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Abstract A promising strategy for establishing direct interspecies electron transfer (DIET)-based syntrophic metabolism during anaerobic digestion (AD) is to stimulate the methanogenic communities with ethanol to rapidly produce biological electrical connections. In this study the strategy was further explored in an engineering-scale up-flow anaerobic sludge blanket (UASB) reactor treating bagasse wastes. The results demonstrated that, under the conditions employed, the ethanol-abundant acidogenic products from ethanol-type fermentation could stimulate the UASB reactor to form the aggregates with a higher conductivity (22.6 ± 0.6 uS/cm) than that in the laboratory-scale UASB reactor treating brewery wastes (8.1 ± 1.7 uS/cm), in which DIET was the primary working mode of interspecies electron exchange. Furthermore, these aggregates also presented a positive response with granular active carbon (GAC) to methanogenesis, suggesting that the DIET-based methanogenic communities were established. As a result, the engineering-scale UASB reactor was capable of maintaining stability in response to the environmental disturbance. Microbial community analysis revealed that specific and substantial enrichments of Petrimonas (21.8% of abundance) and Methanothrix species (64.8%) were detected in the aggregates. These results, and the known ability of Petrimonas species to transfer electrons to elemental sulfur, suggested that Petrimonas species might participate in DIET with Methanothrix species. Graphical abstract Image 1 Highlights • DIET was found in an engineering-scale UASB initially with ethanol-type fermentation. • UASB presented a stable performance in response to acidic and high-OLR impacts. • Ethanol-type products stimulate UASB to form aggregates with a higher conductivity. • Methanogenic metabolism in the aggregates could be further facilitated with GAC. • Microbial communities suggested that Petrimonas might proceed DIET with Methanothrix. [ABSTRACT FROM AUTHOR]

Anaerobic digesters usually become sour during the operation and inhibit methanogenesis. This effect occurs due to the interspecies electron exchange for the syntrophic metabolism of alcohols and volatile fatty acids (VFAs) via interspecies H 2 transfer (IHT). Conductive materials can promote direct interspecies electron transfer (DIET) between the syntrophs and methanogens, providing an alternative to IHT. However, the cooperative mechanism of these two working modes during syntrophic metabolism is unknown, especially during acidic impacts. The results of this study demonstrated that anaerobic digesters supplemented with conductive carbon cloth had a higher capacity to resist the acidic impacts. Conversely, the digesters supplemented with non-conductive cotton cloth (control) were nearly stagnant when the H 2 partial pressure of the anaerobic systems increased. Further experiments at high H 2 partial pressure to inhibit IHT exhibited almost no effect on the syntrophic metabolism in the carbon cloth-supplemented group, in which the methane production approached the stoichiometric production (350 mL CH 4 /g COD removal), while the methane production in the control group ceased. The microbial community analysis revealed that the surface sludge attached to the carbon cloth had the highest abundance of Geobacter and Methanosaeata species known to participate in DIET. These results suggested that the predominant working mode for the interspecies electron exchange might have shifted from IHT to DIET in the presence of the conductive carbon cloth during acidic impacts. DIET primarily occurred on the surface sludge of the carbon cloth and replaced IHT to proceed the syntrophic metabolism and maintained stable anaerobic methanogenesis. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) has been considered as an alternative to interspecies H 2 transfer (IHT) for syntrophic metabolism, but the microorganisms capable of metabolizing the key intermediates, such as propionate and butyrate, via DIET have yet to be described. A strategy of culturing the enrichments with ethanol as a DIET substrate to stimulate the communities for the syntrophic metabolism of propionate and/or butyrate was proposed in this study. The results showed that the syntrophic propionate and/or butyrate degradation was significantly improved in the ethanol-stimulated reactor when propionate/butyrate was the sole carbon source. The conductivity of the ethanol-stimulated enrichments was as 5 folds (for propionate)/76 folds (for butyrate) as that of the traditional enrichments (never ethanol fed). Microbial community analysis revealed that Geobacter species known to proceed DIET were only detected in the ethanol-stimulated enrichments. Together with the significant increase of Methanosaeta and Methanosarcina species in these enrichments, the potential DIET between Geobacter and Methanosaeta or Methanosarcina species might be established to improve the syntrophic propionate and/or butyrate degradation. Further experiments demonstrated that granular activated carbon (GAC) could improve the syntrophic metabolism of propionate and/or butyrate of the ethanol-stimulated enrichments, while almost no effects on the traditional enrichments. Also, the high H 2 partial pressure could inhibit the syntrophic propionate and/or butyrate degradation of the traditional enrichments, but its effect on that of the ethanol-stimulated enrichments was negligible. [ABSTRACT FROM AUTHOR]

Promoting direct interspecies electron transfer (DIET) to enhance syntrophic metabolism may be a strategy for accelerating the conversion of organic wastes to methane, but microorganisms capable of metabolizing propionate and butyrate via DIET under methanogenic conditions have yet to be identified. In an attempt to establish methanogenic communities metabolizing propionate or butyrate with DIET, enrichments were initiated with up-flow anaerobic sludge blanket (UASB), similar to those that were previously reported to support communities that metabolized ethanol with DIET that relied on direct biological electrical connections. In the absence of any amendments, microbial communities enriched were dominated by microorganisms closely related to pure cultures that are known to metabolize propionate or butyrate to acetate with production of H 2 . When biochar was added to the reactors there was a substantial enrichment on the biochar surface of 16S rRNA gene sequences closely related to Geobacter and Methanosaeta species known to participate in DIET. [ABSTRACT FROM AUTHOR]

Increase of methanogenesis in methane-producing microbial electrolysis cells (MECs) is frequently believed as a result of cathodic reduction of CO 2 . Recent studies indicated that this electromethanogenesis only accounted for a little part of methane production during anaerobic sludge digestion. Instead, direct interspecies electron transfer (DIET) possibly plays an important role in methane production. In this study, anaerobic digestion of sludge were investigated in a single-chamber MEC reactor, a carbon-felt supplemented reactor and a common anaerobic reactor to evaluate the effects of DIET on the sludge digestion. The results showed that adding carbon felt into the reactor increased 12.9% of methane production and 17.2% of sludge reduction. Imposing a voltage on the carbon felt further improved the digestion. Current calculation showed that the cathodic reduction only contributed to 27.5% of increased methane production. Microbial analysis indicated that DIET played an important role in the anaerobic sludge digestion in the MEC. [ABSTRACT FROM AUTHOR]

Biochar has been considered as a promising material to enhance the anaerobic digestion of organic wastes, whereas the critical mechanism of biochar promoting interspecies electron transfer is still unclear. The study presented here performed a side-by-side comparison of the three types of biochar (BC300, BC400, and BC500, the pyrolytic temperatures) with the different properties in enhancing the anaerobic digestion of kitchen wastes. The results showed that, in the presence of biochar with the properties of the higher electrical conductance but less redox-active organic functional groups (BC500), the improved performances involved in methane production rates (673.6 mL/d vs 956.8 mL/d) and organic removal efficiencies (69.9% vs 76.4%) were lower than that in the presence of biochar with the properties of the lower electrical conductance but more redox-active organic functional groups (BC400). Biochar (BC400) with the properties of more redox-active organic functional groups enriched the higher abundance of Petrimonas species and Anaerolineaceae capable of electron transfer to the conductive materials than biochar (BC500) with the properties of the higher electrical conductance. The study proposed a strategy of engineering a better anaerobic digestion of kitchen wastes via optimizing the pyrolytic temperature of biochar to form more redox-active organic functional groups as possible. • Biochar with different properties was used in anaerobic digestion of kitchen wastes. • Biochar with the higher conductance did not enhance the better anaerobic digestion. • Biochar with more redox-active functional groups improved the higher performances. • Petrimonas and Anaerolineaceae were specially enriched in the presence of biochar. [ABSTRACT FROM AUTHOR]

