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Comparing theoretical and practical biomass yields calls for revisiting thermodynamic growth models for electroactive microorganisms
Abstract
Research on electroactive microorganisms (EAM) often focuses either on their physiology and the underlying mechanisms of extracellular electron transfer or on their application in microbial electrochemical technologies (MET). Thermodynamic understanding of energy conversions related to growth and activity of EAM has received only a little attention. In this study, we aimed to prove the hypothesized restricted energy harvest of EAM by determining biomass yields by monitoring growth of acetate-fed biofilms presumably enriched in Geobacter, using optical coherence tomography, at three anode potentials and four acetate concentrations. Experiments were concurrently simulated using a refined thermodynamic model for EAM. Neither clear correlations were observed between biomass yield and anode potential nor acetate concentration, albeit the statistical significances are limited, mainly due to the observed experimental variances. The experimental biomass yield based on acetate consumption (YX/ac = 37 ± 9 mgCODbiomass gCODac-1) was higher than estimated by modeling, indicating limitations of existing growth models to predict yields of EAM. In contrast, the modeled biomass yield based on catabolic energy harvest was higher than the biomass yield from experimental data (YX/cat = 25.9 ± 6.8 mgCODbiomass kJ-1), supporting restricted energy harvest of EAM and indicating a role of not considered energy sinks. This calls for an adjusted growth model for EAM, including, e.g., the microbial electrochemical Peltier heat to improve the understanding and modeling of their energy metabolism. Furthermore, the reported biomass yields are important parameters to design strategies for influencing the interactions between EAM and other microorganisms and allowing more realistic feasibility assessments of MET.
... The energy yield values exceeding 100% presented in these tables reflect the intricate nature of bio-electrochemical processes. These processes often exhibit behavior that deviates from both theoretical expectations and mathematical models [34]. The complexity of these factors contributes to unexpected outcomes, resulting in energy recovery values that surpass conventional limits. ...
Bio-electrochemical systems have increasingly become the focus of research due to their potential in environmental biotechnology, particularly in the domains of waste utilization and energy recovery. A prominent method within this domain is the transformation of organic matter into hydrogen via microbial electrolysis cells (MECs). This study offers a thorough analysis of MEC performance, employing exergy analysis and incorporating relevant data from the existing literature. The findings of this research indicate a relationship between process efficiency and effective electron transfer originating from biological oxidation to the cathode reaction, facilitating hydrogen generation. The assessment performed revealed that the exergy efficiency of the process varies by a wide range, depending on conditions such as substrate type and concentration, applied external voltage, and the presence of specific inhibitors. This interplay between substrate concentration, overall efficiency, and energy requirement underlines the complex dynamics of optimizing MEC performance. Our insights provide understanding of the challenges in bio-electrochemical systems, offering implications for their sustainable and efficient use in environmental biotechnology. The theoretical analysis involved assessing the utilization of glucose and glycerol, along with the evaluation of electrical energy consumption and hydrogen yield. Our results demonstrate that a higher applied voltage is associated with greater exergy efficiency. Furthermore, after comparing the use of glucose and glycerol as substrates, our study supports the preferential application of glucose for enhanced efficiency.
Geobacter sulfurreducens is a ubiquitous iron-reducing bacterium in soils, and in engineered systems, it can respire an electrode to produce measurable electric current. Its unique metabolism, heavily dependent on an extensive network of cytochromes, requires a unique cell composition. In this work, we used metallomics, cell fraction and elemental analyses, and transcriptomics to study and analyze the cell composition of G. sulfurreducens. Elemental composition studies (C, H, O, N, and ash content) showed high C:O and H:O ratios of approximately 1.7:1 and 0.25:1, indicative of more reduced cell composition that is consistent with high lipid content. Our study shows that G. sulfurreducens cells have a large amount of iron (2 ± 0.2 μg/g dry weight) and lipids (32 ± 0.5% dry weight/dry weight) and that this composition does not change whether the cells are grown with a soluble or an insoluble electron acceptor. The high iron concentration, higher than similar microorganisms, is attributed to the production of cytochromes that are abundant in transcriptomic analyses in both solid and soluble electron acceptor growth. The unique cell composition of G. sulfurreducens must be considered when growing this microorganism for lab studies and commercial applications. IMPORTANCE Geobacter sulfurreducens is an electroactive microorganism. In nature, it grows on metallic minerals by transferring electrons to them, effectively "breathing" metals. In a manmade system, it respires an electrode to produce an electric current. It has become a model organism for the study of electroactive organisms. There are potential biotechnological applications of an organism that can bridge the gap between biology and electrical signal and, as a ubiquitous iron reducer in soils around the world, G. sulfurreducens has an impact on the global iron cycle. We measured the concentrations of metals, macromolecules, and basic elements in G. sulfurreducens to define this organism's composition. We also used gene expression data to discuss which proteins those metals could be associated with. We found that G. sulfurreducens has a large amount of lipid and iron compared to other bacteria-these observations are important for future microbiologists and biotechnologists working with the organism.
Several studies have reported that current produced by electro-active bacteria (EAB) is dependent on anode potential and substrate concentration. However, information about the relation between biofilm growth and current density is scarce. In this study, biofilm thickness was monitored in-situ and this relation explored at three anode potentials and three acetate concentrations. The highest current densities of 3.7 A.m-2 were obtained for biofilms thinner than 40 µm, even though thicknesses up to 88 µm were measured. Fick's law was used to estimate the acetate penetration depth in the biofilm, acetate diffusion rates in the biofilm, and specific acetate utilization rates. Maximum biofilm thickness of a non-acetate limited biofilm of 55 µm and an acetate diffusion rate of 2.68x10-10 m 2 .s-1 were estimated at-0.2 V vs Ag/AgCl. The results provide information on the target biofilm thickness for which no acetate limitations occur and provide data for modeling works with bio-anodes.
