ABSTRACT
This study reports the development of a textile-based colaminar flow hybrid microbial-enzymatic biofuel cell. Shewanella MR-1 was used as a biocatalyst on the anode, and bienzymatic system catalysts based on glucose oxidase and horseradish peroxidase were applied on an air-breathing cathode to address the overpotential loss in a body-friendly way. A single-layer Y-shaped channel configuration with a double-inlet was adopted. Microchannels of biofuel cells were patterned by silk screen printing with Ecoflex to maintain the flexibility of textile substrates without harm to the human body. The electrodes were fabricated with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate and a mixture of multiwalled carbon nanotubes and single-walled carbon nanotubes by screen printing. The effects of electrode materials, catalyst type, catalyst concentration, and glucose concentration in the catholyte were investigated to optimize the fuel cell performance. The peak power density (44.9 µW cm-2) and maximum current density (388.9 µA cm-2) of the optimized hybrid biofuel cell were better than those of previously reported textile- or paper-substrate microscale single microbial fuel cells. The developed biofuel cell will be a useful platform as a microscale power source that is harmless to the environment and living organisms.
ABSTRACT
Bilirubin oxidases (BODs) [EC 1.3.3.5 - bilirubin: oxygen oxido-reductase] are enzymes that belong to the multicopper oxidase family and can oxidize bilirubin, diphenols, and aryl amines and reduce the oxygen by direct four-electron transfer from the electrode with almost no electrochemical overpotential. Therefore, BOD is a promising bioelectrocatalyst for (self-powered) biosensors and/or enzymatic fuel cells. The advantages of electrochemically active BOD enzymes include selective biosensing, biocatalysis for efficient energy conversion, and electrosynthesis. Owing to the rise in publications and patents, as well as the expanding interest in BODs for a range of physiological conditions, this Review analyzes scientific literature reports on BOD enzymes and current hypotheses on their bioelectrocatalysis. This Review evaluates the specific research outcomes of the BOD in enzyme (protein) engineering, immobilization strategies, and challenges along with their bioelectrochemical properties, limitations, and applications in the fields of (i) biosensors, (ii) self-powered biosensors, and (iii) biofuel cells for powering bioelectronics.
Subject(s)
Biosensing Techniques , Enzymes, Immobilized , Enzymes, Immobilized/chemistry , Oxidoreductases , Oxygen , BilirubinABSTRACT
Microbial electrolysis cells (MECs) are increasingly recognized as a promising technology for converting CO2 to CH4, offering the dual benefits of energy recovery from organic wastewater and CO2 emission reduction. A critical aspect of this technology is the enhancement of the electron-accepting capacity of the methanogenic biocathode to improve CH4 production efficiency. This study demonstrates that adjusting the cathode resistivity is an effective way to control the electric field intensity, thereby enhancing the electron accepting capacity and CH4 production. By maintaining the electric field intensity within approximately 8.50-10.83 mV·cm-1, the CH4 yield was observed to increase by up to two-fold. The improvement in CH4 production under optimized electric field conditions was attributed to the enhancement of the direct accepting capacity of the biocathode. This enhancement was primarily due to an increase in the relative abundance of Methanosaeta by approximately 10 % and an up to 83.78 % rise in the electron-accepting capacity of the extracellular polymeric substance. These insights offer a new perspective on the operation of methanogenic biocathodes and propose a novel biocathode construction methodology based on these findings, thus contributing to the enhancement of MEC efficiency.
