Nuclear Magnetic Resonance Imaging Techniques Research Articles

CHARACTERIZATION OF PORE STRUCTURE AND DYNAMIC SEEPAGE CHARACTERISTICS OF SANDSTONE DETERMINED BY NUCLEAR MAGNETIC RESONANCE (NMR) AND MAGNETIC RESONANCE IMAGING (MRI) TECHNIQUES

The combination of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) to investigate the seepage characteristics of sedimentary rock has been popular research in recent years. This research assessed dynamic seepage features of sandstone and shale samples through NMR and MRI measurements with two respective fluids of different wettability (i.e., distilled water and kerosene). Results show that sandstone and shale possess remarkably different T<sub>2</sub> spectra. The saturation of movable fluid (FFI) of the sandstone and shale samples is 75.09%, 74.92%, and 7.50%, respectively. The seepage T<sub>2</sub> spectra of distilled water in sandstone is predominantly bimodal distribution, whereas kerosene seepage in sandstone presents a single distribution, and those of kerosene seepage in shale show a bimodal distribution. When reaching equilibrium, the time required for kerosene seepage in sandstone is the shortest, followed by water seepage in sandstone, and kerosene seepage in shale is the longest. In addition, the fluid volumes of water and kerosene in sandstone have a strong linear relationship with time, while those of kerosene in shale have a power-function relationship. The dominant channel gradually develops and expands to both sides during the seepage process. Over time, the content of water seepage in sandstone is predominantly concentrated near the inlet, whereas the kerosene content of kerosene seepage in sandstone is primarily centered in the central region. Significant differences in unit discharge exist between the cool and warm color regions of water seepage in sandstone. However, those of kerosene seepage in sandstone are negligible.

Pore-pressure and stress-coupled creep behavior in deep coal: Insights from real-time NMR analysis

Understanding the variations in microscopic pore-fracture structures (MPFS) during coal creep under pore pressure and stress coupling is crucial for coal mining and effective gas treatment. In this manuscript, a triaxial creep test on deep coal at various pore pressures using a test system that combines in-situ mechanical loading with real-time nuclear magnetic resonance (NMR) detection was conducted. Full-scale quantitative characterization, online real-time detection, and visualization of MPFS during coal creep influenced by pore pressure and stress coupling were performed using NMR and NMR imaging (NMRI) techniques. The results revealed that seepage pores and microfractures (SPM) undergo the most significant changes during coal creep, with creep failure gradually expanding from dense primary pore fractures. Pore pressure presence promotes MPFS development primarily by inhibiting SPM compression and encouraging adsorption pores (AP) to evolve into SPM. Coal enters the accelerated creep stage earlier at lower stress levels, resulting in more pronounced creep deformation. The connection between the micro and macro values was established, demonstrating that increased porosity at different pore pressures leads to a negative exponential decay of the viscosity coefficient. The Newton dashpot in the ideal viscoplastic body and the Burgers model was improved using NMR experimental results, and a creep model that considers pore pressure and stress coupling using variable-order fractional operators was developed. The model’s reasonableness was confirmed using creep experimental data. The damage-state adjustment factors ω and β were identified through a parameter sensitivity analysis to characterize the effect of pore pressure and stress coupling on the creep damage characteristics (size and degree of difficulty) of coal.

Evaluation of processing mechanism in Astragali Radix by low-field nuclear magnetic resonance and magnetic resonance imaging.

Astragali Radix (Huangqi) is an important herb medicine that is always processed into pieces for clinical use. Many operations need to be performed before use, among which drying of Astragali Radix (AR) pieces is a key step. Unfortunately, research on its drying mechanism is still limited. Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques were applied to study the moisture state and distribution during drying. The content of bioactive components and texture changes were measured by HPLC and texture analyzer, respectively. The moisture content of the AR pieces decreased significantly during drying, and the time to reach the drying equilibrium were different at different temperatures. The time when at 70°C, 80°C, and 90°C reach complete drying are 180 min, 150 min and 120 min, respectively. 80°C was determined as the optimum drying temperature, and it was observed that the four flavonoids and astragaloside IV have some thermal stability in AR pieces. When dried at 80°C, although the total water content decreased, the free water content decreased from 99.38% to 15.49%, in contrast to the increase in bound water content from 0.62% to 84.51%. The texture parameters such as hardness changed to some extent, with the hardness rising most significantly from 686.23 g to 2656.67 g. Correlation analysis revealed some connection between moisture content and LF-NMR and texture analyzer parameters, but the springiness did not show a clear correlation with most parameters. This study shows that HPLC, LF-NMR, MRI, and texture analyzers provide a scientific basis for elucidating the drying principles of AR pieces. The method is useful and shows potential for extension and application; therefore, it can be easily extended to other natural herb medicines.

