Understanding the Tail Current Behavior of Electroactive Biofilms Realizes the Rapid Measurement of Biochemical Oxygen Demand.

The use of microbial electrochemical sensors, with electroactive biofilms (EABs) as sensing elements, is a promising strategy to timely measure the biochemical oxygen demand (BOD) of wastewater. However, accumulation of Coulombic yield over a complete degradation cycle is time-consuming. Therefore, understanding the correlation between current output and EAB metabolism is urgently needed. Here, we recognized a tail stage (TS) on a current-time curve according to current increase rate─a period with the least electron harvesting efficiency. EAB adopted a series of metabolic compensation strategies, including slow metabolism of residual BOD, suspended growth, reduced cell activity, and consumption of carbon storage polymers, to cope with substrate deficiency in TS. The supplementary electrons provided by the decomposition of glycogen and fatty acid polymers increased the Coulombic efficiencies of TS to >100%. The tail current produced by spontaneous metabolic compensation showed a trend of convergent exponential decay, independent of BOD concentration. Therefore, we proposed the TS prediction model (TSPM) to predict Coulombic yield, which shortened BOD measurement time by 96% (to ∼0.5 h) with deviation <4 mg/L when using real domestic wastewater. Our findings on current output in TS give insights into bacterial substrate storage and consumption, as well as regulation in substrate-deficient environment, and provide a basis for developing BOD sensors.

Fe(0)-Dissimilatory Nitrate Reduction to Ammonium for Autotrophic Recovery of Reactive Nitrogen.

Bioreduction of nitrate to value-added ammonium is a potentially sustainable strategy to recycle nutrients from wastewater. Here, we have proven the feasibility of the reduction of autotrophic nitrate to ammonium with electrons extracted from Fe(0). Using a Geobacter-dominated anodic biofilm as an inoculum, we achieved nitrate-to-ammonium efficiency up to 90 ± 3% with a nitrate reduction rate of 35 ± 1.3 mg N/d/L. An electron acceptor instead of an inoculum greatly influenced the Fe(0)-dissimilatory nitrate reduction to ammonium (DNRA), where nitrite as the electron acceptor provided an effective selective pressure to enrich Geobacter from initial 5 to 56%. The DNRA repressing denitrification was demonstrated by the reverse tendencies of upregulated nrfA and downregulated nirS gene transcription. This finding provides a new route for autotrophic nitrate removal and recycling from water, which has a broader implication on biogeochemical nitrogen and iron cycling.

Design, optimization and application of a highly sensitive microbial electrolytic cell-based BOD biosensor.

Biochemical oxygen demand (BOD) is an important biochemical indicator for determining the degree of water pollution and guiding the design of wastewater treatment processes. BOD sensors based on microbial electrochemical technology can conduct real-time online monitoring of organic matter and have attracted extensive attention. However, research on microbial electrolytic cell (MEC)-type BOD sensors is at the stage of theoretical exploration. Here, we designed and optimized a highly sensitive MEC-type BOD sensor by screening inoculants, comparing electrode materials, and optimizing the reactor configuration. The results showed that effective means to optimize a BOD sensor for fast activation and sensitive testing included the inoculation of the MEC reactor effluent with large amounts of biomass and highly active bacteria, selection of carbon felt electrodes with excellent adsorption and permeability, miniaturization of the reactor, regulation of suitable electrode spacing, and design of the penetrating fluid structure. Then, the optimized sensing system was applied to determine the BOD concentration in model solutions of sodium acetate in a laboratory environment, where it accurately measured BOD concentrations in the range of 10-500 mg/L and maintained good parallelism during long-term operation. Next, the MEC-type BOD sensors were put into practice in the field as an alarm for accidents at an actual sewage plant. The whole BOD sensing system was quickly assembled on site and started up, and it gave an early warning shortly after the concentration of organic matter in the water suddenly increased, thus showing a high potential for engineering applications. This study broadened the domains of application of MEC-type BOD sensors in environmental monitoring, and promoted the development of technological innovation in water ecology and environmental monitoring.

Insight of bacteria and archaea in Feammox community enriched from different soils.