Subject(s)
Bioelectric Energy Sources , Carbon , Carbon Dioxide , Extracellular Polymeric Substance Matrix , Electrolysis , Electrodes , MethaneABSTRACT
Granular activated carbon (GAC) has been widely used at the anode of a microbial fuel cell (MFC) to enhance anode performance due to its outstanding capacitance property. To the best of our knowledge, there haven't been any studies on GAC in the cathode for biofilm development and nitrate reduction in MFC. In this study, by adding GAC to biocathode, we investigated the impact of different GAC amounts and stirring speeds on power generation and nitrate reduction rate in MFC. The denitrification rate was found to be nearly two-times higher in MFCs with GAC (0.046 ± 0.0016 kg m-3 d-1) compared to that deprived of GAC (0.024 ± 0.0012 kg m-3 d-1). The electrotrophic denitrification has produced a maximum power density of 37.6 ± 4.8 mW m-2, which was further increased to 79.2 ± 7.4 mW m-2 with the amount of GAC in the biocathode. A comparative study performed with chemical catalyst (Pt carbon with air sparging) cathode and GAC biocathode showed that power densities produced with GAC biocathode were close to that with Pt cathode. Cyclic voltammetry analysis conducted at 10 mV s-1 between -0.9 V and +0.3 V (vs. Ag/AgCl) showed consistent reduction peaks at -0.6V (Ag/AgCl) confirming the reduction reaction in the biocathode. This demonstrates that the GAC biocathode used in this research is effective at producing power density and denitrification in MFC. Our belief that the nitrate reduction was caused by the GAC biocathode in MFC was further strengthened when SEM analysis showing bacterial aggregation and biofilm formation on the surface of GAC. The GAC biocathode system described in this research may be an excellent substitute for MFC's dual functions of current generation and nitrate reduction.
Subject(s)
Bioelectric Energy Sources , Nitrates/chemistry , Charcoal , Denitrification , Organic Chemicals , ElectrodesABSTRACT
Electroautotrophic microbes at biocathodes in microbial electrolysis cells (MECs) can catalyze the hydrogen evolution reaction with low energy demand, facilitating long-term stable performance through specific and renewable biocatalysts. However, MECs have not yet reached commercialization due to a lack of understanding of the optimal microbial strains and reactor configurations for achieving high performance. Here, we critically analyze the criteria for the inocula selection, with a focus on the effect of hydrogenase activity and microbe-electrode interactions. We also evaluate the impact of the reactor design and key parameters, such as membrane type, composition, and electrode surface area on internal resistance, mass transport, and pH imbalances within MECs. This analysis paves the way for advancements that could propel biocathode-assisted MECs toward scalable hydrogen gas production.
Subject(s)
Bioelectric Energy Sources , Electrodes , Electrolysis , Hydrogen , Hydrogen/metabolism , Bioelectric Energy Sources/microbiology , Hydrogenase/metabolism , Hydrogenase/genetics , Bacteria/metabolism , Bacteria/geneticsABSTRACT
In this study, nickel-cobalt co-modified stainless steel mesh (Ni-Co@SSM) was prepared and used as the biocathode in microbial electrolysis cell (MEC) for sulfamethazine (SMT) degradation. The optimal electrochemical performance of the Ni-Co@SSM was obtained at the electrodeposition time of 600 s, electrodeposition current density of 20 mA cm-2, and nickel-cobalt molar ratio of 1:2. The removal of SMT in MEC with the Ni-Co@SSM biocathode (MEC-Ni-Co@SSM) was 82%, which increased by 30% compared with the conventional anaerobic reactor. Thirteen intermediates were identified and the potential degradation pathways of SMT were proposed. Proteobacteria, Firmicutes, Patescibacteria, Chloroflexi, Bacteroidetes, and Euryarchaeota are the dominant bacteria at the phylum level in the MEC-Ni-Co@SSM, which are responsible for SMT metabolism. Due to the electrical stimulation, there was an increase in the abundance of the metabolic function and the genetic information processing. This work provides valuable insight into utilizing MECs for effective treatment of antibiotic-containing wastewater.
Subject(s)
Nickel , Sulfamethazine , Nickel/analysis , Sulfamethazine/metabolism , Electrodes , Electrolysis , Wastewater , Bacteria/metabolismABSTRACT
The role of metal organic framework (MOF) modified cathode in promoting long chain fatty acid (LCFA) methanation was identified in microbial electrolysis cell coupled anaerobic digestion (MEC-AD) system. The maximum methane production rate of MEC-AD-MOF achieved 49.8 ± 3.4 mL/d, which increased by 41 % compared to MEC-AD-C. The analysis of bio-cathode biofilm revealed that microbial activity, distribution, population, and protein secretion prompted by MOF cathode, which in turn led to an acceleration of electron transfer between the cathode and microbes. Specifically, the relative abundance of acetate-oxidizing bacterium (Mesotoga) in MEC-AD-MOF was 1.5-3.6 times higher than that in MEC-AD-C, with a co-metabolized enrichment of Methanobacterium. Moreover, MOF cathode reinforced LCFA methanation by raising the relative abundance of genes coded key enzymes involved in CO2-reducing pathway, and elevating the tolerance of microbes to LCFA inhibition. These results indicate that MOF can enhance biofilm construction in MEC-AD, thereby improving the treatment performance of lipid wastewater.