In the present study, it is shown that the concentration dependency of undefined mixed culture anodic biofilms does not follow a single kinetic curve, such as the Nernst‐Monod curve. The biofilms adapt to concentration changes, which inevitably have to be applied to record kinetic curves, resulting in strong shifts of the kinetic parameters. The substrate concentration in a continuously operated bioelectrochemical system was changed rapidly via acetate pulses to record Nernst‐Monod curves which are not influenced by biofilm adaptation processes. The values of the maximum current density jmax and apparent half‐saturation rate constant Ks increased from 0.5 to 1 mA cm−2 and from 0.5 to 1.6 mmol L−1, respectively, within approximately 5 h. Double pulse experiments with a starvation phase between the two acetate pulses showed that jmax and Ks decrease reversibly through an adaptation process when no acetate is available. Pseudo‐capacitive charge values estimated from non‐turnover cyclic voltammograms (CV) led to the hypothesis that biofilm adaptation and the observed shift of the Nernst‐Monod curves occurred due to changes in the concentration of active redox proteins in the biofilm. It is argued that concentration‐related parameters of kinetic models for electroactive biofilms are only valid for the operating points where they have been determined and should always be reported with those conditions.
Electro-active bacteria (EAB) can form biofilms on an anode (so-called bioanodes), and use the electrode as electron acceptor for oxidation of organics in wastewater. So far, bioanodes have mainly been investigated under a continuous anode potential, but intermittent anode potential has resulted in higher currents and different biofilm morphologies. However, little is known about how intermittent potential influences the electron balance in the anode compartment. In this study, we investigated electron balances of bioanodes at intermittent anode potential regimes. We used a transparent non-capacitive electrode that also allowed for in-situ quantification of the EAB using optical coherence tomography (OCT). We observed comparable current densities between continuous and intermittent bioanodes, and stored charge was similar for all the applied intermittent times (5 mC). Electron balances were further investigated by quantifying Extracellular Polymeric Substances (EPS), by analyzing the elemental composition of biomass, and by quantifying biofilm and planktonic cells. For all tested conditions, a charge balance of the anode compartment showed that more electrons were diverted to planktonic cells than biofilm. Besides, 27–43% of the total charge was detected as soluble EPS in intermittent bioanodes, whereas only 15% was found as soluble EPS in continuous bioanodes. The amount of proteins in the EPS of biofilms was higher for intermittent operated bioanodes (0.21 mg COD proteins.mg COD biofilm⁻¹) than for continuous operated bioanodes (0.05 mg COD proteins.mg COD biofilm⁻¹). OCT revealed patchy morphologies for biofilms under intermittent anode potential. Overall, this study helped understanding that the use of a non-capacitive electrode and intermittent anode potential deviated electrons to other processes other than electric current at the electrode by identifying electron sinks in the anolyte and quantifying the accumulation of electrons in the form of EPS.
Geobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to −0.3 V vs. Standard Hydrogen Electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, a bc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants lacking cbcBA ceased Fe(III) reduction at −0.21 V vs. SHE, could not transfer electrons to electrodes between −0.21 and −0.28 V, and could not reduce the final 10% – 35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in ∆cbcBA, and deletion of cbcL (an unrelated bc-cytochrome essential near −0.15 V) in ΔcbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.
Wastewater treatment plants (WWTP) are aimed to be transformed from sinks into sources of energy and material. For fostering corresponding engineering efforts and economic assessments, comprehensive knowledge of the energy content of wastewater is required. We show in this proof-of-concept study that these data can be gathered by combining micro- bomb combustion calorimetrywith freeze-drying. Thereby, the methodology formeasuring the combustion enthalpy (ΔcH) of wastewater is significantly improved by decreasing the time demand for the drying process as only tiny amounts of samples are required. Here, the effluent of the primary clarifier of a wastewater treatment plant treating low-strength municipal wastewater was sampled on a weekly basis for 1 year, yielding 53 composite samples that were analyzed for ΔcH and standard wastewater parameters. A robust correlation between the chemical oxygen demand (COD) and ΔcH of −14.9 ± 3.5 kJ gCOD−1 (r = 0.51) was determined, verifying previous results obtained with more laborious and time-demanding methodologies. The global chemical energy potential of the sampled WWTP is presumably higher as the first treatment steps and losses during sample preparation reduced the amount of energy-rich compounds. A stronger correlation was observed between ΔcH and the biochemical oxygen demand (BOD5, r = 0.64), suggesting its usage for predicting the potential of wastewater as feedstock for biotechnological applications. This demonstrates that micro-bomb combustion calorimetry can be applied for deriving precious information on the energy content of wastewater from simple COD measurements.
Biocorrosion first surfaced in the scientific literature when Richard H. Gaines associated corrosion with bacterial activities in 1910. It is also known as microbiologically influenced corrosion (MIC). In general, it covers two scenarios. One is that microbes cause corrosion directly, which usually means microbes secrete corrosive metabolites or microbes harvest electrons from a metal for respiration to produce energy. In the second scenario, microbes are behind the initiation or acceleration of corrosion caused by a pre‐existing corrosive agent such as water and CO2, by compromising the passive film (often a metal oxide film on a metal). MIC is caused by microbial biofilms. It is everywhere around us. This work dissects some notable examples with perspectives.
Invited for this month's cover picture is the group of Prof. Dr. Falk Harnisch at the Helmholtz Centre for Environmental Research (Germany). The cover picture displays a biofilm anode obtained by electrochemically driven enrichment in a one‐chamber reactor that is dominated by Geobacter but also colonized by methanogens that cross feed from cathodic hydrogen. Read the full text of the Article at 10.1002/celc.202000731. “Geobacter enrichment biofilm anodes are cultivated in one‐chamber and two‐chamber reactors. By applying molecular and microscopic methods, it is demonstrated that the reactor configuration and thus, the hydrogen availability shape microbial abundance and composition within these biofilms…“ Learn more about the story behind the research featured on the front cover in this issue's Cover Profile. Read the corresponding Article at 10.1002/celc.202000731.