Anaerobic ammonium oxidation coupled to Fe(III) reduction, known as Feammox, is a newly discovered nitrogen-cycling process, which serves an important role in the pathways of nitrogen loss in the environment. However, the specific types of microorganisms involved in Feammox currently remain unclear. In this study, we selected two groups of soil samples (paddy and mine), from considerably different habitats in South China, to acclimate Feammox colonies. The Paddy Group had a shorter lag period than the Mine Group, while the ammonium transformation rate was nearly equal in both groups in the mature period. The emergence of the Feammox activity was found to be associated with the increased abundance of iron-reducing bacteria, especially Clostridium_sensu_stricto_12, Desulfitobacterium, Thermoanaerobaculum, Anaeromyxobacter and Geobacter. Ammonium oxidizing archaea and methanogens were dominant among the known archaea. These findings extend our knowledge of the microbial community composition of the potential Feammox microbes from soils under different environmental conditions, which broadens our understanding of this important Fe/N transformation process.

Thermal reduced graphene oxide enhanced in-situ H2O2 generation and electrochemical advanced oxidation performance of air-breathing cathode.

Developing highly efficient catalysts with high ORR activity and H2O2 selectivity is an important challenge for producing H2O2 through 2e- oxygen reduction reaction (ORR). In this work, we tuned the reduction degree of graphene oxide by controlling reducing temperature and prepared graphite-TRGO hybrid air breathing cathodes (ABCs). The H2O2 production rate of TRGO-1100 (with highest reduction degree) modified ABC exhibits highest H2O2 generation rate of 20.4 ± 0.8 mg/cm2/h and current efficiency of 94 ± 2%. The charge transfer resistance of TRGO-1100 decreases by 2.5-fold compared with pure graphite cathode. Unreduced GO shows high H2O2 selectivity and low ORR activity, while TRGO shows lower H2O2 selectivity but higher ORR activity. Though the 2e- ORR selectivity of TRGO decreased TRGO with all reduction degrees, the H2O2 production increased in all forming electrodes. Superior performance of TRGO modified ABCs is attributed to high oxygen adsorption and low charge transfer resistance. TRGO possesses super-hydrophobicity and large surface area for oxygen adsorption. Besides, TRGO provides abundant electrochemically active sites to facilitate the electron transfer and formed more mesopores for H2O2 release. Electro-Fenton using TRGO-1100-ABC exhibited great performance for Persistent Organic Pollutants (POPs) degradation, which removed 66% of tetracycline in 5 min.

Long-Term Succession Shows Interspecies Competition of Geobacter in Exoelectrogenic Biofilms.

Geobacter spp. are well-known exoelectrogenic microorganisms that often predominate acetate-fed biofilms in microbial fuel cells (MFCs) and other bioelectrochemical systems (BESs). By using an amplicon sequence variance analysis (at one nucleotide resolution), we observed a succession between two closely related species (98% similarity in 16S RNA), Geobacter sulfurreducens and Geobacter anodireducens, in the long-term studies (20 months) of MFC biofilms. Geobacter spp. predominated in the near-electrode portion of the biofilm, while the outer layer contained an abundance of aerobes, which may have helped to consume oxygen but reduced the relative abundance of Geobacter. Removal of the outer aerobes by norspermidine washing of biofilms revealed a transition from G. sulfurreducens to G. anodireducens. This succession was also found to occur rapidly in co-cultures in BES tests even in the absence of oxygen, suggesting that oxygen was not a critical factor. G. sulfurreducens likely dominated in early biofilms by its relatively larger cell size and production of extracellular polymeric substances (individual advantages), while G. anodireducens later predominated due to greater cell numbers (quantitative advantage). Our findings revealed the interspecies competition in the long-term evolution of Geobacter genus, providing microscopic insights into Geobacter's niche and competitiveness in complex electroactive microbial consortia.

Bioelectrochemical Ammoniation Coupled with Microbial Electrolysis for Nitrogen Recovery from Nitrate in Wastewater.