Subject(s)
Metal-Organic Frameworks , Anaerobiosis , Bioreactors , Methane , Fatty Acids , Electron Transport , Electrolysis , ElectrodesABSTRACT
The performance of biocathode in an enzymatic biofuel cell (EBFC) in the real application is somehow overlooked. Herein, a wearable and flexible lactic-acid/O2 EBFC enhanced with an air-breathing biocathode is designed to solve the limitation of biocathode that arises from the low solubility and slow mass transfer of the dissolved oxygen. To improve the oxygen supply efficiency for the air-breathing biocathode, a superhydrophobic base electrode creating an efficient air-solid-liquid triphase interface is developed. The designed EBFC with an 'island-bridge' configuration is integrated by assembling the current collectors of air-breathing biocathode and bioanode on a commercial laminating film (LF) screen-printed with a noninterfering circuit. It is found that the biocathode/bioanode area ratio should exceed 9:1 so that the designed EBFC (1A//9C) can achieve the optimal performance. This EBFC delivers an open circuit voltage of ca. 0.75 V and outputs a maximum power density of ca. 1.78 mW cm-2. In addition, a scaled-up EBFC (total bioanode area: 1.5 cm2) successfully powers a self-developed low-power device of heartrate in the pulse operation mode when applied on a volunteer's arm.
Subject(s)
Bioelectric Energy Sources , Biosensing Techniques , Wearable Electronic Devices , Humans , Oxygen/chemistry , Electrodes , Glucose/chemistry , Enzymes, Immobilized/chemistryABSTRACT
This study focuses on the development of an air-lift bio-electrochemical reactor (ALBER) with a continuous feeding regime. The objective is to enhance nitrogen removal from synthetic wastewater with a low carbon-to-nitrogen (C/N) ratio. The chemical oxygen demand (COD) and total nitrogen (TN) of the influent wastewater were 500 and 200 mg/L, respectively. The effect of four independent variables, i.e., temperature, hydraulic retention time (HRT), N-NH4+/TN ratio and current density in the range of 16-32 °C, 6-12 h, 25-75%, and 2-10 A/m2, respectively, at three levels on the bio-electrochemical reactor performance were investigated during the bio-electrochemical reactor operation. The Face Center Cube (FCC) of response surface methodology (RSM) was used for design of experiments and model of obtained data. The ALBER achieved the maximum TN removal of 73% (146 mg/l) using external voltage and zeolite/plastic medium at temperature of 16 °C, HRT of 6 h, current density of 2 A/m2 and N-NH4+/TN ratio of 75%. The results indicated that shortening the HRT from 12 to 6 h, reducing the temperature from 32 °C to 24 °C, increasing the current density from 2 to 6 A/m2 and the reduction of nitrate concentration caused an increase in the TN removal. The results indicated that the performance of air-lift bio-electrochemical for nitrogen removal could be attributed to autotrophic denitrification (AD) and simultaneous nitrification/denitrification (SND). The research findings suggest that the ALBER should be further studied for potential use in treating industrial wastewater at low temperatures.
Subject(s)
Denitrification , Wastewater , Nitrogen/analysis , Carbon , Bioreactors , Nitrification , Waste Disposal, Fluid/methodsABSTRACT
A subset of microorganisms that perform respiration can endogenously utilize insoluble electron donors, such as Fe(II) or a cathode, in a process called extracellular electron transfer (EET). However, it is unknown whether similar endogenous EET can be performed by primarily fermentative species like lactic acid bacteria. We report for the first time electron uptake from a cathode by Lactiplantibacillus plantarum, a primarily fermentative bacteria found in the gut of mammals and in fermented foods. L. plantarum consumed electrons from a cathode and coupled this oxidation to the reduction of both an endogenous organic (pyruvate) and an exogenous inorganic electron acceptor (nitrate). This electron uptake from a cathode reroutes glucose fermentation toward lactate degradation and provides cells with a higher viability upon sugar exhaustion. Moreover, the associated genes and cofactors indicate that this activity is mechanistically different from that one employed by lactic acid bacteria to reduce an anode and to perform respiration. Our results expand our knowledge of the diversity of electroactive species and of the metabolic and bioenergetic strategies used by lactic acid bacteria.
