA facilitates the immobilization of chloroplasts and mediate electron transfer between chloroplasts and electrode surfaces. Our results demonstrate that a 120 % photocurrent increment is obtained upon combining chloroplasts with PDHA. Sequentially layering chloroplasts and PDHA on electrode surfaces evinces a 260 % increment in photocurrent responses. We report the highest photocurrent recorded with chloroplasts during PBEC (-48±3 µA cm-2) by pairing chloroplast-PDHA electrodes with diffusible redox mediator, 2,6-dichlorobenzoquinone. The study shows that redox polymer designs for artificially mediating electron transfer from chloroplasts can extend to unbranched conductive polymers in PBEC systems. Figure 1

Photo-bioelectrochemical systems allow for the integration of photosynthetic bacteria at an electrode surface for the conversion of solar energy into electrical current.1 Among various applications, these systems open for the continuous monitoring of toxic compounds in the environment based on their cytotoxic effects on bacteria activity. However, a challenge for the on-field application is the exposure of bacterial cells to a diverse range of condition, requiring robust, versatile microorganisms capable of tolerating dynamic environments. Rhodobacter capsulatus (R. capsulatus) is a purple, photosynthetic bacterium with an outstanding versatile metabolism. Specifically, its versatility is thought to be due to a bacteria phage-like element, the R. capsulatus gene transfer agent (rcGTA), enabling horizontal gene transfer across microorganisms, which results in an expedited evolution to new environmental stresses. The rcGTA has been seen to facilitate resistance to antibiotics2 and provides a mechanism for adaptation to various environmental conditions. Integrating this bacterium with an electrode for photo-bioelectrochemical system development proves to be challenging due to the active redox center’s location inside of the thick cellular membrane. Previous work in our group has succeeded in mediating this redox active center employing monomeric quinones for mediating the extracellular electron transfer to the electrode.3 Current research is focused on engineering redox hydrogels to enhance the extracellular electron transfer in high saline, resulting in equal or higher currents compared to non-saline. To further improve photo-bioelectrocatalysis performance through adaptation of the cells to high saline conditions, we investigated the adaptation mechanism using techniques to study the rcGTA, and bioinformatics to evaluate differential expression of genes in both saline and non-saline conditions. Further research will be focused on harnessing these findings to decrease adaptation time to high salinities and evaluating the bioelectrochemical performance of these adapted strains through chronoamperometry and cyclic voltammetry experiments. The successful completion of this study will contribute towards the design of a photo-bioelectrochemical system for toxic compound detection in high saline conditions, and further increase our knowledge of salt adaptation mechanisms and the gene transfer agent of Rhodobacter capsulatus. References: (1) Grattieri, M.; Minteer, S. D. Decoupling Energy and Power. Nat. Energy 2018, 3 (1), 8–9. https://doi.org/10.1038/s41560-017-0076-x. (2) Lang, A. S.; Zhaxybayeva, O.; Beatty, J. T. Gene Transfer Agents: Phage-like Elements of Genetic Exchange. Nat. Rev. Microbiol. https://doi.org/10.1038/nrmicro2802. (3) Grattieri, M.; Rhodes, Z.; Hickey, D. P.; Beaver, K.; Minteer, S. D. Understanding Biophotocurrent Generation in Photosynthetic Purple Bacteria. ACS Catal. 2019, 9 (2), 867–873. https://doi.org/10.1021/acscatal.8b04464.