Biofilm anodes based on Geobacter enrichment in one‐chamber and two‐chamber reactors are qualitatively and quantitatively investigated. The methanogenic community of biofilms in both types of reactors consists exclusively of hydrogenotrophs, mainly the genus Methanobacterium. As qPCR demonstrates in one‐chamber reactors, the abundance of Geobacter and methanogens increases due to the presence of cathodically produced hydrogen. In two‐chamber reactors, the abundance of methanogens is decreased by 98 % compared to one‐chamber reactors. Adding hydrogen gas to the anodic compartment of two‐chamber reactors recovers the abundance of methanogens and Geobacter. Ratios of methanogens/bacteria are confirmed by using the cofactor F420 autofluorescence of methanogens. The impact of the reactor setup for developing primary microbial electrochemical technologies is discussed, reasoning that two‐chamber reactors have to be generally favored. Yet, their use inheres a trade‐off when cultivating biofilm anodes, as the indicated syntrophic interactions between methanogens and Geobacter are reduced, but pH decrease might influence microbial processes. Beaming with joy: The presence of hydrogen increases the abundance of both Geobacter and methanogens in biofilm anodes, thus communities shine “in the dark” based on F420 cofactor of methanogens. Microbial composition is verified with qPCR and genetic fingerprinting showing a higher degree of purity of Geobacter enrichment biofilms in two‐chamber reactors compared to one‐chamber reactors that is offset by a lower cell number of Geobacter.
Geobacter spp. enrichment biofilms were cultivated in batch using one-chamber and two-chamber bioelectrochemical reactors. Time-resolved substrate quantification was performed to derive physiological parameters as well as incremental coulombic efficiency (i.e., coulombic efficiency during one batch cycle, here every 6h) during early stage biofilm development. The results of one-chamber reactors revealed an intermediate acetate increase putatively due to the presence of acetogens. Total coulombic efficiencies of two-chamber reactors were considerable lower (19.6±8.3% and 49.3±13.2% for 1st and 2nd batch cycle, respectively) compared to usually reported values of mature Geobacter spp. enrichment biofilms presumably reflecting energetic requirements for biomass production (i.e., cells and extracellular polymeric substances) during early stages of biofilm development. The incremental coulombic efficiency exhibits considerable changes during batch cycles indicating shifts between phases of maximizing metabolic rates and maximizing biomass yield. Analysis based on Michaelis-Menten kinetics yielded maximum substrate uptake rates (vmax,Ac, vmax,I) and half-saturation concentration coefficients (KM,Ac,KM,I) based on acetate uptake or current production, respectively. The latter is usually reported in literature but neglects energy demands for biofilm growth and maintenance as well as acetate and electron storage. From 1st to 2nd batch cycle, vmax,Ac and KM,Ac, decreased from 0.0042–0.0051 mmol Ac⁻ h⁻¹ cm⁻² to 0.0031–0.0037 mmol Ac⁻ h⁻¹ cm⁻² and 1.02–2.61 mM Ac⁻ to 0.28–0.42 mM Ac⁻, respectively. Furthermore, differences between KM,Ac/KM,I and vmax,Ac/vmax,I were observed providing insights into the physiology of Geobacter spp. enrichment biofilms. Notably, KM,I considerably scattered while vmax,Ac/vmax,I and KM,Ac remained rather stable indicating that acetate transport within biofilm only marginally affects reaction rates. The observed data variation mandates the requirement of a more detailed analysis with an improved experimental system, e.g., using flow conditions and a comparison with Geobacter spp. pure cultures.
This study analyzes the biofilm growth and long‐term current production of mixed‐culture, electrochemically active biofilms (EABs) on macrostructured electrodes under low‐shear‐force conditions. The channel dimensions were altered systematically in the range 400 μm to 2 mm, and the channel heights were varied between 1 and 4 mm to simulate macrostructures of different scales. Electrodes with finer‐structured surfaces produced higher current densities in the short term owing to their large surface area but were outperformed in the long term because the accumulation of biomass led to limitations of mass transfer into the structures. The best long‐term performance was observed for electrodes with channel dimensions of 1×4 mm, which showed no significant decrease in performance in the long term. Channels with a diameter of 400 μm were overgrown by the biofilm, which led to a transition from 3 D to 2 D behavior, indicating that structures of this scale might not be suitable for long‐term operation under low‐shear‐stress conditions.
Electroactive microorganisms (EAM) harvest energy by reducing insoluble terminal electron acceptors (TEA) including electrodes via extracellular electron transfer (EET). Therefore, compared to microorganisms respiring soluble TEA, an adapted approach is required for thermodynamic analyses. In EAM, the thermodynamic frame (i.e., maximum available energy) is restricted as only a share of the energy difference between electron donor and TEA is exploited via the electron-transport chain to generate proton-motive force being subsequently utilized for ATP synthesis. However, according to a common misconception, the anode potential is suggested to co-determine the thermodynamic frame of EAM. By comparing the model organism Geobacter spp. and microorganisms respiring soluble TEA, we reason that a considerable part of the electron-transport chain of EAM performing direct EET does not contribute to the build-up of proton-motive force and thus, the anode potential does not co-determine the thermodynamic frame. Furthermore, using a modeling platform demonstrates that the influence of anode potential on energy harvest is solely a kinetic effect. When facing low anode potentials, NADH is accumulating due to a slow direct EET rate leading to a restricted exploitation of the thermodynamic frame. For anode potentials ≥ 0.2 V (vs. SHE), EET kinetics, NAD+/NADH ratio as well as exploitation of the thermodynamic frame are maximized, and a further potential increase does not result in higher energy harvest. Considering the limited influence of the anode potential on energy harvest of EAM is a prerequisite to improve thermodynamic analyses, microbial resource mining, and to transfer microbial electrochemical technologies (MET) into practice.