Nitrate-N in wastewaters is hard to be recovered because it is difficult to volatilize with an opposite charge to ammonium. Here, we have proved the feasibility of dissimilatory nitrate reduction to ammonia (DNRA) by the easy-acclimated mixed electroactive bacteria, achieving the highest DNRA efficiency of 44%. It was then coupled with microbial electrolysis to concentrate ammonium by a factor of 4 in the catholyte for recovery. The abundance of electroactive bacteria in the biofilm before nitrate addition, especially Geobacter spp., was found to determine the DNRA efficiency. As the main competitors of DNRA bacteria, the growth of denitrifiers was more sensitive to C/N ratios. The DNRA microbial community contrarily showed a stable and recoverable ammoniation performance over C/N ratios ranging from 0.5 to 8.0. A strong competition of the electrode and nitrate on electron donors was observed at the early stage (15 d) of electroactive biofilm formation, which can be weakened when the biofilm was mature on 40 d. Quantitative PCR showed a significant increase in nirS and nrfA transcripts in the ammoniation process. nirS was inhibited significantly after nitrate depletion while nrfA was still upregulated. These findings provided a novel way to recover nitrate-N using organic wastes as both electron donor and power, which has broader implications on the sustainable wastewater treatment and the ecology of nitrogen cycling.

Revealing Decay Mechanisms of H2O2-Based Electrochemical Advanced Oxidation Processes after Long-Term Operation for Phenol Degradation.

Hydrogen peroxide (H2O2)-based electrochemical advanced oxidation processes (EAOPs) have been widely attempted for various wastewater treatments. So far, stability tests of EAOPs are rarely addressed and the decay mechanism is still unclear. Here, three H2O2-based EAOP systems (electro-Fenton, photoelectro-Fenton, and photo+ electro-generated H2O2) were built for phenol degradation. More than 97% phenol was removed in all three EAOPs in 1 h at 10 mA·cm-2. As a key component in EAOPs, the cathodic H2O2 productivity is directly related to the performance of the system. We for the first time systematically investigated the decay mechanisms of the active cathode by operating the cathodes under multiple conditions over 200 h. Compared with the fresh cathode (H2O2 yield of 312 ± 22 mg·L-1·h-1 with a current efficiency of 84 ± 5% at 10 mA·cm-2), the performance of the cathode for H2O2 synthesis alone decayed by only 17.8%, whereas the H2O2 yields of cathodes operated in photoelectro-generated H2O2, electro-Fenton, and photoelectro-Fenton systems decayed by 60.0, 90.1, and 89.6%, respectively, with the synergistic effect of salt precipitation, •OH erosion, organic contamination, and optional Fe contamination. The lower current decay of 16.1-32.3% in the electrochemical tests manifested that the cathodes did not lose activity severely. Therefore, the significant decrease of H2O2 yield was because the active sites were altered to catalyze the four-electron oxygen reduction reaction, which was induced by the long-term erosion of •OH. Our findings provided new insights into cathode performance decay, offering significant information for the improvement of cathodic longevity in the future.

Unignorable toxicity of formaldehyde on electroactive bacteria in bioelectrochemical systems.

Formaldehyde poses significant threats to the ecosystem and is widely used as a toxicity indicator to obtain electrical signal feedback in electroactive biofilm (EAB)-based sensors. Although many optimizations have been adopted to improve the performance of EAB to formaldehyde, nearly no studies have discussed the toxicity of formaldehyde to EAB. Here, EABs were acclimated with a stable current density (8.9 ± 0.2 A/m2) and then injected with formaldehyde. The current density decreased by 27% and 98% after the injection of 1 and 10 ppm formaldehyde, respectively, compared with that in the control. The ecotoxicity of formaldehyde caused the irreversible loss of current with 3% (1 ppm) and 81% (10 ppm). Confocal laser scanning microscopy and scanning electron microscopy results showed that the redox activity was inhibited by formaldehyde, and the number of dead/broken cells increased from 2% to 40% (1 ppm) and 91% (10 ppm). The contents of the total protein and extracellular polymer substances decreased by more than 28% (1 ppm) and 75% (10 ppm) because of the cleavage reaction caused by formaldehyde. Bacterial community analysis showed that the proportion of Geobacter decreased from 81% to 53% (1 ppm) and 24% (10 ppm). As a result, the current production was significantly impaired, and the irreversible loss increased. Toxicological analysis demonstrated that formaldehyde disturbed the physiological indices of cells, thereby inducing apoptosis. These findings fill the gap of ecotoxicology of toxicants to EAB in a bioelectrochemical system.

