Comparison study on enhancement of phosphorus recovery from low-strength wastewater treated with different magnesium-based electrochemical constructed wetland.

Phosphorus (P) recovery from wastewaters treated with constructed wetlands (CWs) could alleviate the current global P crisis but has not received sufficient attention. In this study, P transformation in different magnesium-based electrochemical CWs, including micro-electrolysis CW (M-CW), primary battery CW (P-CW), and electrolysis CW (E-CW), was thoroughly examined. The results revealed that the P removal efficiency was 53.0%, 75.8%, and 61.9% in the M-CW, E-CW, and P-CW, respectively. P mass balance analysis showed that P electrode deposition was the main reason for the higher P removal in the E-CW and P-CW. Significant differences were found between the E-CW and P-CW, P was distributed primarily on the magnesium plate in the P-CW but was distributed on the carbon plate in the E-CW. The E-CW had excellent P recovery capacity, and struvite was the major P recovery product. More intense magnesium plate corrosion and alkaline environment increased struvite precipitation in the E-CW, with the proportion of 61.6%. The results of functional microbial community analysis revealed that the abundance of electroactive bacteria was positively correlated with the deposition of struvite. This study provided an essential reference for the targeted electrochemical regulation of electric field processes and microorganisms in CWs to enhance P recovery.

Vanadium nitride decorated carbon cloth anode promotes aniline degradation and electricity generation of MFCs by efficiently enriching electroactive bacteria and promoting extracellular electron transfer.

To increase the colonization of electroactive bacteria and accelerate the rate of extracellular electron transfer, a simple coated anode of microbial fuel cell was designed. Here, we took advantage of vanadium nitride (VN) particles to modify the carbon cloth (VN@CC). Compared with bare carbon cloth, the designed VN@CC bioanodes exhibited a larger electrochemically active area, better biocompatibility, and smaller charge transfer impedance. The MFC with VN@CC bioanodes achieved the maximum power density of 3.89 W m-2 and chemical oxygen demand removal rate of 84% when 1000 mg L-1 aniline was degraded, which were about 1.88 and 2.8 times that of CC. The morphology of biofilm and 16s rRNA gene sequence analysis proved that the VN@CC bioanodes facilitated the enrichment of electroactive bacteria (99.02%) and increased the ratio of fast electron transfer in the extracellular electron transfer, thus enhancing the MFC performance of aniline degradation and power output. This work disclosed that it was feasible to increase the overall performance of MFC by enhancing the EET efficiency and presented valuable insights for future work.

Iron reducing sludge as a source of electroactive bacteria: assessing iron reduction in biofilm bacteria, planktonic cells and isolates from a microbial fuel cell.

In this study, bacteria from a microbial fuel cell (MFC) and isolates were evaluated on their Fe3+ reduction capability at different concentrations of iron using acetate as the sole source of carbon. The results demonstrated that the planktonic cells can reach an iron reduction up to 60% at 27 mmol Fe3+. Azospira oryzae (µ 0.89 ± 0.27 d-1) and Cupriavidus metallidurans CH34 (µ 2.34 ± 0.81 d-1) presented 55 and 62% of Fe3+ reduction, respectively, at 16 mmol l-1. Enterobacter bugandensis (µ 0.4 ± 0.01 d-1) 40% Fe3+ at 27 mmol l-1, Citrobacter freundii ATCC 8090 (µ 0.23 ± 0.05 d-1) and Citrobacter murliniae CDC2970-59 (µ 0.34 ± 0.02 d-1) reduced Fe3+ in ~ 50%, at 55 mmol l-1. This is the first report on these bacteria on a percentage of iron reduction. These results may be useful for anode design to contribute to a higher energy generation in MFCs.

Microbial Interactions in Electroactive Biofilms for Environmental Engineering Applications: A Role for Nonexoelectrogens.

