How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

Invited for this month's cover is the collaborative work among Univ. of Milano-Bicocca, Ricerca sul Sistema Energetico S.p.A., Univ. degli Studi di Milano, Univ. of California Irvine, Univ. of New Mexico, CNRS Toulouse. Technische Univ. Braunschweig, Aquacycl LLC, J. Craig Venter Institute, Helmholtz-Centre for Environmental Research. The image shows a sketch of a microbial fuel cell and a target indicating the need of developing common standards for the field of microbial electrochemical technologies. The Full Paper itself is available at 10.1002/cssc.202100294.

How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

A cross-laboratory study on microbial fuel cells (MFC) which involved different institutions around the world is presented. The study aims to assess the development of autochthone microbial pools enriched from domestic wastewater, cultivated in identical single-chamber MFCs, operated in the same way, thereby approaching the idea of developing common standards for MFCs. The MFCs are inoculated with domestic wastewater in different geographic locations. The acclimation stage and, consequently, the startup time are longer or shorter depending on the inoculum, but all MFCs reach similar maximum power outputs (55±22 µW cm-2 ) and COD removal efficiencies (87±9 %), despite the diversity of the bacterial communities. It is inferred that the MFC performance starts when the syntrophic interaction of fermentative and electrogenic bacteria stabilizes under anaerobic conditions at the anode. The generated power is mostly limited by electrolytic conductivity, electrode overpotentials, and an unbalanced external resistance. The enriched microbial consortia, although composed of different bacterial groups, share similar functions both on the anode and the cathode of the different MFCs, resulting in similar electrochemical output.

Response-Surface-Optimized and Scaled-Up Microbial Electrosynthesis of Chiral Alcohols.

A variety of enzymes can be easily incorporated and overexpressed within Escherichia coli cells by plasmids, making it an ideal chassis for bioelectrosynthesis. It has recently been demonstrated that microbial electrosynthesis (MES) of chiral alcohols is possible by using genetically modified E. coli with plasmid-incorporated and overexpressed enzymes and methyl viologen as mediator for electron transfer. This model system, using NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis to convert acetophenone into (R)-1-phenylethanol, is assessed by using a design of experiment (DoE) approach. Process optimization is achieved with a 2.4-fold increased yield of 94±7 %, a 3.9-fold increased reaction rate of 324±67 µm h-1 , and a coulombic efficiency of up to 68±7 %, while maintaining an excellent enantioselectivity of >99 %. Subsequent scale-up to 1 L by using electrobioreactors under batch and fed-batch conditions increases the titer of (R)-1-phenylethanol to 12.8±2.0 mm and paves the way to further develop E. coli into a universal chassis for MES in a standard biotechnological process environment.

Monitoring stratification of anode biofilms in bioelectrochemical laminar flow reactors using flow cytometry.

A laminar flow bioelectrochemical systems (BES) was designed and benchmarked using microbial anodes dominated with Geobacter spp. The reactor architecture was based on modeled flow fields, the resulting structure was 3D printed and used for BES manufacturing. Stratification of the substrate availability within the reactor channels led to heterogeneous biomass distribution, with the maximum biomass found mainly in the initial/middle channels. The anode performance was assessed for different hydraulic retention times while coulombic efficiencies of up to 100% (including also hydrogen recycling from the cathode) and current densities of up to 75 µA cm-2 at an anode surface to volume ratio of 1770 cm2 L-1 after 35 days were achieved. This low current density can be clearly attributed to the heterogeneous distributions of biomass and the stratification of the microbial community structure. Further, it was shown that time and space resolved analysis of the reactor microbiomes per channel is feasible using flow cytometry.

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis.

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d1. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d1-binding ß-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.

Resting Escherichia coli as Chassis for Microbial Electrosynthesis: Production of Chiral Alcohols.

Chiral alcohols constitute important building blocks that can be produced enantioselectively by using nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidoreductases. For NAD(P)H regeneration, electricity delivers the cheapest reduction equivalents. Enzymatic electrosynthesis suffers from cofactor and enzyme instability, whereas microbial electrosynthesis (MES) exploits whole cells. Here, we demonstrate MES by using resting Escherichia coli as biocatalytic chassis for a production platform towards fine chemicals through electric power. This chassis was exemplified for the synthesis of chiral alcohols by using a NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis for synthesis of (R)-1-phenylethanol from acetophenone. The E. coli strain and growth conditions affected the performance. Maximum yields of (39.4±5.7) % at a coulombic efficiency of (50.5±6.0) % with enantiomeric excess >99 % was demonstrated at a rate of (83.5±13.9) µm h-1 , confirming the potential of MES for synthesis of high-value compounds.

Reactors for Microbial Electrobiotechnology.

From the first electromicrobial experiment to a sophisticated microbial electrochemical process - it all takes place in a reactor. Whereas the reactor design and materials used strongly influence the obtained results, there are no common platforms for MES reactors. This is a critical convention gap, as cross-comparison and benchmarking among MES as well as MES vs. conventional biotechnological processes is needed. Only knowledge driven engineering of MES reactors will pave the way to application and commercialization. In this chapter we first assess the requirements on reactors to be used for bioelectrochemical systems as well as potential losses caused by the reactor design. Subsequently, we compile the main types and designs of reactors used for MES so far, starting from simple H-cells to stirred tank reactors. We conclude with a discussion on the weaknesses and strengths of the existing types of reactors for bioelectrochemical systems that are scored on design criteria and draw conclusions for the future engineering of MES reactors.