ABSTRACT
ABSTRACTInaccessibility and expensiveness of vital infrastructures are the main problems in some urban and rural areas to supply fresh water, sustainable energy, and wastewater treatment. An effective solution is the integration of several systems in an environmentally friendly technology of the photosynthetic microbial desalination cell (PMDC). The aim of this study is to assess the process characterisation of an algae-based PMDC, which was loaded with a high-strength mixture of human feces and urine (HFS). The PMDC was also able to efficiently remove COD and total nitrogen of HFS by 50% and 94%, respectively. The maximum power density, voltage, and desalination efficiency of 362.5â mW/m², 175.2â mV, and 60% were accomplished. Adequate parameter adjustment led to a remarkable maximum of 2.25â g/L.d in the ion removal rate. In addition, an energy balance was governed showing that zero or positive net energy in PMDC is feasible by replacing the main energy consumers. Based on the results, this type of MDC had a high efficiency for simultaneous saline water desalination and HFS treatment, which makes it attractive for further studies of upscaling and its application in remote areas.
ABSTRACT
Various types of electroactive microorganisms can be enriched to form biocathodes that reduce charge-transfer resistance, thereby accelerating electron transfer to heavy metal ions with high redox potentials in microbial fuel cells. Microorganisms acting as biocatalysts on a biocathode can reduce the energy required for heavy metal reduction, thereby enabling the biocathode to achieve a lower reduction onset potential. Thus, when such heavy metals replace oxygen as the electron acceptor, the valence state and morphology of the heavy metals change under the reduction effect of the biocathode, realizing the high-efficiency treatment of heavy metal wastewater. This study reviews the mechanisms, primary influencing factors (e.g., electrode material, initial concentration of heavy metals, pH, and electrode potential), and characteristics of the microbial community of biocathodes and discusses the electron distribution and competition between microbial electrodes and heavy metals (electron acceptors) in biocathodes. Biocathodes reduce the electrochemical overpotential in heavy metal reduction, permitting more electrons to be used. Our study will advance the scientific understanding of the electron transport mechanism of biocathodes and provide theoretical support for the use of biocathodes to purify heavy metal wastewater.
ABSTRACT
Bioelectrochemical system is considered as a promising approach for enhanced bio-dechlorination. However, the mechanism of extracellular electron transfer in the dechlorinating consortium is still a controversial issue. In this study, bioelectrochemical systems were established with cathode potential settings at -0.30 V (vs. SHE) for trichloroethylene reduction. The average dechlorination rate (102.0 µM Cl·d-1) of biocathode was 1.36 times higher than that of open circuit (74.7 µM Cl·d-1). Electrochemical characterization via cyclic voltammetry illustrated that electrostimulation promoted electrochemical activity for redox reactions. Moreover, bacterial community structure analyses indicated electrical stimulation facilitated the enrichment of electroactive and dechlorinating populations on cathode. Metagenomic and quantitative polymerase chain reaction (qPCR) analyses revealed that direct electron transfer (via electrically conductive pili, multi-heme c-type cytochromes) between Axonexus and Desulfovibrio/cathode and indirect electron transfer (via riboflavin) for Dehalococcoides enhanced dechlorination process in BES. Overall, this study verifies the effectiveness of electrostimulated bio-dechlorination and provides novel insights into the mechanisms of dechlorination process enhancement in bioelectrochemical systems through electron transfer networks.
Subject(s)
Electrons , Trichloroethylene , Oxidation-Reduction , Electricity , Electrodes , Trichloroethylene/chemistry , Biodegradation, EnvironmentalABSTRACT
A functional bipolar electrode-electrochemiluminescence (BPE-ECL) platform based on biocathode reducing oxygen was constructed for detecting electrochemically active bacteria (EAB) in this paper. Firstly, thiolated trimethylated chitosan quaternary ammonium salt (TMC-SH) layer was assembled on the gold-plated cathode of BPE. TMC-SH contains quaternary ammonium salt branch chain, which can inhibit the growth of microorganisms on the surface or in the surrounding environment while absorbing bacteria. Then, the peristaltic pump was used to flow all of the samples through the cathode, and the EAB was electrostatically adsorbed on the electrode surface. Finally, applying a constant potential to the BPE, bacteria can catalyze electrochemical reduction of O2, and decrease the overpotential of O2 reduction at the cathode, which in turn generates an ECL reporting intensity change at the anode. In this way, live and dead bacteria can be distinguished, and the influence of complex food substrates on detection can be greatly reduced.