Microbial electrochemical systems have gained much attention over the past decade due to their potential for various environmental engineering applications ranging from energy production to wastewater treatment to bioproduction. At the heart of these systems lie exoelectrogens-microorganisms capable of exporting electrons generated during metabolism to external electron acceptors such as electrodes. The bacterial biofilm communities on these electrodes are dominated by exoelectrogens but are nonetheless extremely diverse. So far, within the field, the main focus has been on the electroactive bacteria. However, to broaden our understanding of these communities, it is crucial to clarify how the remaining inhabitants of electrode-respiring biofilms contribute to the overall function of the biofilm. Ultimately, such insights may enable improvement of microbial electrochemical systems by reshaping the community structure with naturally occurring beneficial strains.

Dechlorination of dichloromethane by a biofilter enriched with electroactive bacteria: Performance, kinetics, and microbial community.

Dichloromethane (DCM) is a recalcitrant volatile organic compound that exhibits biological toxicity and bioaccumulation. In this study, gaseous DCM was removed using an electroactive bacterial biofilter (EBB) with graphite rod as the anode and carbon felt as the cathode. The highest removal efficiency (97.09%) was achieved at a cathodic potential of -600 mV (vs. Ag/AgCl). The EBB had a maximum elimination capacity of 79.29 g m-3 h-1 when the inlet load was 96.48 g m-3 h-1. There was no substrate inhibition phenomenon observed in the EBB, and the Michaelis-Menten model was used to describe the kinetics of the EBB. High-throughput sequencing indicated that electroactive genera such as Rhodanobacter sp., Sphingomonas sp., Pseudomonas sp., Chryseobacterium sp., Pseudochrobactrum sp., and Mycobacterium sp. dominated the EBB. The microbial communities were stable and were slightly affected by the DCM inlet concentration. The results can be applied for the effective treatment of recalcitrant volatile organic compounds (VOCs).

Effect of Contact Area and Shape of Anode Current Collectors on Bacterial Community Structure in Microbial Fuel Cells.

Low electrical conductivity of carbon materials is a source of potential loss for large carbonaceous electrode surfaces of MFCs due to the long distance traveled by electrons to the collector. In this paper, different configurations of titanium current collectors were used to connect large surfaces of carbon cloth anodes. The current collectors had different distances and contact areas to the anode. For the same anode surface (490 cm2), increasing the contact area from 28 cm2 to 70 cm2 enhanced power output from 58 mW·m-2 to 107 mW·m-2. For the same contact area (28 cm2), decreasing the maximal distance of current collectors to anodes from 16.5 cm to 7.75 cm slightly increased power output from 50 mW·m-2 to 58 mW·m-2. Molecular biology characterization (qPCR and 16S rRNA gene sequencing) of anodic bacterial communities indicated that the Geobacter number was not correlated with power. Moreover, Geobacter and Desulfuromonas abundance increased with the drop in potential on the anode and with the presence of fermentative microorganisms. Electrochemical impedance spectroscopy (EIS) showed that biofilm resistance decreased with the abundance of electroactive bacteria. All these results showed that the electrical gradient arising from collectors shapes microbial communities. Consequently, current collectors influence the performance of carbon-based anodes for full-scale MFC applications.

Energy Taxis toward Redox-Active Surfaces Decreases the Transport of Electroactive Bacteria in Saturated Porous Media.

The fate and transport of bacteria in porous media are essential for bioremediation and water quality control. However, the influence of biological activities like extracellular electron transfer (EET) and swimming motility toward granular media on cell transport remains unknown. Here, electroactive bacteria with higher Fe(III) reduction abilities were found to demonstrate greater retention in ferrihydrite-coated sand. Increasing the concentrations of the electron donor (1-10 mM lactate), shuttle (0-50 µM anthraquinone-2,6-disulfonate), and acceptor (ferrihydrite, MnO2, or biochar) under flow conditions significantly reduced Shewanella oneidensis MR-1's mobility through redox-active porous media. The deficiency of EET ability or flagellar motion and inhibition of intracellular proton motive force, all of which are essential for energy taxis, enhanced MR-1's transport. It was proposed that EET could facilitate MR-1 to sense, tactically move toward, and attach on redox-active media surface, eventually improving its retention. Positive linear correlations were established among parameters describing MR-1's energy taxis ability (relative taxis index), cell transport behavior (dispersion coefficient and relative change of effluent percentage), and redox activity of media surface (reduction potential or electron-accepting rate), providing novel insights into the critical impacts of bacterial microscale motility on macroscale cell transport through porous media.