High saline contamination events are a growing problem due to the increase in frequency and severity of hurricanes and the accompanying torrential rains.1 Storms affecting coastal areas can flood Superfund sites, which are designated hazardous waste sites managed by the Environmental Protection Agency (EPA), and other dangerous industrial waste sites. These events can result in the release of harmful pollutants into the environment, including heavy metals.2 The dimension of the contaminated area, as well as the cost and labor intensive approaches utilized, make the monitoring through standard laboratory methods complicated.3 Accordingly, a comprehensive screening of a community after a contamination event is not feasible. In this contest, biosensors offer a cost-effective alternative. Microbial biosensors are of particular interest for environmental monitoring due to their stability, wide range of analytes, and the ability to report on physiological toxicity.4 However, high salinities pose stress on the cellular membrane of bacteria cells, and few microbes can survive in salinities higher than the ocean (~3.5% NaCl). Such salinities can frequently occur after a natural disaster, due to stagnant water and subsequent evaporative water loss, demonstrating a need for a halophilic microorganism that can report on toxic contaminants. A bacterium that has these characteristics was previously isolated for electroactivity and further evaluated for use in the microbial analysis of toxic pollutants applicable for a widescale screening process in high saline contamination events.5 The bacterium, Salinivibrio EAGSL, was found to tolerate from 0.1 M to 3 M NaCl and showed anode-respiring capability. The bacterium was isolated as a new strain based on 16s rRNA gene sequencing.6 Therefore bioinformatics was employed for sequencing and assembling the genome to discover traits for NaCl, heavy metal, and general environmental stress factors. Interestingly, bioinformatics analysis contributed to unveiling the mechanism of extracellular electron transfer in this bacterium. The strategies for arsenic analysis included a high throughput 96-well plate cytotoxicity assay and an electrochemical assay for steps towards an online hazardous contaminant detection system. The 96-well plate assay provided a screening method for ~50 samples at once, taking around 6 hours to detect concentrations of 75 μM arsenic. To overcome the need for sampling and analysis, an electrochemical method was also developed for continuous monitoring of toxic contaminants in high saline. The net result is a microbial cytotoxicity assay to evaluate the toxicity of contaminants in a high saline environment. Additionally, this study elucidated the endogenous electron mediation mechanism in this bacterium through bioinformatics and electrochemical characterization, in addition to performance with an exogenous monomeric mediation system for increased current output. References: (1) Witze, A. Why Extreme Rains Are Gaining Strength as the Climate Warms. Nature 2018, 563 (7732), 458–460. https://doi.org/10.1038/d41586-018-07447-1. (2) Hashemi Goradel, N.; Mirzaei, H.; Sahebkar, A.; Poursadeghiyan, M.; Masoudifar, A.; Malekshahi, Z. V.; Negahdari, B. Biosensors for the Detection of Environmental and Urban Pollutions. J. Cell. Biochem. 2018, 119 (1), 207–212. https://doi.org/10.1002/jcb.26030. (3) Yogarajah, N.; Tsai, S. S. H. Detection of Trace Arsenic in Drinking Water: Challenges and Opportunities for Microfluidics. Environ. Sci. Water Res. Technol. 2015, 1 (4), 426–447. https://doi.org/10.1039/C5EW00099H. (4) Grattieri, M.; Minteer, S. D. Self-Powered Biosensors. ACS Sensors 2018, 3 (1), 44–53. https://doi.org/10.1021/acssensors.7b00818. (5) Grattieri, M.; Suvira, M.; Hasan, K.; Minteer, S. D. Halotolerant Extremophile Bacteria from the Great Salt Lake for Recycling Pollutants in Microbial Fuel Cells. J. Power Sources 2017, 356 (2017), 310–318. https://doi.org/10.1016/j.jpowsour.2016.11.090. (6) Alkotaini, B.; Tinucci, S. L.; Robertson, S. J.; Hasan, K.; Minteer, S. D.; Grattieri, M. Alginate-Encapsulated Bacteria for the Treatment of Hypersaline Solutions in Microbial Fuel Cells. ChemBioChem 2018, 19 (11), 1162–1169. https://doi.org/10.1002/cbic.201800142.

The capability of microorganisms to transfer electrons to/from an electrode surface is critical for the development of bioelectrochemical systems. However, the extracellular electron transfer (EET) mechanisms have been elucidated only for few microorganisms, in particular Geobacter sulfurreducens (Sh.) and Shewanella oneidensis (G.S.) respectively. A Microbial fuel cell (MFC) is a bioelectrochemical system that couples the possibility to directly convert chemical energy from civil and industrial wastewater into electrical energy thank to the bioelectrocatalytic activity of bacteria colonizing the electrodes. For on-field application of the technology, a mixed bacterial community will be most likely obtained, since wastewater solutions and biomasses are commonly utilized as inoculum. The presence of mixed-species biofilm can also facilitate the oxidation of complex substrates, since many microorganisms capable of EET can only use low-molecular organic acids and alcohols provided by fermenting bacteria. Accordingly, it is important to understand how microorganisms rather than Sh. and G.S. can contribute to the EET process. Different studies have reported a particularly high presence of Bacteroidetes in the microbial community at the anode and the cathode of MFCs. The biological characterizations have indicated an high percentages of Rikenella Microfusus (R.M.), which belong to the order of Bacteroidales, but only few information about its metabolism are available. R.M. was isolated by fecal materials, it is an obligated anaerobic, nonmotile and gram-negative bacterium, which is capable to oxidize a number of substrates as glucose, mannose, lactose producing acids. Herein, a pure culture of R.M. (ATCC® 29728™) was anaerobically grown and the EET was investigated on carbon cloth electrodes (non-wet proofed, E-TEK) potentiostatically polarized at a positive potential (+0.2 V vs. Ag|AgCl Sat.) and maintained in anaerobic conditions by constant purging of nitrogen gas. For the study, glucose was used to provide the substrate for the bacterium. Electrochemical experimental evidences revealed that flavins might be involved in the EET, facilitating the communication of the bacterium with the electrode surface. Moreover, oxygen influence on the current response was detected, critically affecting the capability of the bacterium to perform EET. The impact of the new finding on future researches devoted to clarify the EET for real application of bioelectrochemical systems will be discussed.

