A New Approach for Evaluating Electron Transfer Dynamics by Using In Situ Resonance Raman Microscopy and Chronoamperometry in Conjunction with a Dynamic Model.

Geobacter sulfurreducens is a good candidate as a chassis organism due to its ability to form thick, conductive biofilms, enabling long-distance extracellular electron transfer (EET). Due to the complexity of EET pathways in G. sulfurreducens, a dynamic approach is required to study genetically modified EET rates in the biofilm. By coupling online resonance Raman microscopy with chronoamperometry, we were able to observe the dynamic discharge response in the biofilm's cytochromes to an increase in anode voltage. Measuring the heme redox state alongside the current allows for the fitting of a dynamic model using the current response and a subsequent validation of the model via the value of a reduced cytochrome c Raman peak. The modeled reduced cytochromes closely fitted the Raman response data from the G. sulfurreducens wild-type strain, showing the oxidation of heme groups in cytochromes until a new steady state was achieved. Furthermore, the use of a dynamic model also allows for the calculation of internal rates, such as acetate and NADH consumption rates. The Raman response of a mutant lacking OmcS showed a higher initial oxidation rate than predicted, followed by an almost linear decrease of the reduced mediators. The increased initial rate could be attributed to an increase in biofilm conductivity, previously observed in biofilms lacking OmcS. One explanation for this is that OmcS acts as a conduit between cytochromes; therefore, deleting the gene restricts the rate of electron transfer to the extracellular matrix. This could, however, be modeled assuming a linear oxidation rate of intercellular mediators.IMPORTANCE Bioelectrochemical systems can fill a vast array of application niches, due to the control of redox reactions that it offers. Although native microorganisms are preferred for applications such as bioremediation, more control is required for applications such as biosensors or biocomputing. The development of a chassis organism, in which the EET is well defined and readily controllable, is therefore essential. The combined approach in this work offers a unique way of monitoring and describing the reaction kinetics of a G. sulfurreducens biofilm, as well as offering a dynamic model that can be used in conjunction with applications such as biosensors.

On-Line Raman Spectroscopic Study of Cytochromes' Redox State of Biofilms in Microbial Fuel Cells.

Bio-electrochemical systems such as microbial fuel cells and microbial electrosynthesis cells depend on efficient electron transfer between the microorganisms and the electrodes. Understanding the mechanisms and dynamics of the electron transfer is important in order to design more efficient reactors, as well as modifying microorganisms for enhanced electricity production. Geobacter are well known for their ability to form thick biofilms and transfer electrons to the surfaces of electrodes. Currently, there are not many "on-line" systems for monitoring the activity of the biofilm and the electron transfer process without harming the biofilm. Raman microscopy was shown to be capable of providing biochemical information, i.e., the redox state of C-type cytochromes, which is integral to external electron transfer, without harming the biofilm. In the current study, a custom 3D printed flow-through cuvette was used in order to analyze the oxidation state of the C-type cytochromes of suspended cultures of three Geobacter sulfurreducens strains (PCA, KN400 and ΔpilA). It was found that the oxidation state is a good indicator of the metabolic state of the cells. Furthermore, an anaerobic fluidic system enabling in situ Raman measurements was designed and applied successfully to monitor and characterize G. sulfurreducens biofilms during electricity generation, for both a wild strain, PCA, and a mutant, ΔS. The cytochrome redox state, monitored by the Raman peak areas, could be modulated by applying different poise voltages to the electrodes. This also correlated with the modulation of current transferred from the cytochromes to the electrode. The Raman peak area changed in a predictable and reversible manner, indicating that the system could be used for analyzing the oxidation state of the proteins responsible for the electron transfer process and the kinetics thereof in-situ.

Dual cathode configuration and headspace gas recirculation for enhancing microbial electrosynthesis using Sporomusa ovata.