Highly sensitive standardized toxicity biosensors for rapid water quality warning.

Microbial electrochemical sensor (MES) using electroactive biofilm (EAB) as the sensing element represents a broad-spectrum technology for early warning of biotoxicity of water samples. However, its commercial application is impeded by limited sensitivity and repeatability. Here, we proposed a layered standardized EAB (SEAB) with enriched Geobacter anodireducens SD-1 in the inner layer and self-matched outer layer. The SEAB sensors showed a 2.3 times higher sensitivity than conventional EAB acclimated directly from wastewater (WEAB). A highly repeatable response sensitivity was concentrated at 0.011 ± 0.0006 A/m2/ppm in 4 replicated batches of SEAB sensors (R2 > 0.95), highlighting their potential for reliable toxicity monitoring in practical applications. In contrast, the sensing performance of all WEAB sensors was unpredictable. SEAB also exhibited a better tolerance towards low concentration of formaldehyde, with only a 4 % loss in viability. Our findings improved the sensitivity and reproducibility of standardized MES for toxicity early warning.

Glucose facilitates the acclimation of organohalide-respiring bacteria.

Organohalide respiring bacteria (OHRB) are the mainstay for bioremediation of organohalide contaminated sites. Enrichment screening of OHRB is prerequisite for the development of high performance dehalogenating bacterial agents. Herein, different domestication strategies were formulated for the main factors (nutrients and inocula) affecting the enrichment of OHRB, and the dehalogenation effect was verified with 2-chlorophenol and per/polyfluoroalkyl substances. The nutrients had a greater impact on the dehalogenation of the systems relative to the inocula, where the combination of glucose and anaerobic sludge (Glu-AS) had a faster degradation rate (26 ± 2.5 µmol L-1 d-1) and more complete dechlorination effectiveness. Meanwhile, the dehalogenation results for perfluorooctanoic acid and trifluoroacetic acid showed the biological defluorination was closely related to the position of fluoride. Further, the microbial community structure profiled the resource competition, metabolic cross-feeding and nutrient dynamic exchange among fermenting bacteria, OHRB and methanogenic bacteria under different domestication strategies as endogenous factors affecting the dehalogenation performance, and speculated a hypothetical model for the interaction of different functional bacteria. Our research contributed guidelines and references for the development of efficient dehalogenating bacterial agents, and provided scientific theoretical and technical support for promoting the maximum efficiency of bioremediation of organohalogenated sites.

Toxicity warning and online monitoring of disinfection by-products in water by electroautotrophic biocathode sensors.

As a result of the 2019 coronavirus pandemic, disinfection byproducts generated by the extensive use of chlorine disinfectants have infiltrated the aquatic environment, severely threatening ecological safety and human health. Therefore, the accurate monitoring of the biotoxicity of aqueous environments has become an important issue. Biocathode sensors are excellent choices for toxicity monitoring because of their special electroautotrophic respiration functions. Herein, a novel electroautotrophic biosensor with rapid, sensitive, and stable response and quantifiable output was developed. Its toxicity response was tested with typical disinfection byproducts dichloromethane, trichloromethane, and combinations of both, and corresponding characterization models were developed. Repeated toxicity tests demonstrated that the sensor was reusable rather being than a disposable consumable, which is a prerequisite for its long-term and stable operation. Microbial viability confirmed a decrease in sensor sensitivity due to microbial stress feedback to the toxicants, which is expected to be calibrated in the future by the standardization of the biofilms. Community structure analysis indicated that Moheibacter and Nitrospiraceae played an important role in the toxic response to chlorine disinfection byproducts. Our research provides technical support for protecting the environment and safeguarding water safety for human consumption and contributes new concepts for the development of novel electrochemical sensors.

Reduction of S0 deposited on electroactive biofilm under an oxidative potential.