A vast array of microorganisms from all three domains of life can produce electrical current and transfer electrons to the anodes of different types of bioelectrochemical systems. These exoelectrogens are typically iron-reducing bacteria, such as Geobacter sulfurreducens, that produce high power densities at moderate temperatures. With the right media and growth conditions, many other microorganisms ranging from common yeasts to extremophiles such as hyperthermophilic archaea can also generate high current densities. Electrotrophic microorganisms that grow by using electrons derived from the cathode are less diverse and have no common or prototypical traits, and current densities are usually well below those reported for model exoelectrogens. However, electrotrophic microorganisms can use diverse terminal electron acceptors for cell respiration, including carbon dioxide, enabling a variety of novel cathode-driven reactions. The impressive diversity of electroactive microorganisms and the conditions in which they function provide new opportunities for electrochemical devices, such as microbial fuel cells that generate electricity or microbial electrolysis cells that produce hydrogen or methane.
Detailed studying of microbial growth in bioelectrochemical systems is required for their proper design and operation. Here we report on the use of Optical Coherence Tomography (OCT) as a tool for in situ and non-invasive quantification of biofilm growth on electrodes (bioanodes). An experimental platform is designed and described in which transparent electrodes are used to allow for real-time, three-dimensional biofilm imaging. The accuracy and precision of the developed method is assessed by relating OCT results to well-established standards for biofilm quantification (COD and Total N) and show high correspondence to these standards. Biofilm thickness as observed by OCT ranged between 3 and 90 µm for experimental durations ranging from 1 to 24 days. This translated to growth yields between 38 and 42 mg CODbiomass/g CODacetate at an anode potential of -0.35 V vs. Ag/AgCl. Time-lapse observations of an experimental run performed in duplicate show high reproducibility in obtained microbial growth yield using the developed method. As such, we identify OCT as a powerful tool for conducting in-depth characterizations of microbial growth dynamics in BESs. Additionally, the presented platform allows concomitant application of this method with various optical and electrochemical techniques.
Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.
In nature, bacteria alternate between two modes of growth: a unicellular life phase, in which the cells are free-swimming (planktonic), and a multicellular life phase, in which the cells are sessile and live in a biofilm, that can be defined as surface-associated microbial heterogeneous structures comprising different populations of microorganisms surrounded by a self-produced matrix that allows their attachment to inert or organic surfaces. While a unicellular life phase allows for bacterial dispersion and the colonization of new environments, biofilms allow sessile cells to live in a coordinated, more permanent manner that favors their proliferation. In this alternating cycle, bacteria accomplish two physiological transitions via differential gene expression: (i) from planktonic cells to sessile cells within a biofilm, and (ii) from sessile to detached, newly planktonic cells. Many of the innate characteristics of biofilm bacteria are of biotechnological interest, such as the synthesis of valuable compounds (e.g., surfactants, ethanol) and the enhancement/processing of certain foods (e.g., table olives). Understanding the ecology of biofilm formation will allow the design of systems that will facilitate making products of interest and improve their yields.
In this article we describe a methodology based on bioenergetic analysis of chemotrophic microbial growth systems that allows for a generalized system description. The main parameters can be estimated based on the identification of (i) the Gibbs energy supplying redox reaction and (ii) the carbon and nitrogen source for microbial growth. The calculated parameter values can be considered as a first approximation and allow for comparison with measured parameter values. Stoichiometric and kinetic parameter values that deviate strongly from the estimated values suggest that a highly specific microbial system is encountered. Herewith, the generalized method may serve as a reference framework for interpretation of stoichiometric and kinetic parameter values describing microbial growth processes.
The respiration of metals by the bacterium Geobacter sulfurreducens requires electrons generated by metabolism to pass from the interior of the cell to electron acceptors beyond the cell membranes. The G. sulfurreducens inner membrane multiheme c-type cytochrome ImcH is required for respiration to extracellular electron acceptors with redox potentials greater than -0.1V vs. SHE, but ImcH is not essential for electron transfer to lower potential acceptors. In contrast, deletion of cbcL, encoding an inner membrane protein consisting of b-type and multiheme c-type cytochrome domains, severely affected reduction of low potential electron acceptors such as Fe(III)-oxides and electrodes poised at -0.1V vs. SHE. Catalytic cyclic voltammetry of a ΔcbcL strain growing on poised electrodes revealed a 50mV positive shift in driving force required for electron transfer out of the cell. In non-catalytic conditions, low-potential peaks present in wild type biofilms were absent in ∆cbcL mutants. Expression of cbcL in trans increased growth at low redox potential and restored features to cyclic voltammetry. This evidence supports a model where CbcL is a component of a second electron transfer pathway out of the G. sulfurreducens inner membrane that dominates when redox potential is at or below -0.1V vs. SHE.
Microbial electrochemistry is the study and application of interactions between living microbial cells and electrodes (i.e. electron conductors, capacitive materials). For a long time this subfield of bioelectrochemistry has been the interest of mainly fundamental researchers. This has considerably changed during the last decade and microbial electrochemistry gained interest from applied researchers and engineers. These researchers took the microbial fuel cell (MFC), which is a system that converts the
chemical energy of organic material in wastewater into electric power, from a concept to a technology.
In addition, a plethora of derivative technologies, such as microbial electrolysis cells (MECs), microbial desalination cells (MDCs), photomicrobial fuel cells (photoMFCs), microbial electrosynthesis (MES), and biocomputing have been developed. The growing number of systems is often referred to in literature under the termini bioelectrochemical system (BES), microbial electrochemical technology (MET), or electrobiotechnology. Within this article we introduce a classification of technologies based on interfacing microbiology and electrochemistry. We argue that BESs comprise all systems based on bioelectrochemistry, with a further layer of termini through the use of METs. Primary METs are based on extracellular electron transfer (direct or mediated), whereas secondary METs include systems in which electrochemistry is connected – at least through ionic contact – with a microbial process via the electrochemical control or adaptation of environmental parameters, such as pH or metabolite concentration level.
