Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.

Humic substances as electron acceptor for anaerobic oxidation of methane (AOM) and electron shuttle in Mn (IV)-dependent AOM.

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. 'Methanoperedens manganicus', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. 'M. manganicus' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. 'M. manganicus', and the total archaea population in the microbial community suggested that Ca. 'M. manganicus' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. 'M. manganicus'. We tested this hypothesis by adding humic acids to a Ca. 'M. manganicus' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

Pyrogenic Carbon Promotes Anaerobic Oxidation of Methane Coupled with Iron Reduction via the Redox-Cycling Mechanism.

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

Multi-heme cytochrome-mediated extracellular electron transfer by the anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens'.

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.

Characterisation of acetogen formatotrophic potential using Eubacterium limosum.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280 ± 56 mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO2 (150 ± 11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. KEY POINTS: First Eubacterium limosum steady-state formatotrophic growth omics dataset High formate specific uptake rate, however carbon dioxide was the major product Formate may be the cause of intracellular stress and biofilm formation.

Polyhydroxyalkanoate-driven current generation via acetate by an anaerobic methanotrophic consortium.

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH4) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by 'Candidatus Methanoperedens nitroreducens' in a CH4-fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH4 directly. In the absence of CH4, current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of 'Ca. M. nitroreducens' to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems.

Electro-fermentation: Sustainable bioproductions steered by electricity.

The market of biobased products obtainable via fermentation processes has steadily increased over the past few years, driven by the need to create a decarbonized economy. To date, industrial fermentation (IF) employs either pure or mixed microbial cultures (MMC), whereby the type of the microbial catalysts and the used feedstock affect metabolic pathways and, in turn, the type of product(s) generated. In many cases, especially when dealing with MMC, the economic viability of IF is still hindered by factors such as the low attained product titer and selectivity, which ultimately challenge the downstream recovery and purification steps. In this context, electro-fermentation (EF) represents an innovative approach, based on the use of a polarized electrode interface to trigger changes in the rate, yield, titer or product distribution deriving from traditional fermentation processes. In principle, the electrode in EF can act as an electron acceptor (i.e., anodic electro-fermentation, AEF) or donor (i.e., cathodic electro-fermentation, CEF), or simply as a means to control the oxidation-reduction potential of the fermentation broth. However, the molecular and biochemical basis underlying EF are still largely unknown. This review provides a comprehensive overview of recent literature studies including both AEF and CEF examples using pure or mixed microbial cultures. A critical analysis of biochemical, microbiological, and engineering aspects which presently hamper the transition of the EF technology from the laboratory to the market is also presented.

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

Unlike gaseous C1 feedstocks for acetogenic bacteria, there has been less attention on liquid C1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW-1 day-1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h-1 ) and cell density (maximum 1.2 ± 0.043 gDCW l-1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases - growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

Strategies to improve viability of a circular carbon bioeconomy-A techno-economic review of microbial electrosynthesis and gas fermentation.

A circular carbon bioeconomy has potential to halt atmospheric accumulation of greenhouse gases causing climate change and sustainably produce chemical, agricultural and fuel products. Here, we report application of a simplified technoeconomic assessment to critically review two approaches in this space - microbial electrosynthesis and gas fermentation. For microbial electrosynthesis, decoupling of surface-dependant abiotic process for electron delivery from volume-dependant biotic carbon fixation, is shown as the only economically viable strategy to scale-up due to comparatively low biofilm electron consumption rate. This is effectively an electrolyser-assisted gas fermentation system. Targeting high-value products, such as protein for human food consumption is one of the few pathways forward for electrolyser-assisted gas fermentation. Alternatively, gas fermentation of reformed biogas presents an interesting and potentially more sustainable implementation pathway to improve economic viability of chemicals. This critical review suggests linking water treatment resource recovery with gas fermentation is attractive for bioplastics and butanol in terms of competitiveness and market demand.

Selective Extraction of Medium-Chain Carboxylic Acids by Electrodialysis and Phase Separation.

Carboxylic acids obtained via the microbial electrochemical conversion of waste gases containing carbon dioxide (i.e., microbial electrosynthesis) can be used in lieu of nonrenewable building-block chemicals in the manufacture of a variety of products. When targeting valuable medium-chain carboxylic acids such as caproic acid, electricity-driven fermentations can be limited by the accumulation of fermentation products in the culturing media, often resulting in low volumetric productivities and titers due to direct toxicity or inhibition of the biocatalyst. In this study, we tested the effectiveness of a simple electrodialysis system in upconcentrating carboxylic acids from a model solution mimicking the effluent of a microbial electrochemical system producing short- and medium-chain carboxylic acids. Under batch extraction conditions, the electrodialysis scheme enabled the recovery of 60% (mol mol-1) of the total carboxylic acids present in the model fermentation broth. The particular arrangement of conventional monopolar ion exchange membranes and hydraulic recirculation loops allowed the progressive acidification of the extraction solution, enabling phase separation of caproic acid as an immiscible oil with 76% purity.