Mixotrophic bacteria for environmental detoxification of contaminated waste and wastewater.

Mixotrophic bacteria provide a desirable alternative to the use of classical heterotrophic or chemolithoautotrophic bacteria in environmental technology, particularly under limiting nutrients conditions. Their bi-modal ability of adapting to inorganic or organic carbon feed and sulfur, nitrogen, or even heavy metal stress conditions are attractive features to achieve efficient bacterial activity and favorable operation conditions for the environmental detoxification or remediation of contaminated waste and wastewater. This review provides an overview on the state of the art and summarizes the metabolic traits of the most promising and emerging non-model mixotrophic bacteria for the environmental detoxification of contaminated wastewater and waste containing excess amounts of limiting nutrients. Although mixotrophic bacteria usually function with low organic carbon sources, the unusual capabilities of mixotrophic electroactive exoelectrogens and electrotrophs in bioelectrochemical systems and in microbial electrosynthesis for accelerating simultaneous metabolism of inorganic or organic C and N, S or heavy metals are reviewed. The identification of the mixotrophic properties of electroactive bacteria and their capability to drive mono- or bidirectional electron transfer processes are highly exciting and promising aspects. These aspects provide an appealing potential for unearthing new mixotrophic exoelectrogens and electrotrophs, and thus inspire the next generation of microbial electrochemical technology and mixotrophic bacterial metabolic engineering. KEY POINTS: • Mixotrophic bacteria efficiently and simultaneously remove C and N, S or heavy metals. • Exoelectrogens and electrotrophs accelerate metabolism of C and N, S or heavy metals. • New mixotrophic exoelectrogens and electrotrophs should be discovered and exploited.

Development of efficient electroactive biofilm in urine-fed microbial fuel cell cascades for bioelectricity generation.

The Microbial fuel cell (MFC) technology harnesses the potential of some naturally occurring bacteria for electricity generation. Digested sludge is commonly used as the inoculum to initiate the process. There are, however, health hazards and practical issues associated with the use of digested sludge depending on its origin as well as the location for system deployment. This work reports the development of an efficient electroactive bacterial community within ceramic-based MFCs fed with human urine in the absence of sludge inoculum. The results show the development of a uniform bacterial community with power output levels equal to or higher than those generated from MFCs inoculated with sludge. In this case, the power generation begins within 2 days of the experimental set-up, compared to about 5 days in some sludge-inoculated MFCs, thus significantly reducing the start-up time. The metagenomics analysis of the successfully formed electroactive biofilm (EAB) shows significant shifts between the microbial ecology of the feeding material (fresh urine) and the developed anodic biofilm. A total of 21 bacteria genera were detected in the urine feedstock whilst up to 35 different genera were recorded in the developed biofilm. Members of Pseudomonas (18%) and Anaerolineaceae (17%) dominate the bacterial community of the fresh urine feed while members of Burkholderiaceae (up to 50%) and Tissierella (up to 29%) dominate the anodic EAB. These results highlight a significant shift in the bacterial community of the feedstock towards a selection and adaptation required for the various electrochemical reactions essential for survival through power generation.

Alginate-Encapsulated Bacteria for the Treatment of Hypersaline Solutions in Microbial Fuel Cells.

A microbial fuel cell (MFC) based on a new wild-type strain of Salinivibrio sp. allowed the self-sustained treatment of hypersaline solutions (100 g L-1 , 1.71 m NaCl), reaching a removal of (87±11) % of the initial chemical oxygen demand after five days of operation, being the highest value achieved for hypersaline MFC. The degradation process and the evolution of the open circuit potential of the MFCs were correlated, opening the possibility for online monitoring of the treatment. The use of alginate capsules to trap bacterial cells, increasing cell density and stability, resulted in an eightfold higher power output, together with a more stable system, allowing operation up to five months with no maintenance required. The reported results are of critical importance to efforts to develop a sustainable and cost-effective system that treats hypersaline waste streams and reduces the quantity of polluting compounds released.