Unlabelled:
Dissimilatory metal-reducing bacteria, such as Geobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption of imcH, encoding an inner membrane c-type cytochrome, eliminated the ability of G. sulfurreducens to reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but the imcH mutant retained the ability to reduce Fe(III) oxides with potentials of ≤-0.1 V versus SHE. The imcH mutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to -0.1 V versus SHE triggered exponential growth. At potentials of ≤-0.1 V versus SHE, both the wild type and the imcH mutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not trigger imcH mutant growth. These results demonstrate that G. sulfurreducens possesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments where Geobacter is found.
Importance:
Insoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein in Geobacter sulfurreducens which is required only for electron transfer to high-potential compounds, and we show that G. sulfurreducens has the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.
Waste biomass is a cheap and relatively abundant source of electrons for microbes capable of producing electrical current outside the cell. Rapidly developing microbial electrochemical technologies, such as microbial fuel cells, are part of a diverse platform of future sustainable energy and chemical production technologies. We review the key advances that will enable the use of exoelectrogenic microorganisms to generate biofuels, hydrogen gas, methane, and other valuable inorganic and organic chemicals. Moreover, we examine the key challenges for implementing these systems and compare them to similar renewable energy technologies. Although commercial development is already underway in several different applications, ranging from wastewater treatment to industrial chemical production, further research is needed regarding efficiency, scalability, system lifetimes, and reliability.
Cited By (since 1996): 60, Export Date: 9 August 2012, Source: Scopus
Harvesting electricity from the environment, organic wastes, or renewable biomass with microbial fuel cells (MFCs) is an appealing strategy, but the destructive sampling required to investigate the anode-associated biofilms has hampered research designed to better understand and optimize microbe–anode interactions. Therefore, a MFC that permits real-time imaging of the anode biofilm with confocal scanning laser microscopy was developed. In this new MFC Geobacter sulfurreducens, an organism closely related to those often found on MFC anodes and capable of high current densities, produced current comparable to that previously reported with other MFC designs. G. sulfurreducens engineered to produce the fluorescent protein mcherry to facilitate real-time imaging produced current comparable to wild-type cells. Introducing C-SNARF-4, a pH-sensitive fluoroprobe, into the anode chamber revealed strong pH gradients within the anode biofilms. The pH decreased with increased proximity to the anode surface and from the exterior to the interior of biofilm pillars. Near the anode surface pH levels were as low as 6.1 compared to ca. 7 in the external medium. Various controls demonstrated that the proton accumulation was associated with current production. Dropping the pH of culture medium from 7 to 6 severely limited the growth of G. sulfurreducens. These results demonstrate that it is feasible to non-destructively monitor the activity of anode biofilms in real time and suggest that the accumulation of protons that are released from organic matter oxidation within anode biofilms can limit current production.
So close, but yet so far: G. sulfurreducens c-type cytochromes become reduced as biofilms grow on electrodes beyond a few cell thicknesses, even if the electrode is poised well above the potential required to oxidize all cytochromes. Cytochrome redox state also lags behind rapid potential changes during voltammetry, but only when the films are multiple cell layers thick, as would be expected if diffusional or exchange-based kinetics controls electron transfer between cytochromes.
Different microbial fuel cell (MFC) configurations have been successfully operated at pilot-scale levels (>100 L) to demonstrate electricity generation while accomplishing domestic or industrial wastewater treatment. Two cathode configurations have been primarily used based on either oxygen transfer by aeration of a liquid catholyte or direct oxygen transfer using air-cathodes. Analysis of several pilot-scale MFCs showed that air-cathode MFCs outperformed liquid catholyte reactors based on power density, producing 234% larger area-normalized power densities and 181% higher volumetric power densities. Reactors with higher electrode packing densities improved performance by enabling larger power production while minimizing the reactor footprint. Despite producing more power than the liquid catholyte MFCs, and reducing energy consumption for catholyte aeration, pilot MFCs based on air-cathode configuration failed to produce effluents with chemical oxygen demand (COD) levels low enough to meet typical threshold for discharge. Therefore, additional treatment would be required to further reduce the organic matter in the effluent to levels suitable for discharge. Scaling up MFCs must incorporate designs that can minimize electrode and solution resistances to maximize power and enable efficient wastewater treatment.
Microbial fuel cells (MFCs) in municipal wastewater treatment plants (M-WWTPs) have garnered increasing interest in terms of attaining energy self-sufficiency due to their theoretical superiority to conventional M-WWTP processes. Despite being widely studied, pertaining literature primarily focuses on the fundamentals and configurations of the MFCs while overlooking their targeted application niche. Therefore, the adoption of MFCs in many niches (i.e., M-WWTPs) has not been adequately reviewed yet. This study aims to critically review the adoption of MFCs for carbon handling in the liquid stream of M-WWTPs with an emphasis on MFC's scalability, use of municipal wastewater (M-WW) as the substrate, and MFC's capital cost. The review includes the scaled-up results and other efforts to engineer MFCs. Three key challenges stymie MFC's adoption in M-WWTPs: low power generation, wide range of reported carbon removal efficiencies, and high capital cost. Accordingly, MFCs should be adopted in M-WWTPs with the goal of energy neutrality, not extra electricity production. To meet the effluent discharge standards, MFCs should be preceded by primary treatment, followed by an anaerobic fluidized bed membrane bioreactor. However, breakthroughs are still required to make this technology cost-efficient and energy-efficient. These efforts should take into consideration the multivariate nature of the MFCs. Additionally, the development of bioelectrochemically assisted anaerobic technologies has shown great prominence as an alternative technology that can be integrated into M-WWTPs. However, validation based on larger-scale applications , cost, and energy estimations is still needed.