Subject(s)
Ammonium Compounds , Biosensing Techniques , Luminescent Measurements , Electrochemical Techniques , ElectrodesABSTRACT
Perchlorate has been described as an emerging pollutant that compromises water sources and human health. In this study, a new electrotrophic perchlorate reducing microorganism (EPRM) isolated from the Atacama Desert, Dechloromonas sp. CS-1, was evaluated for perchlorate removal in water in a bioelectrochemical reactor (BER) with a chemically modified electrode. BERs were operated for 17 days under batch mode conditions with an applied potential of -500 mV vs. Ag/AgCl. Surface analysis (i.e., SEM, XPS, FT-IR, RAMAN spectroscopy) on the modified electrode demonstrated heterogeneous transformation of the carbon fibers with the incorporation of nitrogen functional groups and the oxidation of the carbonaceous material. The BERs with the modified electrode and the presence of the EAM reached high cathodic efficiency (90.79 ± 9.157%) and removal rate (0.34 ± 0.007 mol m-3-day) compared with both control conditions. The observed catalytic enhancement of CS-1 was confirmed by a reduction in the charge transfer resistance obtained by electrochemical impedance spectroscopy (EIS). Finally, an electrochemical kinetic study revealed an eight-electron perchlorate bioreduction reaction at -638.33 ± 24.132 mV vs. Ag/AgCl. Therefore, our results show the synergistic effect of EPRM and chemically modified electrodes on perchlorate removal in a BER.
Subject(s)
Nitrogen , Perchlorates , Humans , Spectroscopy, Fourier Transform Infrared , Nitrogen/metabolism , Electrodes , Oxidation-ReductionABSTRACT
The proliferation of emerging contaminants (ECs) in the environment poses a major threat to the safety of reuse water. However, many ECs exist for which no corresponding control standards have been established. Here, we used polarity reversal to construct a biocathode sensor capable of early warning of ECs biotoxicity in aerobic reuse water with low organic concentrations. The baseline current and sensitivity of the biosensor in response to formaldehyde were enhanced by 25% and 23% using microbial fuel cell effluent as the inoculum. The microbial community explained that the inoculum primarily influenced the performance of the biosensor by modulating species abundance, function and interactions. More importantly, the successfully commissioned biocathode sensor demonstrated rapid warning capability (Response time less than 1.3 h) for ECs such as fluoride, disinfection by-products and antibiotics in an actual landscape reuse system. Further, the sensor could quantify the concentration of a single known contaminant. Our study demonstrated a method for rapid early warning of ECs in an oxygen-rich, low-organics environment, promoting innovative development of monitoring technologies for water ecology and environmental safety.
Subject(s)
Bioelectric Energy Sources , Biosensing Techniques , Water Pollutants, Chemical , Water , Oxygen , Biosensing Techniques/methods , EcologyABSTRACT
A new extremophilic isolate (USS-CCA7) was obtained from an acidic environment (pH â¼ 3.2) in Antarctica phylogenetically related to Acidithiobacillus ferrivorans; its electrotrophic capacities were evaluated in a three-electrode electrochemical cell. Cyclic voltammetry showed cathodic peaks of -428 mV, -536 mV, and -634 mV (vs. Ag/AgCl; pH = 1.7; 3 M KCl) for nitrate, oxygen, and perchlorate, respectively. The catalytic role of this microorganism was also observed by a decrease in the charge transfer resistance registered via electrochemical impedance spectroscopy. Five-day chronoamperometry of culture at pH = 1.7, USS-CCA7 showed a perchlorate removal rate of 19.106 ± 1.689 mgL-1 day-1 and a cathodic efficiency of 112 ± 5.2 %. Growth on electrodes was observed by epifluorescence and scanning electron microscopy. Interestingly, the results showed that toward higher pH, the cathodic peak of perchlorate is reduced in the voltammetric profiles. This study highlights the use of this psychrotolerant acidophile for the bioremediation of harsh perchlorate-pressured terrestrial under acidic conditions.