The inevitable deposition of S0 on the electroactive biofilm (EAB) via anodic sulfide oxidation affects the stability of bioelectrochemical systems (BESs) when an accidental discharge of sulfide occurred, leading to the inhibition of electroacitivity, because the potential of anode (e.g., 0 V versus Ag/AgCl) is ~500 mV more positive than the redox potential of S2-/S0. Here we found that S0 deposited on the EAB can be spontaneously reduced under this oxidative potential independent of microbial community variation, leading to a self-recovery of electroactivity (> 100 % in current density) with biofilm thickening (~210 µm). Transcriptomics of pure culture indicated that Geobacter highly expressed genes involving in S0 metabolism, which had an additional benefit to improve the viability (25 % - 36 %) of bacterial cells in biofilm distant from the anode and the cellular metabolic activity via electron shuttle pair of S0/S2-(Sx2-). Our findings highlighted the importance of spatially heterogeneous metabolism to its stability when EABs encountered with the problem of S0 deposition, and that in turn improved the electroactivity of EABs.

Rapid warning of emerging contaminants in reuse water using biocathode sensors.

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.

Endogenous electric field accelerates phenol degradation in bioelectrochemical systems with reduced electrode spacing.

Reducing the electrode spacing in bioelectrochemical systems (BESs) are widely reported to improve power output, which was mainly attributed to the decrease of ohmic resistance (Rohm) for a long time. Here we found the change of endogenous electric field (EF) intensity was the key to improve electroactivity in response to a reduced electrode spacing, which also accelerated phenol biodegradation. Correlation and principal components analysis revealed that the microbial community of electroactive biofilm (EAB) was independent of Rohm, while the EF intensity was found closely related to most of predominant genera. A strong EF selectively enriched phenol-degrading bacteria Comamonas in suspension and Geobacter in EAB, contributed to the improvement of degradation efficiency. EF also induced the secretion of extracellular polymeric substances, protected EAB from being inactivated by phenol. Our findings highlighted the importance of EF intensity on BESs performance, providing new insights into the design and application of BESs in wastewater treatment.

Anaerobic bioreduction of elemental sulfur improves bioavailability of Fe (III) oxides for bioremediation.

Fe(III) oxides are ubiquitous electron acceptors for anaerobic bioremediation, although their bioavailability was limited due to the passivation of secondary mineralization products. Here we found the solid S0 can be added to improve their bioavailability. Using lepidocrocite (γ-FeOOH), acetate and Geobacter sulfurreducens PCA as representatives of Fe(III) oxides, intermediate of pollutant degradation and microbes, a 6 times higher amount of FeOOH reduction in the presence of S0 was observed with a time needed for S0 reduction shortened by half. The bioreduction of S0 activated the reduction of FeOOH, while the product (conductive FeS) may have bridged electron transfer across the cell membrane and periplasm. The proportion of excessive Fe(II) produced from Fe(III) was quantified as a direct bioreduction (26 %), with an abiotic FeOOH reduction to FeS (20 %) and an FeS-conducted FeOOH bioreduction (54 %), which highlight the key role of gradually formed FeS from S0 in the bioreduction of FeOOH. Our results showed that S0 can be an effective additive for the bioremediation of environments with abundant Fe(III) oxides, which has broader implications for elemental biogeochemical cycling.

Switchover of electrotrophic and heterotrophic respirations enables the biomonitoring of low concentration of BOD in oxygen-rich environment.