Paving the way for bioelectrotechnology: Integrating electrochemistry into bioreactors.

The reactor systems used for microbial electrosynthesis, i.e. bioelectrochemical systems for achieving bioproduction so far reported in literature are relatively small in scale and highly diverse in their architecture and modes of operation. The often diverging requirements of the electrochemical and the biological processes and the interdisciplinarity of the field make the engineering of these systems a special challenge. This has led to multiple, differently optimized approaches of reactor vessels, designs and operating conditions making standardization and normalization or even a systematic engineering almost impossible. Overcoming this lack of standardization, scalability and knowledge-driven engineering is the driving force for this work introducing an upgrade kit for bioreactors transforming these reversibly to bioelectroreactors. The prototypes of the bioreactor upgrade kit were integrated with commercial bioreactor (fermentor) systems and performances compared to a classic, small-scale bioelectrochemical glass cell system. The use of the upgrade kit allowed interfacing with the existing infrastructure of the conventional bioreactors for growing electroactive microorganisms in pure culture conditions, with the added electrochemical control and further process monitoring. The results of growing Shewanella oneidensis MR-1 clearly show that these systems can be used to control, monitor, and scale microbial bioelectrochemical processes, providing better resolution of the data for the tested experimental conditions.

A framework for modeling electroactive microbial biofilms performing direct electron transfer.

A modeling platform for microbial electrodes based on electroactive microbial biofilms performing direct electron transfer (DET) is presented. Microbial catabolism and anabolism were coupled with intracellular and extracellular electron transfer, leading to biofilm growth and current generation. The model includes homogeneous electron transfer from cells to a conductive biofilm component, biofilm matrix conduction, and heterogeneous electron transfer to the electrode. Model results for Geobacter based anodes, both at constant electrode potential and in voltammetric (dynamic electrode potential) conditions, were compared to experimental data from different sources. The model can satisfactorily describe microscale (concentration, pH and redox gradients) and macroscale (electric currents, biofilm thickness) properties of Geobacter biofilms. The concentration of electrochemically accessible redox centers, here denominated as cytochromes, involved in the extracellular electron transfer, plays the key role and may differ between constant potential (300 mM) and dynamic potential (3mM) conditions. Model results also indicate that the homogeneous and heterogeneous electron transfer rates have to be within the same order of magnitude (1.2 s(-1)) for reversible extracellular electron transfer.

Electrifying white biotechnology: engineering and economic potential of electricity-driven bio-production.

Invited for the cover of this issue are the groups of Falk Harnisch at the Helmholtz Centre for Environmental Research (Germany) and his collaboration partners at The University of Queensland (Australia). The image depicts their vision of the world, if "electrification" of white biotechnology comes true. The Concept itself is available at 10.1002/cssc.201402736.

Electrifying white biotechnology: engineering and economic potential of electricity-driven bio-production.

The production of fuels and chemicals by electricity-driven bio-production (i.e., using electric energy to drive biosynthesis) holds great promises. However, this electrification of white biotechnology is particularly challenging to achieve because of the different optimal operating conditions of electrochemical and biochemical reactions. In this article, we address the technical parameters and obstacles to be taken into account when engineering microbial bioelectrochemical systems (BES) for bio-production. In addition, BES-based bio-production processes reported in the literature are compared against industrial needs showing that a still large gap has to be closed. Finally, the feasibility of BES bio-production is analysed based on bulk electricity prices. Using the example of lysine production from sucrose, we demonstrate that there is a realistic market potential as cost savings of 8.4 % (in EU) and 18.0 % (in US) could be anticipated, if the necessary yields can be obtained.

Acoustic detection of cell adhesion on a quartz crystal microbalance.

An acoustic quartz crystal microbalance (QCM) was used to signal and follow the cell­adhesion process of epithelial cells [human embryonic kidney(HEK) 293T and cervical cancer (HeLa) and fibroblasts [African Green Monkey kidney cells (COS-7)] onto gold surfaces. Cells were applied on the sensor and grown under serum-free and serum-supplemented culture media. The sensor resonance frequency (Δf) and motional resistance (ΔR) variations were measured during cell growth to monitor cell adhesion processes. Fingerprints of the adhesion processes, generated using the QCM signal, were found to be specific for each cell type while enabling the identification of the phases of the adhesion process. Under serum-free conditions, the deposition of HEK 293T and HeLa cells was characterized by a decrease of Δf with constant ΔR, whereas for COS­7 cells, this initial deposition was signaled by variations of ΔR at constant Δf. Toward the end of the adhesion process, fingerprints were characterized by a continuous increase of ΔR consistent with the increase in viscoelasticity. The morphology of adherent cells was visualized by fluorescent microscopy, enabling the association of the cell morphology with QCM signals.