Solid-State NMR and MRI Spectroscopy for Li/Na Batteries: Materials, Interface, and In Situ Characterization.

Enhancing the electrochemical performance of batteries, including the lifespan, energy, and power densities, is an everlasting quest for the rechargeable battery community. However, the dynamic and coupled (electro)chemical processes that occur in the electrode materials as well as at the electrode/electrolyte interfaces complicate the investigation of their working and decay mechanisms. Herein, the recent developments and applications of solid-state nuclear magnetic resonance (ssNMR) and magnetic resonance imaging (MRI) techniques in Li/Na batteries are reviewed. Several typical cases including the applications of NMR spectroscopy for the investigation of the pristine structure and the dynamic structural evolution of materials are first emphasized. The NMR applications in analyzing the solid electrolyte interfaces (SEI) on the electrode are further concluded, involving the identification of SEI components and investigation of ionic motion through the interfaces. Beyond, the new development of in situ NMR and MRI techniques are highlighted, including their advantages, challenges, applications and the design principle of in situ cell. In the end, a prospect about how to use ssNMR in battery research from the perspectives of materials, interface, and in situ NMR, aiming at obtaining deeper insight of batteries with the assistance of ssNMR is represented.

Study of Pumpkin Drying Through Magnetic Resonance Imaging

Nuclear Magnetic Resonance (NMR) and Magnetic Resonance Imaging (MRI) techniques are widely used in food science, mainly because they are non-invasive techniques. MRI, as a non-destructive technique, allows the study of intact samples and without any preparation of the samples before analysis. In food processing, the monitoring of distribution and water content is a consolidated analysis technique, frequently used on the market in order to preserve appropriate nutritional and health characteristics of food according to quality standards. In a food matrix, the variation of the water content is related to the changes in the internal structure and in the physico-chemical properties that occur during the transformation process. In this study MRI technique is used to evaluate the variation of the water content as a function of the drying time. Pumpkin samples are analyzed at four different drying temperatures of 50, 60, 65, and 70°C. The transverse relaxation time, T2, is used to assess the hydration level of the samples by comparing the information extracted from MR images with the drying kinetics measured by gravimetric method. Moreover, T2 maps are used to correlate the change in water distribution with the change in T2 values. The results show that the global weight loss curves obtained with the standard gravimetric method and with the MRI data are in excellent agreement. This work indicates that monitoring changes in the T2 profile of food (i.e., pumpkin) is a useful method for evaluating moisture profiles and changes induced on the sample during the drying process.

Confined Brownian Motion Tracked With Motion Blur: Estimating Diffusion Coefficient and Size of Confining Space

Mesoscopic environments and particles diffusing in them are often studied by tracking such particles individually while their Brownian motion explores their environment. Environments may be, e.g., a domain in a cell membrane, an interior compartment of a cell, or an engineered nanopit. Particle trajectories are typically determined from time-lapse recorded movies. These are recorded with sufficient exposure time per frame to be able to detect and localize particles in each frame. Since particles move during this exposure time, particles image with motion blur. This motion blur can compromise estimates of diffusion coefficients and the size of the confining domain if not accounted for correctly. We do that here. We give explicit and exact expressions for the variance of measured positions and the mean-squared displacement of a Brownian particle confined in, respectively, a 1D box, a 2D box, a 2D circular disc, and a 3D sphere. Our expressions are valid for all exposure times, irrespective of the size of the confining space and the value of the diffusion coefficient. They apply also in the common case where the exposure time is smaller than the time-lapse due, e.g., to “dead time” caused by the readout process in the camera. These expressions permit determination of diffusion coefficients and domain sizes for given movies for the simple geometries we consider. More important, the trends observed in our exact results when parameter values are varied are valid also for more complex geometries for which no exact analytical solutions exist. Wherever the underlying physics is the same, the exact quantitative description of its consequences provided here is portable as a qualitative and semi-quantitative understanding of its consequences in general. The results may also be useful for other types of reflected Brownian motion than those occurring in single-particle tracking, e.g., in nuclear magnetic resonance imaging techniques. For use in that particular context, we briefly discuss the effects of confinement on anisotropic Brownian motion imaged with motion blur.