Biochemical oxygen demand (BOD) is a key indicator of water quality. However, there is still no technique to directly measure BOD at low concentrations in oxygen-rich environments. Here, we propose a new scheme using facultative electrotrophs as the sensing element, and confirmed aerobic Acinetobacter venetianus RAG-1 immobilized on electrode was able to measure BOD via the switchover between electrotrophic and heterotrophic respirations. The hybrid binder of Nafion and polytetrafluoroethylene (PTFE) maximized the baseline current (127 ± 2 A/m2) and sensitivity (2.5 ± 0.1 (mA/m2)/(mg/L)). The current decrease and the BOD5 concentration fitted well with a linear model in the case of known contaminants, verified with both lab samples of acetate and glucose (R2>0.96) and in standard curves of real environmental samples collected from the lake and the effluent of wastewater treatment plant (R2>0.98). Importantly, the biosensor tested actual contaminated water samples with an error of 0.4∼10% compared to BOD5 in the case of unknown contaminants. Transcriptomics revealed that reverse oxidative TCA may involve in the electrotrophic respiration of RAG-1 since citrate synthase (gltA) was highly expressed, which was partly downregulated when heterotrophic metabolism was triggered by BOD. This can be returned to electrotroph when BOD was depleted. Our results showed a new way to rapidly measure BOD in oxygen-rich environment, demonstrating the possibility to employ bacteria with two competitive respiration pathways for pollution detection.

Layered Design of a Highly Repeatable Electroactive Biofilm for a Standardized Biochemical Oxygen Demand Sensor.

Microbial electrochemical sensors are promising to monitor bioavailable organics in real environments, but their application is restricted by the unpredictable performance of the electroactive biofilm (EAB), which is randomly acclimated from environmental microflora. With a long-term stable EAB as a template, we successfully designed EAB (DEAB) by the sequential growth of Geobacter anodireducens and automatched microbes, achieving a reproducible high current than those naturally acclimated from wastewater (NEAB). Pre-inoculation of planktonic aerobes as oxygen bioscavengers was necessary to ensure the colonization of Geobacter in the inner layer, and the abundant Geobacter (50%) in DEAB guaranteed 4 times higher current density with a 15-fold smaller variation among 20 replicates than those of NEAB. The sensor constructed with DEAB exhibited a shorter measuring time and a precise biochemical oxygen demand (BOD) measurement with acetate, real domestic wastewater, and supernatant of anaerobic digestion. Here, we for the first time proposed an applicable strategy to standardize EABs for BOD sensors, which is also crucial to ensure a stable performance of all bioelectrochemical technologies.

Bioelectrochemical system for dehalogenation: A review.

Halogenated organic compounds are persistent pollutants, whose persistent contamination and rapid spread seriously threaten human health and the safety of ecosystems. It is difficult to remove them completely by traditional physicochemical techniques. In-situ remediation utilizing bioelectrochemical technology represents a promising strategy for degradation of halogenated organic compounds, which can be achieved through potential modulation. In this review, we summarize the reactor configuration of microbial electrochemical dehalogenation systems and relevant organohalide-respiring bacteria. We also highlight the mechanisms of electrode potential regulation of microbial dehalogenation and the role of extracellular electron transfer in dehalogenation process, and further discuss the application of bioelectrochemical technology in bioremediation of halogenated organic compounds. Therefore, this review summarizes the status of research on microbial electrochemical dehalogenation systems from macroscopic to microscopic levels, providing theoretical support for the development of rapid and efficient in situ bioremediation technologies for halogenated organic compounds contaminated sites, as well as insights for the removal of refractory fluorides.

Two key Geobacter species of wastewater-enriched electroactive biofilm respond differently to electric field.

Electroactive biofilms have attracted increasing attention due to their unique ability to exchange electrons with electrodes. Geobacter spp. are widely found to be dominant in biofilms in acetate-rich environments when an appropriate voltage is applied, but it is still largely unknown how these bacteria are selectively enriched. Herein, two key Geobacter spp. that have been demonstrated predominant in wastewater-enriched electroactive biofilm after long-term operation, G. sulfurreducens and G. anodireducens, responded to electric field (EF) differently, leading to a higher abundance of EF-sensitive G. anodireducens in the strong EF region after cocultivation with G. sulfurreducens. Transcriptome analysis indicated that two-component systems containing sensor histidine kinases and response regulators were the key for EF sensing in G. anodireducens rather than in G. sulfurreducens, which are closely connected to chemotaxis, c-di-GMP, fatty acid metabolism, pilus, oxidative phosphorylation and transcription, resulting in an increase in extracellular polymeric substance secretion and rapid cell proliferation. Our data reveal the mechanism by which EF select specific Geobacter spp. over time, providing new insights into Geobacter biofilm formation regulated by electricity.