[Display omitted] • Possibility of facilitating DIET with different NaCl concentrations was explored. • Additional NaCl at 2 or 4 g/L stimulated growth of Methanosarcina species. • Electrochemical activities of Methanosarcina -dominant communities were increased. • Abundance of genes for electron transfer and CO 2 reduction were increased. • More ATP were yielded to speed up electron transfer and CO 2 reduction in methanogens. The possibility of facilitating direct interspecies electron transfer (DIET) in anaerobic digestion with different concentrations of NaCl was explored. Additional NaCl at 2 or 4 g/L strengthened anaerobic digestion to resist the high-organic loading rate impacts, whereas the higher concentrations of NaCl (6 or 8 g/L) suppressed methanogenesis. Additional MgCl 2 with the same ion strength as NaCl at 2 g/L had no effect on performances. Additional NaCl at 2 or 4 g/L dramatically increased the abundance of Methanosarcina species (20.7%/23.4% vs 8.6%) and stimulated the growth of Sphaerochaeta and Petrimonas species that could transfer electrons to the soluble Fe(III) or elemental sulfur. Electrochemical evidences showed that, additional NaCl at 2 or 4 g/L increased capacitances and decreased charge transfer resistances of Methanosarcina -dominant communities. Metagenomic evidences showed that, additional NaCl at 2 or 4 g/L increased the abundance of genes that encoded the type IV pilus assembly proteins (1.98E-04/1.87E-04 vs 1.85E-04) and cytochrome c -like proteins (5.51E-04/5.60E-04 vs 5.31E-04). In addition, additional NaCl at 2 or 4 g/L increased the abundance of genes for methanophenazine (MP)/MPH 2 transformation (1.04E-05/1.24E-05 vs 8.06E-06) and CO 2 reduction (1.64E-03/1.86E-03 vs 1.06E-03), suggesting a rapid transmembrane transport of electrons and CO 2 reduction in methanogens. Both processes were closely associated with F 420 /F 420 H 2 transformation that required ATP. Additional NaCl at 2 or 4 g/L increased the yield of ATP (256.0/249.3 vs 231.8 nmol/L) that might promote F 420 /F 420 H 2 transformation in methanogens, which overcame the thermodynamic limitations of combining electrons with protons for the reduction of CO 2 to methane and facilitated DIET. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) between Geobacter species and Methanosaeta species is an alternative to interspecies hydrogen transfer (IHT) in anaerobic digester, which however has not been established in anaerobic sludge digestion as well as in bioelectrochemical systems yet. In this study, it was found that over 50% of methane production of an electric-anaerobic sludge digester was resulted from unknown pathway. Pyrosequencing analysis revealed that Geobacter species were significantly enriched with electrodes. Fluorescence in situ hybridization (FISH) further confirmed that the dominant Geobacter species enriched belonged to Geobacter metallireducens. Together with Methanosaeta species prevailing in the microbial communities, the direct electron exchange between Geobacter species and Methanosaeta species might be an important reason for the 'unknown' increase of methane production. Conductivity of the sludge in this electric-anaerobic digester was about 30% higher than that of the sludge in a control digester without electrodes. This study not only revealed for the first time that DIET might be the important mechanism on the methanogenesis of bioelectrochemical system, but also provided a new method to enhance DIET by means of bioelectric enrichment of Geobacter species. [ABSTRACT FROM AUTHOR]

The production of reactive oxygen species (ROS) in the rhizosphere is limited by the low extracellular electron transfer capacity of indigenous microorganisms. In the present study, electrical stimulation was used to promote the generation of rhizospheric ROS by accelerating extracellular electron transfer. The result showed that •OH concentrations in the electrically stimulated group (ES group) exceeded the control group by 15.76 %. Accordingly, the removal rate of the target pollutant (i.e., 2,4-dichlorophenol, and sulfamethoxazole) was 20.01 %−24.80 % higher in the ES group than in the control group. The sediment of the ES group had a higher capacity (30.55 %) and a lower electrical resistance (29.15 %) compared to the control group, which subsequently promoted the dissimilatory iron reduction to produce Fe(II) for triggering a Fenton-like process. The increased extracellular respiratory capacity under electrical stimulation could be attributed to the polarization of C-N and C O bonds, which provided more electron storage sites and thus participated in proton-coupled electron transfer. In addition, the concentration of ATP and co-enzymes (NADH/NAD+ and Complex I/Complex III), reflecting electron exchange within respiratory chains, increased distinctly under electrical stimulation. Applying electrical stimulation seemed feasible to increase ROS production and contaminant degradation in the rhizosphere, deepening the understanding of electrical stimulation to promote the production of ROS in the natural system. [Display omitted] • Rhizospheric ROS production was enhanced by 15.76 % under electrical stimulation. • Electrical stimulation promoted sediment extracellular respiratory capacity. • Polarization of C-N and C O bonds enhanced proton-coupled electron transfer. • Co-enzymes reflecting electron exchange in respiratory chains increased distinctly. • The removal rate of the target pollutants was elevated by 20.01 %−24.80 %. [ABSTRACT FROM AUTHOR]

[Display omitted] • Humic acid delayed secondary mineralization of ferrihydrite during sulfate reduction. • Ferrihydrite-humic acid coprecipitate promoted ASR via enrich more DIR species. • Fh-HA group enriched functional genes related to assimilatory sulfate reduction. Sulfide produced from dissimilatory sulfate reduction can combine with hydrogen to form hydrogen sulfide, causing odor issues and environmental pollution. To address this problem, ferrihydrite-humic acid coprecipitate was added to improve assimilatory sulfate reduction (ASR), resulting in a decrease in sulfide production (190.2 ± 14.6 mg/L in the Fh-HA group vs. 246.3 ± 8.1 mg/L in the Fh group) with high sulfate removal. Humic acid, adsorbed on the surface of ferrihydrite, delayed secondary mineralization of ferrihydrite under sulfate reduction condition. Therefore, more iron-reducing species (e.g. Trichococcus , Geobacter) were enriched with ferrihydrite-humic acid coprecipitate to transfer more electrons to other species, which led to more COD reduction, an increase in electron transfer capacity, and a decrease in the NADH/NAD+ ratio. Metagenomic analysis also indicated that functional genes related to ASR was enhanced with ferrihydrite-humic acid coprecipitate. Thus, the addition of ferrihydrite-humic acid coprecipitate can be considered as a promising candidate for anaerobic sulfate wastewater treatment. [ABSTRACT FROM AUTHOR]

• Link between intracellular electron transfer and expression for e-pili was explored. • Aggregate conductance with ethanol had a strong dependence on temperature/pH. • Ethanol increased expression of genes encoding total type IV pilus assembly proteins. • Ethanol promoted NADH/NAD+ transformation on complex i and ATP synthesis on complex v. Ethanol feeding has been widely documented as an economical and effective strategy for establishing direct interspecies electron transfer (DIET) during anaerobic digestion. However, the mechanisms involved are still unclear, especially on correlation between intracellular electron transfer in electroactive bacteria and their gene expression for electrically conductive pili (e-pili), the most essential electrical connection component for DIET. Upon cooling from room temperature, the conductivity of digester aggregates with ethanol exponentially increased by an order of magnitude (from 45.5 to 125.4 μS/cm), whereas which with its metabolites (acetaldehyde [from 40.5 to 54.4 μS/cm] or acetate [from 32.1 to 50.4 μS/cm]) did not increase significantly. In addition, the digester aggregates only with ethanol were observed with a strong dependence of conductivity on pH. Metagenomic and metatranscriptomic analysis showed that Desulfovibrio desulfuricans was the most dominant and metabolically active bacterium that contained and highly expressed the genes for e-pili. Abundance of genes encoding the total type IV pilus assembly proteins (6.72E-04 vs 1.24E-03, P < 0.05), PilA that determined the conductive properties (2.22E-04 vs 2.44E-04, P > 0.05), and PilB that proceeded the polymerization of pilin (1.56E-04 vs 3.52E-03, P < 0.05) with ethanol was lower than that with acetaldehyde. However, transcript abundance of these genes with ethanol was generally higher than that with acetaldehyde. In comparison to acetaldehyde, ethanol increased the transcript abundance of genes encoding the key enzymes involved in NADH/NAD+ transformation on complex I and ATP synthesis on complex V in intracellular electron transport chain. The improvement of intracellular electron transfer in D. desulfuricans suggested that electrons were intracellularly energized with high energy to activate e-pili during DIET. [Display omitted] [ABSTRACT FROM AUTHOR]