Reduced arsenic availability in paddy soil through Fe-organic ligand complexation mediated by bamboo biochar.

The reuse of arsenic (As)-contaminated paddy fields is a global challenge because long-term flooding would result in As release due to the reductive dissolution of iron minerals. Biochar amendment is a common and effective remediation technique for As-contaminated paddy soil. However, the literature is still lacking in systematic research on the function of biochar in controlling the complexation of released dissolved organic matter (DOM) and iron oxides and its synergistic impact on the availability of As in flooded paddy soil. In the present study, bamboo biochar was prepared at different pyrolysis temperatures (300, 450 and 600 °C), as BB300, BB450 and BB600. Four paddy soil treatments including BB300, BB450, BB600 applications (1% ratio, m/m, respectively) and control (CK, no biochar application) were set and incubated for 60 d in flooding condition. The results showed that As availability represented by adsorbed As species (A-As) was mitigated by BB450 amendment compared with CK. The amendment of BB450 in paddy soil facilitated the complexation of HCl extractable Fe(III)/(II) and DOM and formation of amorphous iron oxides (e.g. complexed Fe species). Moreover, the abundance of Geobacteraceae and Xanthomonadaceae, as common electroactive bacteria, was promoted in the BB450 treated paddy soil in comparison to CK, which assisted to form amorphous iron oxides. The formed amorphous iron oxides then facilitated the formation of ternary complex (As-Fe-DOM) with highly stability, which could be considered as a mechanism for As immobilization after biochar was applied to the flooding paddy soil. Thus, the synergistic effect between amorphous iron oxides and electroactive stains could make main contribution to the passivation of released As in paddy soil under long-term flooding condition. This study provided a new insight for As immobilization via regulating iron-organic ligand complexation amendment with biochar in flooding paddy soil.

Assessment of microbial population in integrated CW-MFC system and investigation of organics and fecal coliform removal pathway.

The current study is focused on understanding the operational mechanism of an integrated constructed wetland-microbial fuel cell (CW-MFC) reactor emphasizing fecal coliform (FC) removal. Few studies are available in the literature investigating the inherent mechanisms of pathogen inactivation in a CW-MFC system. Raw domestic wastewater was treated in three vertical reactors, one planted constructed wetland (R1), one planted CW-MFC (R2), and one unplanted CW-MFC (R3). Spatial analysis of treated effluents showed a considerable amount of organics and fecal coliform removal at the vicinity of the anode in R2. Assessment of the microbial population inside all the reactors revealed that EABs (Firmicutes, Bacteroidetes, and Actinobacteria) were more abundant in R2 compared to R1 and R3. During the activity study, biomass obtained from R2 showed a maximum substrate utilization rate of 1.27 mg COD mgVSS-1 d-1. Kinetic batch studies were carried out for FC removal in all the reactors, and the maximum first order FC removal rate was obtained at the anode of R2 as 2.13 d-1 when operated in closed circuit mode. This value was much higher than the natural die-off rate of FCs in raw wastewater which was 1.16 d-1. Simultaneous bioelectricity monitoring inferred that voltage generation can be correlated to faster FC inactivation, which was probably due to EABs outcompeting other exogenous microbes in a preferable anaerobic environment with the presence of an anode. Reactor R2 was found to be functioning as a symbiotic bio-electrochemical mesocosm.

Electric wiring of bacteria using redox polymers and selective measurement of metabolic activity in the presence of surrounding planktonic bacteria.