A submergible 255 L prototype MFC module was operated under practical conditions with municipal wastewater having a large share in industrial discharges for 98 days to investigate the performance of two of the largest, ever investigated multi-panel stainless steel / activated carbon air cathodes (85×85 cm). At a flow rate of 144 L/d, power density of 78 mW/m2Cat (317 mW/m3) and COD, TSS and TN removal of 41±16 %, 36±16 % and 18±14 %, respectively, were reached. Observed Coulombic efficiency and substrate-specific energy recovery were 29.5±14 % and 0.184±0.125 kWhel/kgCOD,deg, respectively. High salt content of wastewater (TDS = 2.8 g/L) led to severe inorganic fouling causing a drastic decline in power output and energy recovery of more than 90 % in the course of experiments. Mechanical cleaning of the cathodes restored only 22 % (17 mW/m2Cat) of the power output and did not improve nutrient removal or energy recovery.
Microbial anodes are the cornerstone of most electro-microbial processes. Designing 3-dimensional porous electrodes to increase the surface area of the electroactive biofilm they support is a key challenge in order to boost their performance. In this context, the critical review presented here aims to assess whether an optimal range of pore size may exist for the design of microbial anodes. Pore sizes of a few micrometres can enable microbial cells to penetrate but in conditions that do not favour efficient development of electroactive biofilms. Pores of a few tens of micrometres are subject to clogging. Sizes of a few hundreds of micrometres allow penetration of the biofilm inside the structure, but its development is limited by internal acidification. Consequently, pore sizes of a millimetre or so appear to be the most suitable. In addition, a simple theoretical approach is described to establish basis for porous microbial anode design.
The family Geobacteraceae, with its only valid genus Geobacter, comprises deltaproteobacteria ubiquitous in soil, sediments, and subsurface environments where metal reduction is an active process. Research for almost three decades has provided novel insights into environmental processes and biogeochemical reactions not previously known to be carried out by microorganisms. At the heart of the environmental roles played by Geobacter bacteria is their ability to integrate redox pathways and regulatory checkpoints that maximize growth efficiency with electron donors derived from the decomposition of organic matter while respiring metal oxides, particularly the often abundant oxides of ferric iron. This metabolic specialization is complemented by versatile metabolic reactions, respiratory chains, and sensory networks that allow specific members to adaptively respond to environmental cues to integrate organic and inorganic contaminants in their oxidative and reductive metabolism, respectively. Thus, Geobacteraceae are important members of the microbial communities that degrade hydrocarbon contaminants under iron-reducing conditions and that contribute, directly or indirectly, to the reduction of radionuclides, toxic metals, and oxidized species of nitrogen. Their ability to produce conductive pili as nanowires for discharging respiratory electrons to solid-phase electron acceptors and radionuclides, or for wiring cells in current-harvesting biofilms highlights the unique physiological traits that make these organisms attractive biological platforms for bioremediation, bioenergy, and bioelectronics application. Here we review some of the most notable physiological features described in Geobacter species since the first model representatives were recovered in pure culture. We provide a historical account of the environmental research that has set the foundation for numerous physiological studies and the laboratory tools that had provided novel insights into the role of Geobacter in the functioning of microbial communities from pristine and contaminated environments. We pay particular attention to latest research, both basic and applied, that has served to expand the field into new directions and to advance interdisciplinary knowledge. The electrifying physiology of Geobacter, it seems, is alive and well 30 years on.
The outstanding capacity of Geobacter sulfurreducens cells to directly connect their inner electron transport chain to a polarized electrode have prompted the application of a wide arrange of electrochemical techniques to explore their metabolism and current production possibilities. In this work we use very basic electrochemical assays as open circuit potential, voltammetric and chronopotentiometric measurements to obtain fundamental information on bacterial electrochemical characteristics of importance to interpret bacterial functioning. Specifically, we obtained information on G. sulfurreducens biofilm capacity to store charge in three major molecular reservoirs bridging the gap between NADH and the electrode, demonstrating, at the same time, the value of OCP measurements as a reporter of internal redox state of cells.
Abstract | Electron transfer between microorganisms and an electrode — even across long
distances — enables the former to live by coupling to an electronic circuit. Such a system
integrates biological metabolism with artificial electronics; studying these systems adds to our
knowledge of charge transport in the chemical species involved, as well as, perhaps most
importantly, to our knowledge of charge transport and chemistry at the cell–electrode interfaces.
This understanding may lead to microbial electrochemical systems finding widespread
application, particularly in the energy sector. Bioelectrochemical systems have already shown
promise for electricity generation, as well as for the production of biochemical and chemical
feedstocks, and with improvement are likely to give rise to viable applications.
The core of primary microbial electrochemical technologies (METs) is the ability of the electroactive microorganisms to interact with electrodes via extracellular electron transfer (EET), allowing wiring of current flow and microbial metabolism. Geobacter sulfurreducens and Shewanella oneidensis are the model organisms for understanding and engineering EET. Many other microorganisms are reported being electroactive but are often sparsely characterized. Based on a literature survey 94 species are ascribed as electroactive. Their apparent diversity raises questions on the natural importance and distribution of the EET capacity, that is, of the ecological niche of microbial electroactivity. To identify this potential niche the environmental preferences and natural habitat characteristics of all electroactive species were combined with their metabolic, growth and EET characteristics and an extensive meta-analysis performed. The results indicate that there is not a single ecological niche for electroactive microorganisms. Significantly more electroactive species presumably exist in nature as well as already existing strain collections but due to current cultivation techniques their EET potential is not leveraged. Thus, in the light of specific traits required for industrial application, microbial resource mining based on ecological knowledge bears a great potential for broadening the foundation of microbial electrochemistry as well as for future developments of primary METs.