Possibility of boosting conversion of waste activated sludge to methane via facilitating direct interspecies electron transfer with glycerol was explored. Methane yields with 5 %/8 % glycerol were 1.18/1.31 folds higher than that without glycerol. After removing methane yields from glycerol, methane yields with 5 %/8 % glycerol was still 17 %/19 % higher than that without glycerol. Electron transfer coefficients for charge/discharge in sludge with 8 % glycerol were 1.12/1.39 folds higher than that without glycerol. Analysis of conductivity-temperature responses found that conductivity of sludge with 8 % glycerol exhibited an exponential increase when temperature was declined, similar to that with 8 % ethanol. However, conductivity of sludge with 8 % glycerol was one order of magnitude higher than that with 8 % ethanol. Electrochemical Fourier transform infrared spectra showed that reversible changes of bands associated with c -type cytochrome in sludge with 8 % glycerol were more intense than that with 8 % ethanol. Analysis of microbial communities showed that glycerol feeding increased the abundance of Methanospirillum hungatei JF-1, and specially enriched Tangfeifania diversioriginum , Syntrophomonas zehnderi OL-4 and Trichococcus pasteurii. These results indicated that glycerol feeding facilitated the better direct interspecies electron transfer than ethanol feeding that had been previously documented during anaerobic digestion of waste activated sludge. [Display omitted] [ABSTRACT FROM AUTHOR]

• Dispersive attraction of MIL-88B(Fe) enhanced CO 2 adsorption on cathodic biofilm. • CC/MIL-88B(Fe) electrode accelerated methane production in anaerobic digestion. • E-pili and cytochrome C were highly expressed in the CC/MIL-88B(Fe) biofilm. • MIL-88B(Fe) boosted the establishment of DIET in MEC-based methanogenesis systems. Direct interspecies electron transfer (DIET), as a new metabolic pathway for methanogenesis, is difficult to establish in common anaerobic digesters due to the lack of electroactive methanogens, syntrophic electroactive bacteria, and conductive proteins. In this study, a synthetic CC/MIL-88B electrode was used to accelerate the establishment of DIET in a microbial electrolysis cell (MEC)-based anaerobic digester. Results showed that the CC/MIL-88B electrode had a strong dispersive attraction generated by its three-dimensional aromatic ring structure, which benefited the capture of CO 2 in the biofilm attached to the electrode and further increased the reduction of CO 2 to produce methane via DIET. Electrochemical and metaproteomics analysis indicated that the MIL-88B(Fe) stimulated the secretion of conductive proteins (such as e-pili and cytochrome C). The abundances of electroactive methanogen Methanosarcina and other electroactive bacteria increased to 4.89 and 1.37 times, respectively. In addition, the expression of the key enzymes related to DIET-based methanogenesis (Fpo and Rnf) of Methanosarcina and the pathway of CO 2 -reduction methanogenesis were enhanced significantly. These results indicated that the synthetic CC/MIL-88B electrode helped the establishment of DIET in the MEC system, which provided a new perspective to establish DIET during anaerobic digestion by accelerating the utilization of CO 2. [ABSTRACT FROM AUTHOR]

Conversion of organic wastes to methane via anaerobic digestion has been practiced for more than 160 years. However, the technical bottlenecks derived from the thermodynamic limitations of interspecies hydrogen/formate transfer (IHT/IFT) between syntrophs and methanogens resulting in the slow fermentation rate, low methane conversion efficiency and poor stability of system still limit its large-scale application. No engineering strategy to speed up IHT/IFT for dramatically changing the performances of anaerobic digestion has been developed. However, recent studies documented that simply pretreating organic wastes to produce ethanol could effectively accelerate and stabilize anaerobic digestion. This may be linked to the promotion of direct interspecies electron transfer (DIET), an alternative working mode that no longer requires hydrogen or formate as an electron carrier. Further information on how this strategy affects the function of methanogenic communities to clarify its advantages is required. In this study, we summarized the evidence that pretreating organic wastes to produce ethanol enhanced anaerobic digestion, discussed the link between ethanol and DIET, and emphasized how this strategy would be better applied in the future. [Display omitted] • Evidence that ethanol-type fermentation enhanced anaerobic digestion was summarized. • Link between ethanol and direct interspecies electron transfer was discussed. • Engineering a better anaerobic digestion by ethanol-type fermentation was emphasized. [ABSTRACT FROM AUTHOR]

[Display omitted] • Embedded CDs accelerated the cross-wall electron transfer of microbial respiration. • Enhanced microbial respiration promoted microbial advanced oxidation of aniline. • CDs served as the electron transfer bridge to induce oxygen reduction extracellularly. • CDs polarized amide groups in the cell wall and enhanced PCET in cell respiration. Microbial advanced oxidation, a crucial pathway for pollutant decomposition in the natural environment, is typically limited by microbial extracellular respiration. This is because the electro-inert cell walls of common microorganisms, such as Bacillus subtilis , hinder the cross-wall electron transfer necessary for reducing ferric species and inducing Fenton-like reactions. In this study, carbon quantum dots (CDs) were fed to promote the extracellular respiration of B. subtilis to strengthen the microbial advanced oxidation of aniline. The results showed that the CDs were embedded in the cell wall of bacterial cells to improve the extracellular electron transfer, which increased the •OH production (by 60.25%) and the aniline degradation. Meanwhile, the •OH generation mainly increased outside of the cell, which was beneficial for the degradation of pollutants and for relieving the damage of the radical to bacterial cells. The CDs induced the polarization of amide groups in the cell walls to maintain actively proton-coupled electron transfer to increase ATP generation by 39.28%. Feeding CDs provided a potential strategy to strengthen the extracellular respiration of common microorganisms and increase pollutant degradation via microbial advanced oxidation in the environment. [ABSTRACT FROM AUTHOR]

• Ethanol-type fermentation with magnetite during AD of kitchen waste was established. • Magnetite stimulated the secretion and improved electron transfer capacity of EPS. • Syntrophomonas transfer electron to Methanospirillum and Methanobacterium via EPS. • The addition of magnetite improved system stability by establishment of DIET. Magnetite, a common mineral that is abundant in the soils and sediments, has been widely documented to enhance the anaerobic digestion of organic wastes, whereas the mechanisms of magnetite promoting interspecies electron transfer are still unclear. In this study, under the conditions (ethanol-type fermentation) employed, magnetite stimulated the secretion of extracellular polymeric substances (EPS). Analysis of three-dimensional excitation emission matrix revealed that these EPS secreted in the presence of magnetite were primarily comprised of the redox-active organic functional groups. Electrochemical analysis showed that the EPS secreted with magnetite had the higher electron-accepting and electron-donating capacity than the EPS without magnetite. Syntrophomonas species capable of extracellularly transferring electron were enriched with supplementing magnetite. Together with the increased abundance of Methanospirillum and Methanobacterium species that could proceed direct interspecies electron transfer (DIET), the anaerobic digestion was likely improved due to the establishment of DIET with supplementing magnetite. As a result, anaerobic digestion of kitchen wastes was evidently enhanced. With decreasing the solid retention time to 30 d, the methane production rate only slightly declined to 18 ± 0.8 mL/g-VSS/d in the magnetite-supplemented digester, while almost no methane was detected in the digester without magnetite. [ABSTRACT FROM AUTHOR]