Potential use of low-field nuclear magnetic resonance to determine the drying characteristics and quality of Arctium lappa L. in hot-blast air

The present study investigated the effect of drying temperature (50, 60, 70, and 80 °C) on the drying kinetics and water properties of burdock slices. The changes in water mobility and water distribution were characterized using 1H low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques. The Page model satisfactorily described the hot-air drying curves of burdock. The drying kinetic curves showed that the second stage of drying corresponded exactly with when the relaxation times of free and immobile water decreased to the equilibrium points. MRI showed that water diffused from the center to the surface during drying. Overall, the LF-NMR, color and HPLC data showed that, at 60 °C, free water in burdock dissipated slowly, its color stability was higher, and its chlorogenic acid content was higher than at 70 and 80 °C. Significant correlations were found between moisture content, color and NMR parameters.

Effect of Different Cooking Methods on Proton Dynamics and Physicochemical Attributes in Spanish Mackerel Assessed by Low-Field NMR.

The states of protons within food items are highly related to their physical attributes. In this study, the effect of cooking methods including boiling, steaming, roasting and frying on proton dynamics, physicochemical parameters and microstructure of Spanish mackerel was assessed by low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques. The treatment of cooking resulted in a significant reduction of proton mobility and declined freedom of protons. The state changes of protons can be monitored easily in an intuitive and non-destructive manner during various cooking process. The treatments of boiling, steaming, roasting and frying resulted in different cooking loss and similar water-holding capability. A significant increase of total carbonyl content and thiobarbituric acid reactive substances was found, while a decrease of the values for free thiols and surface hydrophobicity was observed. The analysis of circular dichroism spectroscopy and cryo-scanning electron microscopy showed significant structural change. The correlation coefficients of Rcal2 and Rcv2 from partial least squares (PLS) regression models were more than 0.980, suggesting good correlation between LF-NMR data and hardness, resilience, springiness, chewiness, gumminess, and adhesiveness. Good recoveries and a relatively small coefficient of variation (CV) were obtained from the PLS regression models, indicating good reliability and accuracy in predicting texture parameters for mackerel samples.

Rheo-NMR in food science-Recent opportunities.

For over 25years, nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques have been used to study materials under mechanical deformation. Collectively, these methods are referred to as Rheo-NMR. In many cases, it provides spatially and temporally resolved maps of NMR spectra, intrinsic NMR parameters (such as relaxation times), or motion (such as diffusion or flow). Therefore, Rheo-NMR is complementary to conventional rheological measurements. This review will briefly summarize current capabilities and limitations of Rheo-NMR in the context of material science and food science in particular. It will report on recent advances such as the incorporation of torque sensors or the implementation of large amplitude oscillatory shear and point out future opportunities for Rheo-NMR in food science.

Water status and distribution in shiitake mushroom and the effects of drying on water dynamics assessed by LF-NMR and MRI

Water status and distribution in shiitake mushroom and the effects of drying temperature on water migration were investigated by low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) technique. Three water components assigned to bound, immobilized water and free water were observed in fresh shiitake mushroom by LF-NMR, and higher moisture content was found in the lamella and outer layer of stalk by MRI. During drying, the transverse relaxation time and peak area of immobilized and free water in pileus significantly decreased. Higher drying temperature could promote the changes in water mobility and population. MRI results revealed the water evaporated from external surface of shiitake mushroom, then the water in inner region migrated to the surface during the drying. The drying properties, color and shrinkage of shiitake mushroom were also measured. The strong correlation between moisture content and total peak area of water with a coefficient of 0.9792 revealed the potential of LF-NMR as a rapid and nondestructive method to monitor drying degree of shiitake mushroom.