Bioelectrocalorimetry allows assessing the heat (enthalpy) balance and revealing the microbial electrochemical Peltier heat of biofilm electrodes. This effect accounts for a heat formation of 27 ± 6 kJ mol−1 of electrons transferred and represents a significant energy loss for primary microbial electrochemical technologies.
The goal of this work was to develop a microbiosensor to measure acetate concentration profiles inside biofilms in situ. The working principle of the microbiosensor was based on the correlation between the acetate concentration and the current generated during acetate oxidation by Geobacter sulfurreducens. The microbiosensor consisted of a 30-µm carbon microelectrode with an open tip as a working electrode, with G. sulfurreducens biofilm on the tip and a pseudo Ag/AgCl reference electrode, all enclosed in a glass outer case with a 30-µm tip diameter. The microbiosensor showed a linear response in the 0-1.6 mM acetate concentration range with a 79±8 µM limit of detection (S/N=2). We quantified the stirring effect and found it negligible. However, the interfering effect of alternative electron donors (lactate, formate, pyruvate, or hydrogen) was found to be significant. The usefulness of the acetate microbiosensor was demonstrated by measuring acetate concentration depth profiles within a G. sulfurreducens biofilm. The acetate concentration remained at bulk values throughout the biofilm when no current was passed, but it decreased from the bulk values to below the detection limit within 200 μm when current was allowed to pass. The zero acetate concentration at the bottom of the biofilm showed that the biofilm was acetate-limited.
General expressions for mass, elemental, energy, and entropy balances are derived and applied to microbial growth and product formation. The state of the art of the application of elemental balances to aerobic and heterotrophic growth is reviewed and extended somewhat to include the majority of the cases commonly encountered in biotechnology. The degree of reduction concept is extended to include nitrogen sources other than ammonia. The relationship between a number of accepted measures for the comparison of substrate yields is investigated. The theory is illustrated using a generalized correlation for oxygen yield data. The stoichiometry of anaerobic product formation is briefly treated, a limit to the maximum carbon conservation in product is derived, using the concept of elemental balance. In the treatment of growth energetics the correct statement of the second law of thermodynamics for growing organisms is emphasized. For aerobic heterotrophic growth the concept of thermodynamic efficiency is used to formulate a limit the substrate yield can never surpass. It is combined with a limit due to the fact that the maximum carbon conservation in biomass can obviously never surpass unity. It is shown that growth on substrates of a low degree of reduction is energy limited, for substrates of a high degree of reduction carbon limitation takes over. Based on a literature review concerning yield data some semiempirical notions useful for a preliminary evolution of aerobic heterotrophic growth are developed. The thermodynamic efficiency definition is completed by two other efficiency measures, which allow derivation of simple equations for oxygen consumption and heat production. The range of validity of the constancy of the rate of heat production to the rate of oxygen consumption is analyzed using these efficiency measures. The energetic of anaerobic growth are treated—it is shown that an approximate analysis in terms of an enthalpy balance is not valid for this case, the evaluation of the efficiency of growth has to be based on Gibbs free energy changes. A preliminary analysis shows the existence of regularities concerning the free energy conservation on anaerobic growth. The treatment is extended to include the effect of growth rate by the introduction of a linear relationship for substrate consumption. Aerobic and anaerobic growth are discussed using this relationship. A correlation useful in judging the potentialities for improvement in anaerobic product formation processes is derived. Finally the relevance of macroscopic principles to the modeling of bioengineering systems is discussed.
Microbes can respire on metals. This seemingly simple finding is one of the major discoveries that were made in the field of microbiology in the last few decades. The importance of this observation is evident. Metals are highly abundant on our planet. Iron is even the most abundant element on Earth and the forth most abundant element in the Earth's crust. Hence, in some environments iron, but also other metals or metalloids, are the dominant respiratory electron acceptors. Their reduction massively drives the carbon cycle in these environments and establishes redox cycles of the metallic electron acceptors themselves. These redox cycles are not only a driving force for other biotic reactions but are furthermore necessary for initiating a number of geochemically relevant abiotic redox conversions. Although widespread and ecologically influential, electron transfer onto metals like ferric iron or manganese is biochemically challenging. The challenge is to transfer respiratory electrons onto metals that occur in nature at neutral pH in the form of metal oxides or oxihydroxides that are effectively insoluble. Obviously, it is necessary that the microbes specially adapt in order to catalyze the electron transfer onto insoluble electron acceptors. The elucidation of these adaptations is an exciting ongoing process. To sum it up, dissimilatory metal reduction has wide-spread implications in the field of microbiology, biochemistry and geochemistry and its discovery was one of the major reasons to establish a novel scientific field called geomicrobiology. Recently, the discovery of potential applications of dissimilatory metal reducers in bioremediation or current production in a microbial fuel cell further increased the interest in studying microbial metal reduction. © 2012 Springer-Verlag Berlin Heidelberg. All rights are reserved.
Real time measurement of volatile fatty acids (VFA) in anaerobic digestion (AD) is crucial for process monitoring and management. It is essential for establishing demand-driven biogas production, however to date it is not commercially available. We propose a novel microbial electrochemical sensor for in-line measurement of acetate and other VFA in AD. The measurement range as well as the measurement resolution and dynamic behavior of the biosensor were investigated under flow conditions. A measurement range of 0.5 to 5 mmol L-1 acetate with a resolution (R) of 0.25 and 1 mmol L-1 was determined. For application in AD the achieved measurement range is still too low, therefore measures to improve it as well as further application properties of the sensor are discussed.