Biochar, with redox moieties or conjugated π-bond, can act as electron shuttle or conductor to facilitate electron transfer of syntrophic metabolism to enhance anaerobic digestion. High pyrolysis temperature (>500 ℃) is usually required to prepare conductive biochar, which however may cause biochar to loss redox moieties such as quinone/hydroquinone that are capable of serving as electron shuttle. Considering that magnetite is an excellent conductor which has been applied in improving syntrophic metabolism of anaerobic digestion, a novel magnetite-contained biochar was prepared using iron-rich Fenton sludge as raw material in this study. Amorphous iron oxides of Fenton sludge were transformed into magnetite at 400 ℃ of pyrolysis, while redox quinone/hydroquinone moieties of biochar were preserved well. Correspondingly, this magnetic biochar owned both high capacitance and excellent conductivity. When supplementing the biochar into an anaerobic digestion system, methane production was significantly enhanced. This study also offered a new approach to recycle Fenton sludge that is regarded as hazardous material. ga1 • After the pyrolysis, iron oxides and organic matters in Fenton sludge were transformed into magnetite and biochar. • The Fenton sludge derived biochar owns both high capacitance and excellent conductivity. • This novel biochar facilitated electron transfer between syntrophic microorganism. • An alternative method was provided to dispose Fenton sludge. [ABSTRACT FROM AUTHOR]

• Dry anaerobic digestion might be operated well with zero-valent iron addition. • The higher yields of CH 4 achieved with adding 10 g/L ZVI. • The electron transfer capacity and electroactive proteins were improved. • Methanothrix and Methanosarcina as DIET-partners were enriched by ZVI addition. Although commonly viewed as a promising method, dry anaerobic digestion is not been widely applied to dispose of food wastes, especially in developing countries because of its insufficiency in handling with lower mass transfer and high acidic accumulation of the system. Zero valent iron (ZVI) has been found to demonstrate superior performance such as enhancing methane production. However, up to date, the mechanism of ZVI remains unclear. In this study, adding 5 g/L ZVI could improve interspecies hydrogen transfer (IHT) to enhance the dry anaerobic digestion of food wastes, but was unable to resist the shocks of high organic loading. With increasing ZVI dosage to 10 g/L, the performances of digestion systems were improved to maintain the systems stable. With 10 g/L of ZVI addition, electron transfer capacity of the sludge increased by 5.4 folds, and electroactive proteins of sludge increased by 2.3 folds. Microbial community analysis also indicated that the relative abundances of Methanothrix and Methanosarcina performing direct interspecies electron transfer were enriched to 67.5% and 27.2% with 10 g/L ZVI addition, respectively. These results suggested that direct interspecies electron transfer could be established with a proper dosage of ZVI that served as a conductive material to connect electron exchange among microorganisms. Thus, ZVI played a dual role including improving interspecies hydrogen transfer and promoting direct interspecies electron transfer to keep the systems efficient to treat high-solid food wastes. [ABSTRACT FROM AUTHOR]

Anaerobic co-digestion (AcoD) has been widely applied to the disposal of waste activated sludge (WAS), since it optimizes the C/N ratio and decreases the buffer capacity, which dose not solve the problem involved in the slow methanogenic metabolism that limits methanogenesis yet. In this study, a strategy of initially fermenting the polysaccharide-rich organic wastes to produce the ethanol-contained fermentation liquid (EFL) that was then mixed with WAS for further AcoD was proposed, with the aim of establishing direct interspecies electron transfer (DIET). The results showed that, the methane production rates and organic removal efficiencies in the digesters treating WAS mixed with EFL were significantly higher than that in the single-phase AcoD system about 30% and 6–7%, respectively, but the improved performances in the digesters treating WAS mixed with the neutral-pH fermentation liquid (FL) were not significant, which resulted in a low-efficiency energy recovery. The conductivity of sludge fed with WAS mixed with EFL were about 3–5 folds higher than that with the neutral-pH FL, suggesting that the DIET-based metabolism was established. Together with the special enrichment of Fe(III)-reducing genus involved in Rhodoferax and high-abundance Methanospirillum capable of producing the electrical pili, a novel DIET between Rhodoferax and Methanospirillum was inferred. Image 1 • DIET-based metabolism was established during AcoD of WAS with EFL. • Digester with ethanol-type FL presented a high-efficiency methanogenesis. • Rhodoferax known as Fe(III)-reducing genus was specifically enriched with ethanol. • Results suggested that Rhodoferax might proceed a novel DIET with Methanospirillum. [ABSTRACT FROM AUTHOR]

• New strategy for co-fermentation of corn straw and waste activated sludge. • Ethanol produced of CS increased the methane yield in AcoD digester based on DIET. • Compared to commercial ethanol producing, this strategy has more economic benefits. • The VSS removal rate was higher than ethanol production from CS and common AD of WAS. Commercial ethanol production from straw is a series of complex processes that are energy-intensive and uneconomical. Corn straw was used as a bioethanol source to mix with waste activated sludge for improving anaerobic co-digestion (AcoD) in this study. Ethanol production from the straw after yeast fermentation was 1400–2200 mg COD/L, accounting for about 0.1% of the fermentative effluent, but methane production of the yeast-group increased by 36% compared to that of control-group with no ethanol production in advance. Volatile suspended solid removal achieved 60%, obviously higher than common anaerobic digestion (AD). Multiple lines of evidence including sludge conductance, effects of activated carbon on methanogenesis and microbial community demonstrated that ethanol from the straw fermentation stimulated direct interspecies electron transfer to be established in the digesters. The results suggested that using ethanol produced from straw was a cost-effective novel way for energy recovery from disposal of agricultural and municipal wastes. [ABSTRACT FROM AUTHOR]

Zero-valent iron (ZVI) has been widely reported to strengthen anaerobic digestion; however, ZVI passivation prevents its continuous function. In this study, a dynamic magnetic field (DMF) was imposed on a ZVI-added anaerobic digester treating food waste to promote microbial interspecies electron transfer to enhance methane production. Results showed that methane production with DMF was 15.3% higher than that without DMF. Meanwhile, the Fe(II) concentration from ZVI increased by 39.46% with DMF, but the electron equivalent was far less than the increased methane production even if all electrons from the ZVI were used for hydrogenotrophic methanogenesis. In agreement, the relative abundances of hydrogenotrophic methanogens were less changed after DMF. Notably, an induced electric current ranging from −0.017–0.021 μA was detected with DMF. With the DMF-induced current as electrical stimulation, the sludge conductivity and capacitance increased by 242% and 18%, respectively. In-situ electrochemical tests on the functional groups of the sludge showed that the protein-like substances were polarized by the induced electromotive force to enhance the proton-coupled electron transfer in the sludge. Together with the enriched electrotrophic methanogens, the enhanced microbial interspecies electron exchange was the main reason for the improved performance of anaerobic digestion with DMF. [Display omitted] [ABSTRACT FROM AUTHOR]

Anaerobic digestion (AD) is one of the paramount technologies for organic wastes treatment. However, there still remain intractable issues including low digestibility and sluggish reaction kinetics in its practical implementation, leading to limited bio-product production. Recently, carbon-based additives (CAs) have aroused great attentions for enhanced AD performance by establishing direct interspecies electron transfer (DIET) and adjusting the functional microorganisms due to their large specific surface area and high conductivity. However, such inherent physicochemical capabilities of CAs and its specific role in AD have not been fully exploited. In this review, we detailedly summarize the research advances of biochar and activated carbon (AC) as additives in AD system, which are combed in terms of structure construction of CAs, macro-behaviors of CAs, micro-mechanisms of CAs and hybrid strategy. Finally, perspectives regarding future modification and application of CAs in AD system are also discussed. • The latest research advances of CAs in AD system are summarized. • The CAs amendment of AD replies on its inherent physicochemical capabilities. • The role of CAs is combed on macro-behaviors, micro-mechanisms and hybrid strategy. • The future development directions of CAs are proposed. [ABSTRACT FROM AUTHOR]

ZVI was reported to enrich H 2 -utilizing methanogens that enhanced interspecies H 2 transfer, while Fe(III) oxide served as a conductive material to promote direct interspecies electron transfer (DIET). However, the interaction of these two modes in anaerobic digestion has not been clarified yet. In this study, when adding Fe 3 O 4 and ZVI simultaneously into an anaerobic digester, the abundance of hydrogenotrophic methanogens decreased drastically compared to ZVI-added digester and Fe-free digester. However, the methane production of ZVI + Fe 3 O 4 added digester were 68.9% higher than Fe-free digester and 20.0% higher than ZVI-added digester, respectively. Sludge reduction rate of these three digesters also showed similar results. These indicated that hydrogenotrophic methanogenesis was not the main reason for methanogenesis in Fe 3 O 4 -added digester. Instead, Syntrophomonas and Methanosaeta were specially enriched in Fe 3 O 4 -added digesters, which implied that the potential DIET between Syntrophomonas and Methanosaeta was likely a crucial reason for accelerating anaerobic digestion of waste sludge. [ABSTRACT FROM AUTHOR]