Simulation and visualization experiment of manganese ion diffusion and damage to gel in a porous media-gel system

Abstract A new visualization method for studying the damage to gel structure caused by high salinity ions is explored by using the characteristics of suppression image signal of Mn2+ and nuclear magnetic resonance (NMR) imaging technique. The diffusion and distribution characteristics of Mn2+ in porous media-gel system were studied based on manganese chloride static diffusion and gel flooding experiments, and the gel’s nuclear magnetic image and displacement pressure were tested. The results show that the diffusion of Mn2+ conforms to the Fick diffusion law in porous media-gel system, and the diffusion speed of Mn2+ increases and the area of gel image decreases gradually with the increase of concentration, and the image of gel decreases faster and the pressure drop of water drive is larger in flooding experiment of manganese chloride with higher concentration. Reaction-diffusion model with the reaction of Mn2+ with gel was established to study the concentration distribution characteristics of Mn2+. The model is validated by comparing the results with magnetic resonance imaging (MRI) experiments and the diffusion coefficient of Mn2+ equals 1.6 mm2/h, and the minimum concentration of Mn2+ to impact gel NMR image signals is 2.5 g/L. The above results show that the diffusion of Mn2+ into the gel in the rock core inhibits the imaging signal of the gel and damages its strength, and the greater the concentration is, the greater the influence. Increase of adsorption amount of gel and reaction rate, reduction of diffusion time, and addition of ion adsorption isolator all can reduce the impact of Mn2+ on the gel.

Influence of Freezing–Thawing Cycle on Water Dynamics of Turbot Flesh Assessed by Low-Field Nuclear Magnetic Resonance and Magnetic Resonance Imaging

Abstract Turbot is a valuable commercial species due to its high nutrient content. Moisture is an important indicator of meat spoilage. This study elucidated distinctive water dynamics in turbot flesh in the freezing–thawing process by nondestructive low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques. T 2 relaxation spectra were utilized to describe the mobility and content of different types of water. Principal component analysis (PCA) revealed a clear discrimination of various freezing–thawing cycles. T 1- and T 2-weighted MRI provided further visualization of internal information for turbot flesh. Microscopic examination clearly identified protein denaturation and structural shrinkage. Furthermore, NMR parameters and conventional physicochemical parameters of color, shear force and thiobarbituric acid-reactive substances showed good correlations. To sum up, the study revealed that LF-NMR and MRI are promising techniques to portray the relationship between the water dynamics and changes of turbot quality properties during the freezing–thawing process.

Water Dynamics and Physicochemical Analysis of Two Different Varieties of Apple Jam (Fuji) and (Yinduqing) by LF- NMR and MRI

Abstract Apple jam is one of the favorite foods consumed worldwide. This study investigated the effect of storage time and sugar concentration on water dynamics in Fuji and Yinduqing, using low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) techniques. The samples were taken for jam preparation with various concentrations of sugar solution (0, 25, 50 and 75-oBrix) and stored at a room temperature of 20 °C for 16 days. The LF-NMR relaxation time T 2 distribution displayed bound, intermediate and free water, respectively. The lowest T 2 range reflected lowest sugar hydrogen mobility depending on total soluble solids (TSS) and water content. A significant correlation (r > 0.9) existed between LF-NMR analysis and physicochemical parameters (water content, color, pH and TSS). MRI displayed uniform water distributions in samples with low sugar concentration. Fluorescence microscopy was used to evaluate sugar crystals and microstructural changes during processing. The study demonstrated the viability of LF-NMR method in determining water dynamics in the apple jams upon processing and storage that may be implemented for quality analysis in other jam products.

Mechanical properties, corrosion performance and cell viability studies on newly developed porous Fe-Mn-Si-Pd alloys

Porous Fe-30Mn6Si1Pd (wt.%) alloys were prepared by a simple press and sinter process from ball-milled Fe, Mn, Si and Pd powders blended with 10 wt%, 20 wt% and 40 wt% NaCl to obtain different degrees of porosity. For comparison purposes, a bulk fully-compact Fe-30Mn6Si1Pd alloy was produced by arc-melting and subsequent copper-mold suction-casting. While the porous Fe-30Mn6Si1Pd alloys only consist of γ-austenite, their fully-compact counterpart comprises ε-martensite and γ-austenite phases. In all cases, the low magnetic susceptibility response assures good compatibility with nuclear magnetic resonance and magnetic resonance imaging techniques. Furthermore, a reduction of the Young's modulus, from 55 to 7 GPa, was attained by introducing porosity. The biodegradation performance was evaluated by static immersion and electrochemical corrosion tests in Hank's solution. The influence of immersion time on composition, microstructure, mechanical and magnetic properties was assessed. While introducing porosity renders alloys with suitable mechanical and magnetic properties, it also has a detrimental effect in terms of cell viability. Hence, the porosity level needs to be controlled in order to obtain alloys with an optimized performance.∖