Solar energy is a highly exploitable energy source for renewable energy technologies; thus, a great deal of recent research has focused on the ability to generate electrical energy from solar energy (photoelectrochemistry). This has resulted in a wide variety of photoactive biomaterials being studied at electrode surfaces, such as isolated photosystems, thylakoid membranes as well intact photosynthetic organisms. Chloroplasts are photosynthetic organelles that harness solar energy in higher plants and algae. They are the vital sources of carbon-based fuels and have self-repair mechanisms against photo-damage; however, chloroplasts are less studied for harnessing solar energy. The electrochemical communication of chloroplasts at electrode surfaces is challenging since they are shielded inside thick electrochemically-insulating cell membranes. Therefore, the majority of studies surrounding chloroplasts in photobioelectrochemical systems (PBESs) utilize soluble exogenous electron transfer (ET) mediators. The use of soluble electron mediators, however, is not feasible for large-scale application. Redox polymers are polymeric backbones that have been modified with redox-active moieties and can be used as an immobilization matrix as well as a non-diffusive electron mediator for extracellular-ET from biomaterials to the electrode. Nevertheless, a successful electrochemical study on chloroplasts with any redox polymer is yet to be reported. Here, we report the photoelectrochemical wiring of chloroplasts on a naphthoquinone-based redox polymer. Intact chloroplasts were immobilized on Toray carbon paper electrodes and illuminated under a fiber optic light to simulate sunlight. Cyclic voltammetric and amperometric experiments demonstrate the ability to obtain photo-excited electrons that are produced from water oxidation by intact chloroplasts. In the presence of a diffusive mediator as well as the redox polymer, maximum photobiocurrents obtained from the chloroplasts are as high as 28±5 µA cm-2.

Due to the high cost of wastewater treatement, new and alternative low cost technologies need to be investigated. Bioelectrochemical systems and particularly microbial fuel cells (MFCs) seem to address positively this problem. In MFCs the organic waste is utilized as a fuel that is oxidised from microorganisms through their metabolism generating electricity. The main problem related with MFCs utilization is the small electricity production and high cost of the electrodes materials. These are the major reasons why MFCs are not commercialized in a large scale yet. In order to overcome the high cost, that is mainly related to the high price of the noble metals used as catalysts at the cathode, inexpensive catalysts for oxygen reduction reaction (ORR) should be explored. These catalysts belong to the group of non platinum based catalyst that are proved as very active towards ORR. This work focused on the utilization of a low cost non-PGM (Fe-Aminoantipyrine) catalyst explored in the design of oxygen reducing cathode for MFCs application. The activity of this catalyst was characterized electrochemically in a three-electrode configuration varying the pH of the electroyte. Then the cathodes were introduced in double chamber MFC (Figure 1) with the cathode completely immersed in the solution. The two compartments (125 ml) of the MFC were separated by proton-exchange membrane (Nafion 211). The anode composed of carbon brush (6x4 cm projected surface area) pre-colonized with mixed cultured bacteria. The anode chamber was filled with 50% in volume PBS (50 mM) and 50% in volume of activated sludge (pH=7.5±0.1). The non-PGM cathode (2.3 x 2.3 cm geometric area) was immersed in solution with different pHs (6, 7.5, 9 and 11) and purged with air for oxygen supply. The MFCs performance was investigated at different pHs in order to simulate possible industrial wastes with pHs different than neutral. The catalyst used in this work was synthesized by modified sacrificial support method which was developed at UNM1. In general, the metal precursor (iron nitrate) and nitrogen-containing low-molecular weight organic precursor (4-aminoantipiryne) are deposited on the surface of fumed silica (surface area ~120 m-2 g-1). the obtained composite material is heat treated in nitrogen atmosphere at T=950°C. After heat treatment fumed silica was removed by excess amount of HF. Potentiodynamic polarizations curve of the anode and the cathode separately were carried out using platinum mesh as a counter and a Ag/AgCl (3M KCl) as reference electrodes with scan rate 0.2 mV/s. MFC overall polarization curves were measured using a potentiostat with a scan rate of 1 mV/s. Power curves were determined using Ohm`s law (P= V x I). The electrochemical measurements of the cathode as a result of differences in the electrolyte pH showed highest electrocatalytic activity of the