In this study, Fe(OH) 3 was dosed into anaerobic reactor (R1) to stimulate the electron transfer process during the decoloration treatment. As results indicated, the chemical oxygen demand (COD) removal and decoloration efficiency were increased by 61.7% and 32.0% than that of the control reactor (R2), respectively. The cyclic-voltammetric analysis of the effluent demonstrated that the cycles of Fe(III)/Fe(II) and 1-imino-2-napthoquinone/1-amino-2-napthol (oxidative/reductive state of the decoloration intermediate product) had been established in R1. The concentration of cytochrome c and the conductivity of suspended sludge in R1 were also 3.2 and 2.1 times higher than that in R2. All experiments above indicated that the electron transfer process between substrates and azo bonds was accelerated efficiently. Furthermore, the abundance of iron reducing bacteria was higher than that of R2, indicating that dissimilatory iron reduction as a reason for the Fe(III)/Fe(II) cycle played an important role in the anaerobic decoloration treatment. [ABSTRACT FROM AUTHOR]

• Exposure to a dynamic magnetic field enhanced the bio-methanogenesis in sediment. • Dynamic magnetic field induced electromotive force in sediment as electrical signals. • Electromotive force polarized amide groups to improve the sediment electro-activity. • Polarized proteins benefited proton-coupled electron transfer in respiration chains. • Enhanced proton out-flow in respiration chains promoted methanogenesis in sediment. As a primary driving force of global methane production, methanogens like other living organisms are exposed to an environment filled with dynamic electromagnetic waves, which might induce electromotive force (EMF) to potentially influence the metabolism of methanogens. However, no reports have been found on the effects of the induced electromotive force on methane production. In this study, we found that exposure to a dynamic magnetic field enhanced bio-methanogenesis via the induced electromotive force. When exposed to a dynamic magnetic field with 0.20 to 0.40 mT of intensity, the methane emission of the sediments increased by 41.71%. The respiration of methanogens and bacteria was accelerated by the EMF, as the ratios of F 420 H 2 /F 420 and NAD+/NADH of the sediment increased by 44.12% and 55.56%, respectively. The respiratory enzymes in respiration chains might be polarized with the EMF to accelerate the proton-coupled electron transfer to enhance microbial metabolism. Together with the enriched exoelectrogens and electrotrophic methanogens, as well as the increased sediment electro-activities, this study indicated that the EMF could enhance the electron exchange among extracellular respiratory microorganisms to increase the methane emission from sediments. [Display omitted] [ABSTRACT FROM AUTHOR]

• Electrical stimulation enhanced the electroactivity of EPS and promotes EET. • Polarized amide groups boosted the dipole moment of α-helix peptide of EPS protein. • Polarization decreased the IP and E LUMO -E HOMO of C-terminal of the α-helix peptide. • α-helix peptide was easier to as ET site mediate ET under electrical stimulation. Electroactivity is an important parameter to assess the ability of the extracellular polymeric substance (EPS) of microorganisms to participate in extracellular respiration. Many reports have found that the electroactivity of microbial sludge could be enhanced with electrical stimulation, but the reason remains unclear. The results of this study showed that the current generation of the three microbial electrolysis cells increased by 1.27–1.76 times during 49 days of electrical stimulation, but the typical electroactive microorganisms were not enriched. Meanwhile, the capacitance and conductivity of EPS of sludge after the electrical stimulation increased by 1.32–1.83 times and 1.27–1.32 times, respectively. In-situ FTIR analysis indicated that the electrical stimulation could lead to the polarization of amide groups in the protein, likely affecting the protein structure related to the electroactivity. Accordingly, the dipole moment of the α-helix peptide of protein of sludge increased from 220 D to 280 D after the electrical stimulation, which was conducive to electron transfer in the α-helix peptide. Moreover, the vertical ionization potential and E LUMO -E HOMO energy gap of the C-terminal in the α-helix peptide decreased from 4.43 eV to 4.10 eV and 0.41 eV to 0.24 eV, respectively, which indicated that the α-helix was easier to serve as the electron transfer site of electron hopping. These results meant that the enhancement of the dipole moment of the α-helix peptide unchoked the electron transfer chain of the protein, which was the main reason for the increased electroactivity of EPS protein. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • AOM coupled with EET to electrodes was accelerated with magnetite. • Current density was increased by approximately 6–9 folds with magnetite. • Biofilms with magnetite were highly conductive compared to that without magnetite. • Biofilms with magnetite were displayed with high-concentration redox-active proteins. • Magnetite increased the abundance of genes encoding Rnf and HdrABC. Methane-dependent bioelectrochemcial systems (BESs) have been recently considered as a promising approach for mitigating methane emission, energy harvesting as well as water environment remediation. However, the mechanisms of extracellular electron transfer (EET) from anaerobic methanotrophic (ANME) archaea/methanogens to electrodes are still poorly understood, which limits the further improvement of methane oxidation and current generation. The study presented here showed that, in the presence of magnetite, anodic biofilms primarily comprised of ANME archaea/methanogens were highly conductive, compared to that without magnetite. Further examination with scanning electrochemical microscopy and electrochemical Fourier transform infrared spectra found that, anodic biofilms with magnetite were redox-based conductive, with a higher concentration of redox-active proteins displayed on their surface than that without magnetite. Metagenomic analysis showed that, the difference in the abundance of genes encoding the cytochrome c (CYC)-like proteins in the anodic biofilms with or without magnetite was not significant. However, magnetite specially increased the abundance of genes encoding the membrane-bound proteins, Rnf, and periplasmic heterodisulfide reductase, HdrABC, contained in ANME archaea/methanogens, providing a possibility that magnetite sped up methane oxidation as well as electron transfer from inner membrane to outer membrane in AOM coupled with EET to electrodes. As a result, the current density in methane-dependent BES with magnetite was increased by approximately 6–9 folds. [ABSTRACT FROM AUTHOR]

[Display omitted] • NH 4 +-N removal efficiency increased by 23.53 % with Fe(OH) 3 in the Anammox reactor. • Fe(OH) 3 -added reactor had higher Feammox activity than Fe(OH) 3 -free reactor. • Magnetite and wustite was produced by Feammox in the reactor added with Fe(OH) 3. • Outer membrane cytochrome c of Fe(OH) 3 -added Anammox could participate in EET. • Redox enzymes of cytochrome c of Candidatus Brocadia were enriched by Fe(OH) 3. It is widely found that the ratio of nitrite consumption to ammonia consumption (ΔNO 2 --N/ΔNH 4 +-N) in Anammox systems is lower than its stoichiometry of 1.32, but the reason is unclear. In this study, ΔNO 2 --N/ΔNH 4 +-N of an Anammox system decreased to 0.80 with the addition of Fe(OH) 3 , which was significantly lower than a control system without the addition of Fe(OH) 3. The lower ratio or more NH 4 + removal was likely related to the reduction of Fe(OH) 3 that served as extracellular electron acceptor to anaerobically oxidize NH 4 +, namely Feammox. Chemical or biological oxidation of the produced Fe2+ formed the Fe(III)/Fe(II) cycle to likely cause the continuous Feammox in the Anammox system. Isotopic analysis verified that the Anammox sludge collected from the Fe(OH) 3 -added reactor produced more 30N 2 by Feammox. Raman analysis showed that the outer membrane cytochrome c of Fe(OH) 3 -added Anammox sludge could participate in the extracellular electron transfer during Feammox. Metagenomic analysis further confirmed that the gene encoding redox enzymes of cytochrome c of Candidatus Brocadia were enriched with adding Fe(OH) 3. The results suggested that the Fe(OH) 3 induced Anammox bacteria to perform extracellular electron transfer, allowing the Anammox system to effectively remove NH 4 + via the Feammox pathway. [ABSTRACT FROM AUTHOR]