Substrate availability drives mixed culture fermentation of glucose to lactate at steady state.

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions.

Enhancing methane oxidation in a bioelectrochemical membrane reactor using a soluble electron mediator.

BACKGROUND: Bioelectrochemical methane oxidation catalysed by anaerobic methanotrophic archaea (ANME) is constrained by limited methane bioavailability as well as by slow kinetics of extracellular electron transfer (EET) of ANME. In this study, we tested a combination of two strategies to improve the performance of methane-driven bioelectrochemical systems that includes (1) the use of hollow fibre membranes (HFMs) for efficient methane delivery to the ANME organisms and (2) the amendment of ferricyanide, an effective soluble redox mediator, to the liquid medium to enable electrochemical bridging between the ANME organisms and the anode, as well as to promote EET kinetics of ANME. RESULTS: The combined use of HFMs and the soluble mediator increased the performance of ANME-based bioelectrochemical methane oxidation, enabling the delivery of up to 196 mA m-2, thereby outperforming the control system by 244 times when HFMs were pressurized at 1.6 bar. CONCLUSIONS: Improving methane delivery and EET are critical to enhance the performance of bioelectrochemical methane oxidation. This work demonstrates that by process engineering optimization, energy recovery from methane through its direct oxidation at relevant rates is feasible.

Microbial electrochemical sensors for volatile fatty acid measurement in high strength wastewaters: A review.

In this review, the use of MESe are evaluated in the monitoring of volatile fatty acids (VFAs) during the anaerobic digestion of high strength wastewater, with a focus on slaughterhouse wastewater. VFAs are identified as a key intermediary in anaerobic digestion, hence their accumulation could be used to infer possible process instability of anaerobic digesters. Current sample measurement for VFAs through off-line laboratory analysis can be costly, time consuming, and require specialist skills. Consequently, microbial electrochemical sensors (MESe) are currently being investigated as a low-cost alternative method for in-line VFA measurement. In this paper, the fundamental operation of MESe is summarised, including the exploration of several factors that would impact the operation of MESe in real wastewater applications. It is found that, in the context of wastewater sensing, MESe technology has been unable to bridge the gap between the laboratory and real-world anaerobic digesters effectively. Important issues surrounding biofouling, sensitivity, and detection range are explored and prioritised in this review, and an overview of potential research pathways is provided. These include the potential to further explore alternate electrode materials, use of ion exchange membranes, and development of other sensor components, as further described in the review.

Microbial electrosynthesis system with dual biocathode arrangement for simultaneous acetogenesis, solventogenesis and carbon chain elongation.

A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2-C4 carboxylic acids and alcohols from carbon dioxide. Each cathode chamber provides ideal pH conditions to favor acetogenesis/carbon chain elongation (pH = 6.9), and solventogenesis (pH = 4.9), respectively, without the requirement of external acid/base dosing.

Bioelectrochemical Denitrification for the Treatment of Saltwater Recirculating Aquaculture Streams.

Maintaining low concentrations of nitrogen compounds (ammonium, nitrate and nitrite) in recirculating aquaculture waters is extremely important for a larger and healthier fish production, as well as for water discharge purposes. Although ammonium removal from aquaculture streams is usually done within a nitrifying step, nitrate removal via denitrification is still partially limited by the low organic matter availability. Therefore, an easy-to-operate autotrophic denitrifying bioelectrochemical system is herein proposed for the treatment of seawater aquaculture streams. The nitrate-containing synthetic stream flows sequentially through a biological denitrifying cathode (placed at the lower portion of a tubular reactor) and an abiotic anode (generating electrons and oxygen from water splitting, at the upper portion). Experimental results with synthetic seawater showed that the system reached denitrification rates of 0.13 ± 0.01 kg N m-3 day-1, operating with minimum ammonium and nitrite accumulation, as well as minimum chlorine formation in the abiotic anode, despite the high chloride concentration. There results support the technical potential for simultaneous bioelectrochemical denitrification and partial re-oxygenation of aquaculture waters either for recirculation or discharge purposes.

Anodic electro-fermentation: Anaerobic production of L-Lysine by recombinant Corynebacterium glutamicum.

Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidizing conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/hr. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electro-fermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine.

Microbial nanowires - Electron transport and the role of synthetic analogues.

Electron transfer is central to cellular life, from photosynthesis to respiration. In the case of anaerobic respiration, some microbes have extracellular appendages that can be utilised to transport electrons over great distances. Two model organisms heavily studied in this arena are Shewanella oneidensis and Geobacter sulfurreducens. There is some debate over how, in particular, the Geobacter sulfurreducens nanowires (formed from pilin nanofilaments) are capable of achieving the impressive feats of natural conductivity that they display. In this article, we outline the mechanisms of electron transfer through delocalised electron transport, quantum tunnelling, and hopping as they pertain to biomaterials. These are described along with existing examples of the different types of conductivity observed in natural systems such as DNA and proteins in order to provide context for understanding the complexities involved in studying the electron transport properties of these unique nanowires. We then introduce some synthetic analogues, made using peptides, which may assist in resolving this debate. Microbial nanowires and the synthetic analogues thereof are of particular interest, not just for biogeochemistry, but also for the exciting potential bioelectronic and clinical applications as covered in the final section of the review. STATEMENT OF SIGNIFICANCE: Some microbes have extracellular appendages that transport electrons over vast distances in order to respire, such as the dissimilatory metal-reducing bacteria Geobacter sulfurreducens. There is significant debate over how G. sulfurreducens nanowires are capable of achieving the impressive feats of natural conductivity that they display: This mechanism is a fundamental scientific challenge, with important environmental and technological implications. Through outlining the techniques and outcomes of investigations into the mechanisms of such protein-based nanofibrils, we provide a platform for the general study of the electronic properties of biomaterials. The implications are broad-reaching, with fundamental investigations into electron transfer processes in natural and biomimetic materials underway. From these studies, applications in the medical, energy, and IT industries can be developed utilising bioelectronics.

Effect of the anode potential on the physiology and proteome of Shewanella oneidensis MR-1.

Shewanella species respire using iron and manganese oxides as well as electrodes as solid terminal electron acceptors. Shewanella oneidenis MR-1 exploits mediated as well as direct extracellular electron transfer (EET) modes to transfer electrons at different formal potentials. These different EET modes at different potentials may utilise alternate electron transfer pathways. Therefore, we investigated how different anode potentials, providing different maximum microbial energy gains impacted S. oneidensis microbial physiology. Using quantitative proteomics, comparative analysis of the cellular variations to different anode potentials was performed. A label-free proteomic mass spectrometric analysis method, SWATH-MS, was used to gather quantitative information to determine physiological changes of Shewanella oneidensis MR-1 grown at different anodic potentials. S. oneidensis was cultured and grown in electrochemical cells at the set anode potentials of +0.71V, +0.21V & -0.19V versus SHE reference electrode, while the current production was monitored. At maximum current, electrodes were removed and whole-cell proteins extracted. Subsequent SWATH-MS analysis revealed information on 740 identified proteins across the three electrode potentials. For the first time, we show the abundance of S. oneidensis electron transfer proteins differs with electrode potential.

Erratum to: Anoxic metabolism and biochemical production in Pseudomonas putida F1 driven by a bioelectrochemical system.

[This corrects the article DOI: 10.1186/s13068-016-0452-y.].

Anode potential influences the structure and function of anodic electrode and electrolyte-associated microbiomes.

Three bioelectrochemical systems were operated with set anode potentials of +300 mV, +550 mV and +800 mV vs. Standard Hydrogen Electrode (SHE) to test the hypothesis that anode potential influences microbial diversity and is positively associated with microbial biomass and activity. Bacterial and archaeal diversity was characterized using 16 S rRNA gene amplicon sequencing, and biofilm thickness was measured as a proxy for biomass. Current production and substrate utilization patterns were used as measures of microbial activity and the mid-point potentials of putative terminal oxidases were assessed using cyclic voltammetry. All measurements were performed after 4, 16, 23, 30 and 38 days. Microbial biomass and activity differed significantly between anode potentials and were lower at the highest potential. Anodic electrode and electrolyte associated community composition was also significantly influenced by anode potential. While biofilms at +800 mV were thinner, transferred less charge and oxidized less substrate than those at lower potentials, they were also associated with putative terminal oxidases with higher mid-point potentials and generated more biomass per unit charge. This indicates that microbes at +800 mV were unable to capitalize on the potential for additional energy gain due to a lack of adaptive traits to high potential solid electron acceptors and/or sensitivity to oxidative stress.