Subject(s)
Drainage , Perchlorates , Antarctic Regions , Microscopy, Electron, Scanning , ElectrodesABSTRACT
Biofuel cells (BFCs) with enzymatic electrocatalysts have attracted significant attention, especially as power sources for wearable and implantable devices; however, the applications of BFCs are limited owing to the limited O2 supply. This can be addressed by using air-diffusion-type bilirubin oxidase (BOD) cathodes, and thus the further development of the hierarchical structure of porous electrodes with highly effective specific surface areas is critical. In this study, a porous layer of gold is deposited over magnesium-oxide-templated carbon (MgOC) to form BOD-based biocathodes for the oxygen reduction reaction (ORR). Porous gold structures are constructed via electrochemical deposition of gold via dynamic hydrogen bubble templating (DHBT). Hydrogen bubbles used as a template and controlled by the Coulomb number yield a porous gold structure during the electrochemical deposition process. The current density of the ORR catalyzed by BOD without a redox mediator on the gold-modified MgOC electrode was 1.3 times higher than that of the ORR on the MgOC electrode. Furthermore, the gold-deposited electrodes were modified with aromatic thiols containing negatively charged functional groups to improve the orientation of BOD on the electrode surface to facilitate efficient electron transfer at the heterogeneous surface, thereby achieving an ORR current of 12 mA cm-2 at pH 5 and 25 °C. These results suggest that DHBT is an efficient method for the fabrication of nanostructured electrodes that promote direct electron transfer with oxidoreductase enzymes.
Subject(s)
Bioelectric Energy Sources , Carbon , Carbon/chemistry , Gold/chemistry , Oxygen/chemistry , Electrodes , Hydrogen , Enzymes, Immobilized/chemistryABSTRACT
Microbial fuel cells (MFCs) have been considered a promising technology for Cr6+ removal, but they are limited by Cr6+-reducing biocathodes with low extracellular electron transfer (EET) and poor microbial activity. In this study, three kinds of nano-FeS hybridized electrode biofilms, obtained through synchronous biosynthesis (Sy-FeS), sequential biosynthesis (Se-FeS) and cathode biosynthesis (Ca-FeS), were applied as biocathodes for Cr6+ removal in MFCs. The Ca-FeS biocathode exhibited the best performance due to the superior properties of biogenic nano-FeS (e.g., more synthetic amount, smaller particle size, better dispersion). The MFC with the Ca-FeS biocathode achieved the highest power density (42.08 ± 1.42 mW/m2) and Cr6+ removal efficiency (99.18 ± 0.1 %), which were 1.42 and 2.08 times as high as those of the MFC with the normal biocathode, respectively. The synergistic effects of nano-FeS and microorganisms enhanced the bioelectrochemical reduction of Cr6+, first realizing deep reduction of Cr6+ to Cr0 in biocathode MFCs. This significantly alleviated the cathode passivation caused by Cr3+ deposition. In addition, the hybridized nano-FeS as "armor" layers protected the microbes from toxic attack by Cr6+, improving the biofilm physiological activity and extracellular polymeric substances (EPS) secretion. The hybridized nano-FeS as "electron bridges" facilitated the microbial community to form a balanced, stable and syntrophic ecological structure. This study proposes a novel strategy through the cathode in-situ biosynthesis of nanomaterials to fabricate hybridized electrode biofilms with enhanced EET and microbial activity for toxic pollutant treatment in bioelectrochemical systems.
Subject(s)
Bioelectric Energy Sources , Nanoparticles , Chromium/chemistry , ElectrodesABSTRACT
Bioelectrochemical systems which employ microbes as electrode catalysts to convert chemical energy into electrical energy (or conversely), have emerged in recent years for water sanitation and energy recovery. Microbial biocathodes, and especially those reducing nitrate are gaining more and more attention. The nitrate-reducing biocathodes can efficiently treat nitrate-polluted wastewater. However, they require specific conditions and they have not yet been applied on a large scale. In this review, the current knowledge on nitrate-reducing biocathodes will be summarized. The fundamentals of microbial biocathodes will be discussed, as well as the progress towards applications for nitrate reduction in the context of water treatment. Nitrate-reducing biocathodes will be compared with other nitrate-removal techniques and the challenges and opportunities of this approach will be identified.