Bio-methanogenic digestion of incineration leachate is hindered by high OLRs, which can lead to build-up of VFAs, drops in pH and ultimately in reactor souring. It was hypothesized that incorporation of carbon cloth into reactors treating leachate would promote DIET and enhance reactor performance. To examine this possibility, carbon cloth was added to laboratory-scale UASB reactors that were fed incineration leachate. As expected, the carbon-cloth amended reactor could operate stably with a 34.2% higher OLR than the control (49.4 vs 36.8 kg COD/(m 3 d)). Microbial community analysis showed that bacteria capable of extracellular electron transfer and methanogens known to participate in DIET were enriched on the carbon cloth surface, and conductivity of sludge from the carbon cloth amended reactor was almost twofold higher than sludge from the control (9.77 vs 5.47 μS/cm), suggesting that microorganisms in the experimental reactor may have been expressing electrically conductive filaments. [ABSTRACT FROM AUTHOR]

• Fe (VI) pretreatment increased MCFAs production from WAS by 58.6 times. • OH, ·O 2 − and 1O 2 were the main ROS for destroying EPS and cytoderm. • Fe (VI) facilitated the formation and targeted conversion of intermediates. • Fe (VI) screened key microbes and up-regulated functional genes expression. • Fe (VI) avoided methanogens inhibitor addition and stimulated vivianite synthesis. The rupture of cytoderm and extracellular polymeric substances (EPS), and competitive inhibition of methanogens are the main bottlenecks for medium-chain fatty acids (MCFAs) production from waste activated sludge (WAS). This study proposes a promising ferrate (Fe (VI))-based technique to enhance MCFAs production from WAS through accelerating WAS disintegration and substrates transformation, and eliminating competitive inhibition of methanogens, simultaneously. Results shows that the maximal MCFAs production attains 8106.3 mg COD/L under 85 mg Fe/g TSS, being 58.6 times that of without Fe (VI) pretreatment. Mechanism exploration reveals that Fe (VI) effectively destroys EPS and cytoderm through electron transfer, reactive oxygen species generation (i.e., OH, O 2 − and 1O 2) and elevated alkalinity, resulting in the transfer of organics from solid to soluble phase and from macromolecules to intermediates. Generation and transformation of intermediates analyses illustrate that Fe (VI) facilitates hydrolysis, acidification and chain elongation (CE) but suppresses methanogenesis, promoting the targeted conversion of intermediates to MCFAs. Also, Fe (VI) pretreatment provides potential electron shuttles for chain elongation. Microbial community and functional genes encoding key enzymes analysis indicates that Fe (VI) screens key microorganisms and up-regulates functional genes expression involved in CE pathways. Overall, this technology avoids methanogens inhibitor addition and stimulates vivianite synthesis during MCFAs production from WAS. [Display omitted] [ABSTRACT FROM AUTHOR]

The aim of this work was to study the methanogenic metabolism of dog food, a food waste surrogate, in laboratory-scale reactors with different carbon-based conductive materials. Carbon cloth, carbon felt, and granular activated carbon all permitted higher organic loading rates and promoted faster recovery of soured reactors than the control reactors. Microbial community analysis revealed that specific and substantial enrichments of Sporanaerobacter and Methanosarcina were present on the carbon cloth surface. These results, and the known ability of Sporanaerobacter species to transfer electrons to elemental sulfur, suggest that Sporanaerobacter species can participate in direct interspecies electron transfer with Methanosarcina species when carbon cloth is available as an electron transfer mediator. [ABSTRACT FROM AUTHOR]

[Display omitted] • The nitrogen removal rate of Anammox sludge increased by 39 % with adding Fe(OH) 3. • Feammox generated 30N 2 was observed in an Anammox consortia. • Fe(OH) 3 spurred the EET related enzyme activity of Anammox sludge. • Electron-storage capacity of Anammox sludge increased with Fe(OH) 3. Feammox has been applied to wastewater biological nitrogen removal. However, few studies have reported that Fe(III)(hydr)oxides induced Anammox consortia to remove NH 4 + via the Feammox pathway. In this study, Fe(OH) 3 was added to Anammox systems to investigate its effect on nitrogen removal via Feammox. The specific Anammox activity increased by 39 % by Fe(OH) 3. Ammonia oxidation was observed to occur along with Fe(III) reduction and Fe(II) generation, which was further confirmed by the isotope test with feeding 15NH 4 +-N to detect 30N 2. The cyclic voltammetry test showed that electron-storage capacity of Anammox sludge increased with Fe(OH) 3. In situ Fourier transform infrared spectroscopy suggested that Fe(OH) 3 enhanced the polarization of functional groups of outer membrane cytochrome of Anammox consortia to benefit extracellular electron transfer. This study demonstrated that Fe(OH) 3 could induce Anammox consortia to perform extracellular respiration to enhance NH 4 +-N removal in the Anammox sludge system. [ABSTRACT FROM AUTHOR]

• Fenton sludge as an Fe(III) source of dissimilatory iron reduction improved anaerobic treatment. • Fenton sludge could be consumed during anaerobic digestion to a certain extent. • Continuously feeding Fenton sludge did not deteriorate anaerobic treatment. • Iron content in the digester decreased when stopping feeding Fenton sludge. • Feeding Fenton sludge increased the electron transfer capacity of microorganisms. Fenton sludge generated from the Fenton process contains a large number of ferric species and organic pollutants, which need to be properly treated before discharge. In this study, Fenton sludge as an Fe(III) source for dissimilatory iron reduction (DIR) was continuously added with increasing dosage into an anaerobic digester to enhance the treatment. Results showed continuously feeding Fenton sludge to the anaerobic digester did not deteriorate the performance and increased methane production and COD removal rate by 2.2 folds and 14.0%, respectively. The Fe content of sludge in the digester increased from 40.25 mg/g (dry weight) to 131.53 mg/g after continuously feeding for 77days, and then declined to 109.17 mg/g when the feeding was stopped. Mass balance analysis showed that 20.5 to 48.4% of Fe in the Fenton sludge was released to the effluent. After experiment, the ratio of reducible Fe species to the total Fe was 75.1%, which maintained the high activity in DIR. Microbial community analysis showed that iron-reducing bacteria were enriched with the addition of Fenton sludge and the sludge in the digester had a higher conductivity and capacitance to strengthen the electron transfer of DIR. All results suggested that feeding Fenton sludge into anaerobic digesters was a feasible method to dispose of Fenton sludge as well as to enhance the performance of anaerobic digestion. [Display omitted] [ABSTRACT FROM AUTHOR]

• Appling intermittent voltage improved methane yield with half energy consumption. • Accelerated IET accounted for the enhanced anaerobic digestion with electro-assist. • Intermittently applied voltage improved sludge electro-activity to promote IET. • Polarized amide groups after electro-treatment increased sludge electro-activity. Intermittent voltage supply has been reported to improve the performance of electro-assisted anaerobic digestion but has not been well understood. In this study, an intermittent voltage of 0.6 V (1 day on-1 day off) was applied in an electro-assisted anaerobic digester to explore its effects. Compared to those without the voltage, the methane yield increased nearly by 20.0%, and organic decomposition increased by 9.5% with the intermittent voltage, which was similar to those with the continuous voltage. The amide groups of the sludge protein after the electro-treatment were polarized to enhance electron transfer and electron storage of protein-like substances of the sludge. Although the voltage was supplied intermittently, the increased conductivity and capacitance of the sludge and EPS could effectively transport electrons between exoelectrogens and electrotrophs (such as Firmicutes and Methanothrix) to promote the anaerobic digestion. This study explained the essence of electrochemical enhancement of anaerobic digestion from the perspective of molecular structure, that is, the polarization of functional groups by voltage could improve the sludge electro-activity to maintain effective interspecies electron transfer in the periodic voltage supply. [ABSTRACT FROM AUTHOR]