Non-electroactive bacteria (n-EAB), constituting the majority of known bacteria to date, have been underutilized in electrochemical conversion technologies due to their lack of direct electron transfer to electrodes. In this study, we established an electric wiring between n-EAB (gram-positive Bacillus subtilis and gram-negative Escherichia coli) and an extracellular electrode via a ferrocene-polyethyleneimine-based redox polymer (Fc-PEI). Chronoamperometry recordings indicated that Fc-PEI can transfer intracellular electrons to the extracellular electrode regardless of the molecular organization of PEI (linear or branched) and the membrane structure of bacteria (gram-positive or -negative). As fluorescence staining suggested, Fc-PEI improves the permeability of the bacterial cell membrane, enabling electron carriers in the cell to react with Fc. In addition, experiments with Fc-immobilized electrodes without PEI suggested the existence of an alternative electron transfer pathway from B. subtilis to the extracellular Fc adsorbed onto the cell membrane. Furthermore, we proposed for the first time that the bacteria/Fc-linear PEI modified structure enables selective measurement of immobilized bacterial activity by physically blocking contact between the electrode surface and planktonic cells co-existing in the surrounding media. Such electrodes can be a powerful analytical tool for elucidating the metabolic activities of specific bacteria wired to the electrode even within complex bacterial communities.

Intermittent electrostimulation-modified direct interspecies electron transfer for enhanced methanogenesis in anaerobic digestion of sulfate-rich wastewater.

Methane recovery and organics removal in sulfate (SO42-)-rich wastewater anaerobic digestion are hindered by electron competition between methanogenesis and sulfidogenesis. Here, intermittently electrostimulated bioelectrodes were developed to facilitate direct interspecies electron transfer (DIET)-driven syntrophic methanogenesis, increasing substrate competition among methanogenic archaea (MA). By optimising the electrochemical environment, MA was able to employ electron transfer more efficiently than sulfate-reducing bacteria (SRB), resulting in significant methane accumulation (58.1 ± 1.0 mL-CH4/m3reactor) and COD removal (90.5 ± 0.5 %) at lower COD/SO42- ratio. Intermittent electrostimulation improved the metabolic pathway for electroactive bacteria to utilize acetate and direct electrons to electrotrophic MA, decreasing SRB abundance and affecting the sulfate reduction pathway. Intermittently electrostimulated biofilms significantly increased gene levels of key enzymes in electron transport for cytochrome and e-pili biosynthesis, crucial for DIET, demonstrating enhanced DIET-driven syntrophic methanogenesis. This study provides a strategic approach to optimize methanogenesis in sulfate-rich wastewater anaerobic digestion.

Review of cathodic electroactive bacteria: Species, properties, applications and electron transfer mechanisms.

Cathodic electroactive bacteria (C-EAB) which are capable of accepting electrons from solid electrodes provide fresh avenues for pollutant removal, biosensor design, and electrosynthesis. This review systematically summarized the burgeoning applications of the C-EAB over the past decade, including 1) removal of nitrate, aromatic derivatives, and metal ions; 2) biosensing based on biocathode; 3) electrosynthesis of CH4, H2, organic carbon, NH3, and protein. In addition, the mechanisms of electron transfer by the C-EAB are also classified and summarized. Extracellular electron transfer and interspecies electron transfer have been introduced, and the electron transport mechanism of typical C-EAB, such as Shewanella oneidensis MR-1, has been combed in detail. By bringing to light this cutting-edge area of the C-EAB, this review aims to stimulate more interest and research on not only exploring great potential applications of these electron-accepting bacteria, but also developing steady and scalable processes harnessing biocathodes.

Nonelectroactive clostridium obtains extracellular electron transfer-capability after forming chimera with Geobacter.

Extracellular electron transfer (EET) of microorganisms is a major driver of the microbial growth and metabolism, including reactions involved in the cycling of C, N, and Fe in anaerobic environments such as soils and sediments. Understanding the mechanisms of EET, as well as knowing which organisms are EET-capable (or can become so) is fundamental to electromicrobiology and geomicrobiology. In general, Gram-positive bacteria very seldomly perform EET due to their thick non-conductive cell wall. Here, we report that a Gram-positive Clostridium intestinale (C.i) attained EET-capability for ethanol metabolism only after forming chimera with electroactive Geobacter sulfurreducens (G.s). Mechanism analyses demonstrated that the EET was possible after the cell fusion of the two species was achieved. Under these conditions, the ethanol metabolism pathway of C.i was integrated by the EET pathway of G.s, by which achieved the oxidation of ethanol for the subsequent reduction of extracellular electron acceptors in the coculture. Our study displays a new approach to perform EET for Gram-positive bacteria via recruiting the EET pathway of an electroactive bacterium, which suggests a previously unanticipated prevalence of EET in the microbial world. These findings also provide new perspectives to understand the energetic coupling between bacterial species and the ecology of interspecies mutualisms.