High-rate production of acetate and other value-added products from the reduction of CO2 in microbial electrosynthesis (MES) using acetogens can be achieved with high reducing power where H2 appears as a key electron mediator. H2 evolution using metal cathodes can enhance the availability of H2 to support high-rate microbial reduction of CO2. Due to the low solubility of H2, the availability of H2 remains limited to the bacteria. In this study, we investigated the performances of Sporomusa ovata for CO2 reduction when dual cathodes were used together in an MES, one was regular carbon cathode, and the other was a titanium mesh that allows higher hydrogen evolution. The dual cathode configuration was investigated in two sets of MES, one set had the usual S. ovata inoculated graphite rod, and another set had a synthetic biofilm-imprinted carbon cloth. Additionally, the headspace gas in MES was recirculated to increase the H2 availability to the bacteria in suspension. High-rate CO2 reduction was observed at -0.9 V vs Ag/AgCl with dual cathode configuration as compared to single cathodes. High titers of acetate (up to ∼11 g/L) with maximum instantaneous rates of 0.68-0.7 g/L/d at -0.9 V vs Ag/AgCl were observed, which are higher than the production rates reported in the literatures for S. ovata using MES with surface modified cathodes. A high H2 availability supported the high-rate acetate production from CO2 with diminished electricity input.

Advances in cathode designs and reactor configurations of microbial electrosynthesis systems to facilitate gas electro-fermentation.

In gas fermentation, a range of chemolithoautotrophs fix single-carbon (C1) gases (CO2 and CO) when H2 or other reductants are available. Microbial electrosynthesis (MES) enables CO2 reduction by generating H2 or reducing equivalents with the sole input of renewable electricity. A combined approach as gas electro-fermentation is attractive for the sustainable production of biofuels and biochemicals utilizing C1 gases. Various platform compounds such as acetate, butyrate, caproate, ethanol, butanol and bioplastics can be produced. However, technological challenges pertaining to the microbe-material interactions such as poor gas-liquid mass transfer, low biomass and biofilm coverage on cathode, low productivities still exist. We are presenting a review on latest developments in MES focusing on the configuration and design of cathodes that can address the challenges and support the gas electro-fermentation. Overall, the opportunities for advancing CO and CO2-based biochemicals and biofuels production in MES with suitable cathode/reactor design are prospected.

Design and implementation of a low cost bio-printer modification, allowing for switching between plastic and gel extrusion.

Due to the high cost of bioprinters they are not feasible for proof of concept experiments or educational purposes. Furthermore, the more affordable DIY methods all disable the plastic printing capability of the original printer. Here we present an affordable bio-printing modification that is easy to install and maintains the original capabilities of the printer. The modification used mostly 3D printed parts and is based on the popular, open-source Prusa i3 3D printer. The modifications are kept as simple as possible and uses standard slicing software, allowing for installation by less experienced builders. By using disposable syringes and easily sterilizable parts, an aseptic bioprinting setup can be achieved, depending on the environment. It also allows for 2 component printing as well as UV curing. The bio-printing and curing capabilities were shown by printing and curing an artificial biofilm of an electro-active bacteria, Geobacter sulfurreducens, onto a carbon-cloth electrode which was used in a microbial fuel cell.

Numerical modeling and verification of a sonobioreactor and its application on two model microorganisms.

Ultrasound has many uses, such as in medical imaging, monitoring of crystallization, characterization of emulsions and suspensions, and disruption of cell membranes in the food industry. It can also affect microbial cells by promoting or slowing their growth and increasing the production of some metabolites. However, the exact mechanism explaining the effect of ultrasound has not been identified yet. Most equipment employed to study the effect of ultrasound on microorganisms has been designed for other applications and then only slightly modified. This results in limited control over ultrasound frequency and input power, or pressure distribution in the reactor. The present study aimed to obtain a well-defined reactor by simulating the pressure distribution of a sonobioreactor. Specifically, we optimized a sonotrode to match the bottle frequency and compared it to measured results to verify the accuracy of the simulation. The measured pressure distribution spectrum presented the same overall trend as the simulated spectrum. However, the peaks were much less intense, likely due to non-linear events such as the collapse of cavitation bubbles. To test the application of the sonobioreactor in biological systems, two biotechnologically interesting microorganisms were assessed: an electroactive bacterium, Geobacter sulfurreducens, and a lignocellulose-degrading fungus, Fusarium oxysporum. Sonication resulted in increased malate production by G. sulfurreducens, but no major effect on growth. In comparison, morphology and growth of F. oxysporum were more sensitive to ultrasound intensity. Despite considerable morphological changes at 4 W input power, the growth rate was not adversely affected; however, at 12 W, growth was nearly halted. The above findings indicate that the novel sonobioreactor provides an effective tool for studying the impact of ultrasound on microorganisms.