[Display omitted] • The MCFAs yield and selectivity were respectively enhanced by 155.4% and 66.7%. • Fe 3 O 4 enhanced hydrolysis, acidification and CE processes at different degrees. • Fe 3 O 4 improved CE through improving electron transfer. • Dissimilatory iron reduction/Fe2+ had little impacts on chain elongation process. • Fe 3 O 4 screened key functional microbes for MCFAs production. Medium chain fatty acids (MCFAs) production from waste activated sludge (WAS) is restricted by poor biodegradability of WAS and low electron transfer efficiency. Herein, a novel ferroferric oxide (Fe 3 O 4) technique was proposed. Results indicated that the MCFAs yield and selectivity were respectively enhanced by 155.4% and 66.7% in the Fe 3 O 4 -mediated WAS. Mechanistic studies disclosed that Fe 3 O 4 promoted substrates degradation through conducting dissimilatory iron reduction (DIR) and stimulating hydrolase activity, providing precursors for chain elongation (CE). Generally, Fe 3 O 4 improved the key processes for MCFA production at different degrees, i.e., hydrolysis, acidification and CE. Interestingly, MCFAs yield enhancement was primarily ascribed to facilitated electron transfer rather than DIR or produced ferrous iron, which could be supported by the analyses of electrochemical properties, electron transfer system activity and morphology. Further, Fe 3 O 4 shifted the key microorganisms in favor of MCFAs production. Overall, this strategy could improve MCFAs production, sludge dewatering and phosphorus removal, concurrently. [ABSTRACT FROM AUTHOR]

• Potential of DIET within aggregates with ethanol, glycol, and glycerol was explored. • Aggregates were electrically conductive, with a metallic-like conductivity. • Genes for e-filaments and c -type cytochromes were highly expressed within aggregates. • Methanothrix were dominant and metabolically active in carbon dioxide reduction. • Possibility of alcohols comprised of hydroxy groups stimulating DIET was provided. The possibility of stimulating direct interspecies electron transfer (DIET) within aggregates of methanogenic digesters respectively with ethanol, glycol, and glycerol as a primary substrate was investigated to better understand the mechanisms of alcohol compounds stimulating DIET. Aggregates fed with ethanol, glycol, and glycerol were electrically conductive (10.4–19.4 uS/cm), with a temperature dependence of metallic-like conductivity. Close examination of transmission electron microscope images observed the potential interspecies connected networks assembled by filaments within these aggregates. Further investigations via metatranscriptomics found that, genes for electrically conductive pili (e-pili) (Log 2 FPKM, 9.39–10.96) and c -type cytochromes (8.90–9.64) were highly expressed within aggregates. Glycerol-fed aggregates exhibited the highest gene expression for e-pili, while glycol-fed aggregates exhibited the highest gene expression for c -type cytochromes. Methanothrix species were dominant and metabolically active within aggregates. Genes encoding the enzymes involved in carbon dioxide reduction were highly expressed in Methanothrix species, suggesting that they participated in DIET. In addition, transcript abundance of genes encoding alcohol dehydrogenase and NADH-quinone oxidoreductase in alcohol dehydrogenation closely associated with NADH/NAD+ transformation within glycol- and glycerol-fed aggregates was generally higher than that within ethanol-fed aggregates. These results, and the fact that NADH/NAD+ transformation was very linked to the ATP synthesis complex that further supported the formation of extracellular electrical connection components, e-pili and membrane-bound multi-heme c -type cytochromes (MHCs), provided a possibility that alcohol compounds comprised of hydroxy groups could stimulate DIET and more hydroxy groups comprised were better for this stimulation. [Display omitted] [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the primary mechanism of magnetite promoting anaerobic degradation of aromatic compounds under the low-concentration sulphate conditions. Under influent conditions of benzoate at 50 mM-chemical oxygen demand (COD) and sulphate at 15 mM, magnetite promoted benzoate degradation (77.1% vs 56.3%), while the effluent sulphate concentration was slightly higher than that without magnetite (1.6 mM vs 0.7 mM), inconsistent with functional gene prediction that both sulphate respiration and sulphur compound respiration were relatively more active in the presence of magnetite. Remarkably, X-ray diffraction showed that, signal related to Fe 3 O 4 faded away and finally was replaced by FeSO 4 and FeS, indicating that magnetite participated in benzoate degradation coupled to sulphate reduction via dissimilatory Fe(III) reduction. Further X-ray photoelectron spectroscopy showed that, signal related to S0 was only detected with magnetite, suggesting the possibility of re-oxidation of sulphide to elemental sulphur coupled to Fe(III) reduction. This was further supported by the increase in abundance of Desulfuromonas acetexigens capable of growing on Fe(III). In addition, magnetite specially enriched the chemolithotrophic sulphur-disproportionating microbes, Desulfovibrio aminophilus , which might proceed the disproportionation of elemental sulphur to sulphate and sulphide to achieve a sulphur cycle for benzoate degradation. [Display omitted] • Magnetite enhanced benzoate degradation under low-concentration sulphate conditions. • Signal related to magnetite in X-ray diffraction spectra was no longer detected. • Elemental sulphur was dominant reduced sulphur species in presence of magnetite. • Both sulphate/Fe(III)-reducing and sulphur-disproportionating microbes were enriched. • Magnetite mediated a sulphur cycle rather than direct interspecies electron transfer. [ABSTRACT FROM AUTHOR]

[Display omitted] • Startup of a methane-dependent denitrification reactor was shortened with magnetite. • Magnetite mediated high-capacitance/low-resistance electrically connected community. • Both methane-oxidizing and nitrate-reducing microbes were enriched with magnetite. • Methane oxidation with magnetite was insensitive to external addition of hydrogen. • They might participate in DIET via magnetite substituting for c -type cytochromes. Denitrifying anaerobic methane oxidation (DAMO) not only mitigates methane emission from wastewater treatment, but compensates for the deficiency of current nitrogen removal process. However, the doubling time of DAMO microbes is usually more than two weeks, resulting in a long-period startup of a methane-dependent denitrification reactor especially with anaerobic sludge as inoculum. The study presented here found that, in the presence of magnetite, the nitrate removal rate reached 17.6 ± 0.4 mgNO 3 −-N/L/d after the 120-day experiments, which was approximately 2.4 folds higher than that in the absence of magnetite (7.3 ± 0.1 mgNO 3 −-N/L/d). The nitrate removal rate in the presence of magnetite reached 7–8 mgNO 3 −-N/L/d at day 80–85, which shortened the startup periods of approximately 35–40 days. Microbial morphology based on scanning electron microscope and transmission electron microscope showed that, the surface of cells was deposited by magnetite and they might be connected via magnetite. Further investigations via cyclic voltammetry and electrochemical impedance spectroscopy showed that magnetite mediated an electrically connected community with a higher capacitance and lower resistance than that in the absence of magnetite. Microbial community analysis showed that, in the presence of magnetite, the potential DAMO bacteria (Methylacidimicrobium and Methylotetracoccus species), methanogens (Methanothrix species) as well as nitrate-reducing bacteria (Bellilinea and Dechloromonas species) were enriched. They might participate in direct interspecies electron transfer (DIET) via magnetite substituting for c -type cytochromes as the electrical connection components, since methane oxidation in the presence of magnetite was insensitive to the external addition of potential intermediates, such as hydrogen. [ABSTRACT FROM AUTHOR]

Anaerobic decolorization of azo dye in sulfate-containing wastewater has been regarded as an economical and effective method, but it is generally limited by the high concentration of azo dye and accumulation of toxic intermediates. To address this problem, Fe 3 O 4 was added to one of the anaerobic reactors to investigate the effects on system performances. Results showed that AO7 removal rate, COD removal rate, and sulfate reduction were enhanced with the addition of Fe 3 O 4 under various influent AO7 concentrations (153 mgCOD/L – 1787 mgCOD/L). According to the proposed pathway for the degradation of AO7, more intermediates (2-hydroxy-1,4-naphthoquinone, phthalide, 4-methylphenol) were produced in the presence of Fe 3 O 4. The electron transfer capacity of sludge was also increased since Fe 3 O 4 could stimulate to secrete humic acid-like organics in EPS. Microbial analysis showed that iron-reducing bacteria like Clostridium and Geobacter were also enriched, which were capable of azo dye and aromatic compounds degradation. [Display omitted] • Decolorization of AO7 and sulfate reduction was enhanced with Fe 3 O 4. • Electron transfer capacity was enhanced by stimulating secretion of EPS. • Iron reducing bacteria was enriched to produce more easily degradable intermediates. • Redox mediators in EPS proceeded electron transfer between sulfide and azo dye. [ABSTRACT FROM AUTHOR]