Water dynamics of turbot flesh during frying, boiling, and stewing processes and its relationship with color and texture properties: Low-field NMR and MRI studies

Water dynamics in turbot flesh during frying, boiling and stewing was assessed by nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Protons associated with different types of water were distinguished by T2 relaxation spectra, and significant differences of water mobility were detected. Principle component analysis revealed a clear discrimination between samples of different cooking methods. MRI provided further visualization of internal information for turbot flesh during cooking. The linear regression analysis demonstrated a strong correlation between the NMR parameter A23 and b* value (R2 = .972) during frying process, A23 and L* (R2 = .963), T22 and b* (R2 = .991) during boiling process, T22 and b* (R2 = .883) during stewing process. Moreover, a good correlation was found between A21 and cohesiveness (R2 = .924) during the frying process, A23 and resilience (R2 = .879) during boiling process, as well as between A23 and chewiness (R2 = .946) during stewing process. Practical applications Water mobility and distribution can significantly affect turbot flesh physical and biochemical status leading to change the texture, color, tenderness and taste during cooking process. Therefore, it is very important to develop a fast, nondestructive method to monitor the water dynamics, reduce the cooking time, save energy and visualize the structure change of turbot flesh during cooking. In this study, a rapid and nondestructive NMR and MRI method were developed to analyze the water dynamics of turbot flesh during frying, boiling and stewing for the first time. The water states, distribution and mobility were analyzed by the NMR and MRI. Different cooking methods displayed different water dynamics in turbot flesh, which could be identified when NMR combined with chemometrics principal component analysis. The facile method by using both NMR and MRI for analysis of turbot flesh may have great potential for rapid and nondestructive analysis of other food processing.

Dynamics of water mobility and distribution in Sur clam (Mactra chinensis) during dehydration and rehydration processes assessed by low-field NMR and MRI

Drying method effects on water dynamics in Sur clam during dehydration and rehydration were studied using the non-destructive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Three water populations were observed in Sur clam samples with different migration behavior when they were dried by hot air at 60 °C for 8 h versus sun-dried for 53 h. Good correlation between the A2 parameter of NMR and moisture ratio MR was found, and the Page model exhibited the maximum coefficient R2 value with the minimum root mean squared error. Notably, the NMR and MRI characterization showed that the water release of hot-air dried clams was different from that of the sun-dried samples, and the principal component analysis (PCA) of NMR relaxation data offered a stable classification for the hot-air dried and sun-dried clams. Similarly, the rehydration of hot-air dried and sun-dried clams was also assessed by NMR and MRI revealing that the absorbed water was mainly the immobilized and free water. The proper rehydration time was 180 and 120 min, respectively, for the hot-air and sun dried clams. The rehydrated clams could be distinguished from the boiled samples before dehydration process through the PCA of NMR relaxation data, but undistinguishable between the rehydrated hot-air dried and sun-dried clams. These results demonstrated that NMR and MRI are effective methods for non-destructive analysis of Sur clams during dehydration and rehydration processes.

A Method to Analyze the Protein Denaturation of Whole Quail Egg Based on in situ NMR and MRI

Abstract The paper aims to study the change of protein denaturation in whole quail egg during heating using in situ nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. The NMR relaxometry demonstrated that the protein denaturation occurred within 64–66 °C. The quail eggs after 8 min heating at 65 °C were successfully recognized by NMR combined with principal component analysis due to protein denaturation. The protein denaturation dynamics study revealed that the protein denaturation occurred at 8, 6, 4, 2 min for the quail eggs heated at 65, 70, 75, 100 °C, respectively. In addition, the protein denaturation of quail egg was also detected by the T 1 weighted MRI imaging, confirming that the dramatic changes occurred at 8 min when the eggs were heated at 65 °C. All these results showed that the NMR with MRI provided an effective way to assess the protein denaturation of quail eggs.