catalyst at lower pHs. This confirms our previous observation for the dependance of the Fe-AAPyr activity from the electrolyte pH2. The polarization and power curves (Figure 2) of the whole MFCs followed the same trend as the cathode polarization curves showing the dominating role of the cathode over the MFCs performance. The MFCs with low pH of the catholyte demonstrated the highest power (200 μW) and the highest open circuit voltage (OCV = 850 mV). This study demonstrated the applicability of non-PGM catalyst for the development of cathodes for MFC application. Further studies with improved MFCs design should be performed. Long-term operation test will be carried out investigating the influence of the wastewater pollutants on the cathode and subsequently the whole MFC operation and output. References A. Serov, U. Martinez, A. Falase, P. Atanassov, Electrochem. Comm. 22 (2012) 193-196. S. Brocato, A. Serov, P. Atanassov Electrochim. Acta, 87 (2013) 361-365

In this contribution, a meromictic lake situated in the north of Italy is descripted from a physico-chemical point of view. The characteristics of this lake makes the application of microbial fuelcells (MFC) technology a challenging task. Many are the particularities that produce a unique environment for microbial fuelcells: A) Conformation of the lake basin: the glacial origin of the lake, together with the steep walls of the basin and the depth of 120 m, creates the conditions for the evolution of a meromicticcondition (Fig 1). A first layer of water (about 50 m) is continuously recirculated and oxygenated; a second layer finds the depletion of oxygen; a third layer is constituted of stationary water. B) Water analyses: analysis of the water composition at various depths reveals stratifications of different metals, especially of manganese. C) Physico-chemical variations: based on home-built sensors, oxygen, pH, sulphide, conductivity, and temperature profiles were obtained, demonstrating the unique correlations between these parameters (Fig. 2). D) compositionof the rocks around the lake: carbonatic dolomite mineral, with small percentages of manganese, is susceptible to leaching and acidic dissolution. For the first time, the presence of a well-defined zone appearing as a “white cloud” was revealed near the interfacial region of the oxygen depletion. This cloud is rich in Mn. A mechanism is herein proposed for the white cloud formation by means of analysis of Mn lacustral concentrations as well as of redox and pH variations. This phenomenon is referred to the first chemical dissolution of the rocks by the slightly acidic monimolimion zone. Mn(II) solution diffuses upwards and meets the zone of oxygen depletion, where pH increases. Mn(OH)2 (white hydroxide) starts to floculate, and buildup of the Mn salts takes place. Moving downwards, the hydroxide meets again the acidic zone, thus dissolving. Moving upwards, the white hydroxide encounters oxygen and an higher redox potential. At these pH (about 8), transformation of Mn(OH)2 to Mn3O4is quick and produces a reddish-brown powder, easy to precipitate. From time to time, hydrodynamics and convective diffusion lead to mixing of the water of the differen layers, and the brown color is visible even on the surface of the lake. The presence of these layers, characterized by different chemistry, pH and redox potential (more than 500 mV of potential difference), might be exploited to achieve electrical current generation and Mn depollution of the waters. The role of microrganism in the cycling of manganese is now under investigation aiming to the experiemntation of bio-electrochemical systems. The electrolysis catalyzed by bacteria naturally present in the lake is able to produce large amounts of Mn2O3 from Mn(II) hydroxide. A lab-scale experiment demonstrates that this process is viable. As-produced Mn2O3precipitates on the electrode surface in a non-insulating porous layer, with the final result of collecting Mn and clear the water from the white cloud effect. In this scenario, microbial fuel cells (MFC) might produce power by utilizing the microbial capacity of catalyzing the oxygen reduction at the cathode, and the Mn(OH)2 oxidation at the anode. Another type of MFC might develop power by reduction of Mn2O3. Furthermore, microbial electrolysis cells (MEC) could produce valuable products. In our opinion, the white cloud layer is a product of long-term building up of the Mn(II) concentration, and the process of water clarification may be achieved by a non-intrusive, long-term, application of the bacterial catalyzed electrochemical systems described. Fig. 1: Scheme of a typical meromictic lake, with the three main characteristic stratifications: mixolimion, chemocline, and monimolimion and Depth-profiles of the Idro lake, by sensor probes: A) redox potential; B) sulphide pontentiometric signal; C) temperature; D) O2 content.