Despite some success with microbial fuel cells and microbial electrolysis cells in recovering resources from wastes, challenges with their scale and yield need to be resolved. Waste streams from biorefineries e.g. bioethanol and biodiesel plants and wastewaters are plausible substrates for microbial electrosynthesis (MES). MES integration can help biorefineries achieving the full polygeneration potentials, i.e. recovery of metals turning apparently pollutants from biorefineries into resources, production of biofuels and chemicals from reuse of CO2 and clean water. Symbiotic integration between the two systems can attain an economic and environmental upside of the overall system. We envision that electrochemical technologies and waste biorefineries can be integrated for increased efficiency and competitiveness with stillage released from the latter process used in the former as feedstock and energy resource recovered from the former used in the latter. Such symbiotic integration can avoid loss of material and energy from waste streams, thereby increasing the overall efficiency, economics and environmental performance that would serve towards delivering the common goals from both the systems. We present an insightful overview of the sources of organic wastes from biorefineries for integration with MES, anodic and cathodic substrates and biocatalysts. In addition, a generic and effective reaction and thermodynamic modelling framework for the MES has been given for the first time. The model is able to predict multi-component physico-chemical behaviour, technical feasibility and best configuration and conditions of the MES for resource recovery from waste streams.
Geobacter-dominated biofilms can be selected under stringent conditions that limit the growth of competing bacteria. However, in many practical applications, such stringent conditions cannot be maintained and the efficacy and stability of these artificial biofilms may be challenged. In this work, biofilms were selected on low-potential anodes (-0.36V vs Ag/AgCl, i.e. -0.08V vs SHE) in minimal acetate or ethanol media. Selection conditions were then relaxed by transferring the biofilms to synthetic wastewater supplemented with soil as a source of competing bacteria. We tracked community succession and functional changes in these biofilms. The Geobacter-dominated biofilms showed stability in their community composition and electrochemical properties, with Geobacter sp. being still electrically active after six weeks in synthetic wastewater with power densities of 100±19mW·m(-2) (against 74±14mW·m(-2) at week 0) for all treatments. After six weeks, the ethanol-selected biofilms, despite their high taxon richness and their efficiency at removing the chemical oxygen demand (0.8g·L(-1) removed against the initial 1.3g·L(-1) injected), were the least stable in terms of community structure. These findings have important implications for environmental microbial fuel cells based on Geobacter-dominated biofilms and suggest that they could be stable in challenging environments.
Copyright © 2015 Elsevier B.V. All rights reserved.
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 (300mM) 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 (1.2s(-1)) for reversible extracellular electron transfer.
Copyright © 2015 Elsevier B.V. All rights reserved.
Current outputs of microbial fuelcells (MFCs) are too low for most perceived practical applications. Most efforts for further optimization have focused on modifications of fuelcell architecture or electrode materials, with little investigation into the properties of microorganisms that are most essential for maximal current production. Geobacter sulfurreducens produces the highest current densities of any known pure culture; is closely related to the Geobacter species that often predominate in anode biofilms harvesting electricity from organic wastes; and produces highly conductive anode biofilms. Comparison of biofilm conductivities and current production in different strains of G. sulfurreducens revealed a direct correlation between biofilm conductivity and current density. Electrochemical impedance spectroscopy measurements demonstrated that higher biofilm conductivity not only reduced resistance to electron flow through the biofilm, but also lowered the activation energy barrier for electron transfer between the biofilm and the anode. These results demonstrate the crucial role of biofilm conductivity in achieving high current density in MFCs and suggest that increasing biofilm conductivity can boost MFC performance.
Through their ability to directly transfer electrons to electrodes, Geobacter sp. are key organisms for microbial fuel cell technology. This study presents a simple method to reproducibly select Geobacter-dominated anode biofilms from a mixed inoculum of bacteria using graphite electrodes initially poised at -0.25, -0.36 and -0.42V vs. Ag/AgCl. The biofilms all produced maximum power density of approximately 270mWm(-2) (projected anode surface area). Analysis of 16S rRNA genes and intergenic spacer (ITS) sequences found that the biofilm communities were all dominated by bacteria closely related to Geobacter psychrophilus. Anodes initially poised at -0.25V reproducibly selected biofilms that were dominated by a strain of G. psychrophilus that was genetically distinct from the strain that dominated the -0.36 and -0.42V biofilms. This work demonstrates for the first time that closely related strains of Geobacter can have very different competitive advantages at different anode potentials.
The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilm-electrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7 nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03 s(-1) . In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2 s(-1) . The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.
Thermodynamic analysis may be applied in order to predict microbial growth yields roughly, based on an empirical correlation of the Gibbs energy of the overall growth reaction or Gibbs energy dissipation. Due to the well-known trade-off between high biomass yield and high Gibbs energy dissipation necessary for fast growth, an optimal range of Gibbs energy dissipation exists and it can be correlated to physical characteristics of the growth substrates. A database previously available in the literature has been extended significantly in order to test such correlations. An analysis of the relationship between biomass yield and Gibbs energy dissipation reveals that one does not need a very precise estimation of the latter to predict the former roughly. Approximating the Gibbs energy dissipation with a constant universal value of −500 kJ C-mol−1 of dry biomass grown predicts many experimental growth yields nearly as well as a carefully designed, complex correlation available from the literature, even though a number of predictions are grossly out of range. A new correlation for Gibbs energy dissipation is proposed which is just as accurate as the complex literature correlation despite its dramatically simpler structure.
The limitation of pH inside electrode-respiring biofilms is a well-known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode-respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm.
The capability of the experimental systems used in two-chambered microbial fuel cell experimentation was tested in terms of repeatability and reproducibility. The optimal number of replicates needed to discriminate between responses of technical interest, both in open-circuit and closed-circuit experiments was studied. For N = 4 replicates, these differences were set to 9.0% CODR units, 261 mV and 63 mg/L in VFAs for open-circuit experiments and 3.6%, 30.2 mV and 45 mg/L in closed circuit experiments. Cycling operation with several reactor refills using fresh wastewater and keeping the same biofilm between cycles almost has no influence in CODR and VFAs but voltage standard deviation reduces by one half between the first and fourth cycle. This study takes part by the option of increasing the number of replicates because although it may have lower repeatability, the amount of data generated per unit time is larger than running the experiments in cycles.