Cortical Brain Functions – The Brodmann Legacy in the 21st Century

AbstractIn 1909, Korbinian Brodmann described 52 functional brain areas, 43 of them found in the human brain. More than a century later, his devoted functional map was incremented by Glasser et al in 2016, using functional nuclear magnetic resonance imaging techniques to propose the existence of 180 functional areas in each hemisphere, based on their cortical thickness, degree of myelination (cortical myelin content), neuronal interconnection, topographic organization, multitask answers, and assessment in their resting state. This opens a huge possibility, through functional neuroanatomy, to understand a little more about normal brain function and its functional impairment in the presence of a disease.

Microemulsions of Record Low Amphiphile Concentrations Are Affected by the Ambient Gravitational Field.

It is shown that the ternary system heavy water-heptane-hexadecyl hexaethylene oxide (C16E6) has a stable bicontinuous microemulsion phase down to an exceptionally low concentration at the balanced temperature of 26.8 °C. It is further demonstrated that the ambient gravitational field has an influence on the observed phase equilibria for typical sample sizes (∼1 cm). Direct measurements using a nuclear magnetic resonance imaging technique demonstrate that sample compositions vary with the height in the vials. It is furthermore found that some samples show four phases at equilibrium in apparent violation of Gibbs' phase rule. It is pointed out that Gibbs' phase rule strictly applies only when effects of gravity are negligible. A further consequence of the ambient gravitational field is that, for the system studied, the microemulsion one-phase samples are not observed, when using standard size vials, that is, sample heights on the order of a centimeter. Quantitative determinations of concentration profiles can be used to determine parameters of the free-energy density for the system.

(Invited) Limitations in Electrochemically Active Biofilms

Almost a decade has passed since the rediscovery of microbial fuel cell research, yet the power generation of these devices has not advanced. This is mainly due to the research being focused solely on the improvement of power generation rather than on a fundamental understanding of electron transfer processes and their limitations in the biofilms grown on the anode and cathode. The chemical and electrochemical gradients in these biofilms play a critical role in electron transfer rates between cells and a solid electron acceptor or donor. Our research group has developed and used novel microelectrodes and in situ Nuclear Magnetic Resonance (NMR) imaging techniques to investigate electron transfer mechanisms in biofilms. The newly developed microelectrodes were successfully operated on polarized surfaces. Both NMR and microelectrode technologies allowed us to quantify metabolic and chemical variations in biofilms while the cells respired on polarized electrode surfaces. We combined our techniques with two new techniques – rotating disk electrode (RDE) and electrochemical quartz crystal balance (eQCM). The addition of both these techniques allowed us to differentiate mass transfer limitations from intra-biofilm electron transfer processes. When we attempted to identify limitations in anodic biofilms, first, we addressed the importance of local acetate concentrations within G. sulfurreducens biofilms using a novel acetate microelectrode which has better resolution than NMR. The microelectrode work confirmed our previous NMR findings and generated profiles on the order of µM. We should note that the microelectrode method developed is significantly more economical to use than NMR and can facilitate rapid in situ measurements of acetate in anoxic biofilms. Second, we integrated our previous RDE method with our eQCM methods to show that, on both a per current basis and a per mass basis, biofilm capacitance is a better indicator of biofilm performance than conductance.In particular, we compared the capacitance/conductance relationship of G. sulfurreducens biofilms to those of both polyaniline films and diffusing flavin systems. The results indicated that the tandem increase in capacitance and conductance is unique to the extracellular electron transfer mechanisms operating in G. sulfurreducens biofilms. Our local potential and current measurements indicated that the upper layers of the biofilms are always under a reducing condition. This knowledge also confirmed that the amount of cytochromes in the biofilm is not high enough or the biofilm conductivity is not sufficient to generate an oxidizing condition near the top of a biofilm. Finally, we extended the role of excess surface area in the scale-up of bioelectrochemical systems by examining biomass accumulation under this condition. We found that biomass continues to accumulate on the excess surface area electrodes with net arithmetic growth rates and acetate consumption during steady-state current. The linear growth rates constrained by ion transport limitations that we found may be important factors in understanding the current generation using high-surface-area 3D electrodes with advection. In conclusion, we demonstrated the importance of the metabolic inactivity of layers within G. sulfurreducens biofilms using microelectrodes and NMR. In addition, we verified these findings using electrochemical impedance spectroscopy (EIS) by demonstrating the pseudocapacitive behavior of biofilm impedance. We also demonstrated the feasibility of directly measuring biofilm inefficiencies via intra-biofilm electron transfer rates and local current depth profiles.