Marine Biofilm Engineered to Produce Current in Response to Small Molecules.

Engineered electroactive bacteria have potential applications ranging from sensing to biosynthesis. In order to advance the use of engineered electroactive bacteria, it is important to demonstrate functional expression of electron transfer modules in chassis adapted to operationally relevant conditions, such as non-freshwater environments. Here, we use the Shewanella oneidensis electron transfer pathway to induce current production in a marine bacterium, Marinobacter atlanticus, during biofilm growth in artificial seawater. Genetically encoded sensors optimized for use in Escherichia coli were used to control protein expression in planktonic and biofilm attached cells. Significant current production required the addition of menaquinone, which M. atlanticus does not produce, for electron transfer from the inner membrane to the expressed electron transfer pathway. Current through the S. oneidensis pathway in M. atlanticus was observed when inducing molecules were present during biofilm formation. Electron transfer was also reversible, indicating that electron transfer into M. atlanticus could be controlled. These results show that an operationally relevant marine bacterium can be genetically engineered for environmental sensing and response using an electrical signal.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from -150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.

Living electrochemical biosensing: Engineered electroactive bacteria for biosensor development and the emerging trends.

Bioelectrical interfaces made of living electroactive bacteria (EAB) provide a unique opportunity to bridge biotic and abiotic systems, enabling the reprogramming of electrochemical biosensing. To develop these biosensors, principles from synthetic biology and electrode materials are being combined to engineer EAB as dynamic and responsive transducers with emerging, programmable functionalities. This review discusses the bioengineering of EAB to design active sensing parts and electrically connective interfaces on electrodes, which can be applied to construct smart electrochemical biosensors. In detail, by revisiting the electron transfer mechanism of electroactive microorganisms, engineering strategies of EAB cells for biotargets recognition, sensing circuit construction, and electrical signal routing, engineered EAB have demonstrated impressive capabilities in designing active sensing elements and developing electrically conductive interfaces on electrodes. Thus, integration of engineered EAB into electrochemical biosensors presents a promising avenue for advancing bioelectronics research. These hybridized systems equipped with engineered EAB can promote the field of electrochemical biosensing, with applications in environmental monitoring, health monitoring, green manufacturing, and other analytical fields. Finally, this review considers the prospects and challenges of the development of EAB-based electrochemical biosensors, identifying potential future applications.

Carbon nanotubes encapsulating FeS2 micropolyhedrons as an anode electrocatalyst for improving the power generation of microbial fuel cells.

The low power density originating from poor electroactive bacteria (EAB) adhesion and sluggish extracellular electron transfer (EET) at the anode interface, is a major impediment preventing the practical implementation of microbial fuel cells (MFCs). Tailoring the surface properties of anodes is an effective and powerful strategy for addressing this issue. In this study, we successfully fabricated an efficient anode electrocatalyst, consisting of carbon nanotubes encapsulating iron disulfide (FeS2@CNT) micropolyhedrons, using simple hydrothermal and freeze-drying methods, which not only strengthened the anode interaction with EAB but also promoted the EET process at the anode interface. As expected, the MFCs with a FeS2@CNT anode yielded an outstanding power density of 1914 mWm-2 at a current density of 4350 mA m-2, which significantly exceeded those of pure CNT (1096.2mW m-2, 2703.3 mA m-2) and carbon cloth (426.8mWm-2, 965.6 mA m-2) anodes. The high-power output can be attributed to the synergistic effect between FeS2 and CNTs, endowing the anode with biocompatibility for biofilm adhesion and colonization, nutrient diffusion, and the presence of abundant Fe and S active sites for EET mediation. Owing to the low cost, facile fabrication process, and excellent electrocatalytic performance toward the redox reactions in biofilms, the synthesized FeS2@CNT electrocatalyst is a promising material for high-performance and cost-effective MFCs with commercial applications.