The study presented here was to evaluate the effects of combining biological ethanol-type fermentation pretreatment (BEFP) with biochar on the growth of novel species capable of direct interspecies electron transfer (DIET) and methanogenesis in anaerobic co-digestion (AcoD) of kitchen wastes (KWs) and waste activated sludge (WAS). The results showed that, after BEFP, the genera capable of extracellular electron transfer to Fe(III) oxides or the elemental sulfur, such as Geobacter , Sphaerochaeta and Sporanaerobacter species, were detected, which however were not detected in the seed sludge. In the presence of biochar, their abundance was further increased, suggesting that biochar stimulated their growth. With biochar, methane production rate increased by about 44% and the effluent concentration of total organic substrates further declined, compared with that without biochar. With biochar, methane production efficiency reached 241.6 mL/g-COD, more than 30% higher than that without biochar (185.0 mL/g-COD), suggesting that more energy from the oxidation of organic substrates was converted into methane. Analysis of Fourier transform infrared spectroscopy (FT-IR) and three-dimensional excitation emission matrix (3D-EEM) showed that decomposition of complex organic compounds in KWs and WAS was enhanced, since the novel species might proceed DIET with methanogens and participate in the metabolism of complex organic compounds. [ABSTRACT FROM AUTHOR]

Kitchen wastes (KW) have been widely investigated for bio-ethanol production, while no study utilizes KW as ethanol source to stimulate the methanogenic communities to perform direct interspecies electron transfer (DIET), since the excess acidity contained after the biological ethanol-type fermentation pretreatment (BEFP) can seriously inhibit the DIET-based syntrophic metabolism. In this study, a strategy that utilized waste activated sludge (WAS) as co-substrate to relieve the excess acidity after BEFP during anaerobic co-digestion (AcoD) was proposed. The results showed that, under the mixed ratio of 1:2 and 1:5 (KW:WAS, volume ratio), both methane production and organic compound removal evidently increased, compared with that treating the sole WAS. Conversely, under the other mixed ratios (sole KW, 5:1, 2:1 and 1:1), no methane but the evident hydrogen production was detected, and syntrophic metabolism of organic acids and alcohols was prevented. Three-dimensional excitation emission matrix (3D-EEM) analysis showed that the protein-like organic compounds contained in both KW and WAS were effectively degraded. Furthermore, the maximum methane production potential from WAS during AcoD (260.5 ± 4.1 and 264.3 ± 2.7 mL/g-COD) was higher than that treating sole WAS (250.8 ± 0.1 mL/g-COD). Microbial community analysis showed that, some genera capable of metabolizing the complex organic compounds with the reduction of the elemental sulfur or equipped with the electrically conductive pili were specially enriched during AcoD under the mixed ratio of 1:2 and 1:5. They might proceed DIET with methanogens, such as Methanosarcina and Methanospirillum species, to maintain the syntrophic metabolism effective and stable, since the abundance of both Methanosarcina and Methanospirillum species evidently increased. Image 1 • Kitchen wastes (KW) as bio-ethanol source were used to establish DIET. • Waste activated sludge (WAS) relieved excess acidity in KW after ethanol production. • Degradation of both KW and WAS was enhanced during anaerobic co-digestion. • Some sulfur-reducing genera and genera equipped with e-pili were specially enriched. • Abundance of both Methanosarcina and Methanospirillum significantly increased. [ABSTRACT FROM AUTHOR]

Suspended biofilm carriers mediating direct interspecies electron transfer (DIET)-based syntrophic metabolism is a promising strategy to enhance anaerobic digestion and methane production by associating the advantages of conductive suspended biofilm carriers and anaerobic integrated floating fixed-film and activated sludge (An-IFFAS) process. However, the current knowledge of DIET using conductive suspended biofilm carrier is still limited. In this study, novel electron mediator suspended biofilm carriers had been prepared by introducing a series of graphite powders (3 wt%, 5 wt% and 7 wt%) into high-density polyethylene (HDPE), and applied in An-IFFAS reactors. Results showed that An-IFFAS reactors filled with graphite-modified carriers could enhance the degradation of organic matters and the production of methane significantly in comparison with the control reactor filled with conventional HDPE carriers at organic loading rates (OLRs) of 5.9–23.7 kg COD/m3/d. Microbial analysis proved that 7 wt% graphite-modified carrier improved approximately 4.2% abundance of Geobacter and 7.3% abundance of electrotrophic methanogens (Methanothrix) that exchange electron via DIET comparing with that of HDPE carriers, respectively. These findings demonstrated that electron mediator suspended biofilm carrier was able to potentially proceed DIET and enhance the efficiency of anaerobic digestion and recover CH 4 -related energy. Image 1 • Electron mediator carrier had the capability of AD-enhancing. • Electron mediator carrier-filled reactors gained higher methane productivity. • More Geobacter and electrotrophic methanogens were enriched in electron mediator carrier reactors. • Graphite established DIET on the surface of novel carriers. [ABSTRACT FROM AUTHOR]

• Fe 2 O 3 supported on conductive carbon cloth was added to anaerobic reactors. • The cumulative methane production increased by 15.4%. • Fe 2 O 3 stimulated the secretion of electron shuttle components. • Methanothrix were dominant over Methanobacterium. • Direct interspecies electron transfer mode was established. In this study, a Fe 2 O 3 supported on conductive carbon cloth (FC) was prepared and supplemented into anaerobic digestion reactors to improve propionate degradation. In the FC-supplemented reactors, the cumulative methane production and propionate degradation increased by 15.4% and 19.67% compared with those of the control, respectively. Less methane production with H 2 /CO 2 as the sole substrate in the culture taken from the FC reactors suggested that interspecies hydrogen transfer in the FC reactors was weaker. These results suggested that direct interspecies electron transfer (DIET) was established in the FC reactors to improve the performance. Fe 2 O 3 increased the secretion of electron shuttle components of extracellular polymeric substances to increase electron exchange capacity of biomass of the FC reactors, which further facilitated the DIET. Analysis on microbial communities confirmed that the abundance of microorganisms-related DIET in the FC reactors was higher than that in the control. [ABSTRACT FROM AUTHOR]

• Citrate was used to solubilize the Fe(OH) 3 to improve the phenol degradation. • Citrate declined reduction potential of Fe(OH) 3 to facilitate the enrichment of IRBs. • Co-occurrence of citrate and Fe(OH) 3 accelerated anaerobic conversion of phenol to methane. The improved performances during anaerobic degradation of phenol to methane with Fe(OH) 3 were usually inapparent, due to its lower solubility (unaccessible to dissimilatory iron reduction) and more positive reduction potential of Fe(III)/Fe(II) (unfavorable for enriching Fe(III)-reducing bacteria [IRBs]). In this study, citrate, the organic chelates, were used to solubilize Fe(III) with the aim of improving the phenol degradation and declining the reduction potential of Fe(III)/Fe(II). Results showed that, in the co-occurrence of citrate and Fe(OH) 3 , the degradation rates of phenol were about 1.3-fold rapider than that with sole Fe(OH) 3. Analysis of cyclic voltammetry demonstrated that the reduction potential of Fe(III)/Fe(II) in the form of Fe(OH) 3 (−0.41 to −0.28 V vs Ag/AgCl) declined to −0.61 to −0.41 V. As a result, the Fe(III)-reducing genera, such as Petrimonas and Shewanella , which held a great potential of proceeding syntrophic metabolism via direct interspecies electron transfer (DIET), were significantly enriched. [ABSTRACT FROM AUTHOR]