Wide distribution of extracellular electron transfer functionality in natural proteinaceous organic materials for microbial reductive dehalogenation.

Extracellular electron transfer materials (EETMs) in the environment, such as humic substances and biochar, are formed from the humification/heating of natural organic materials. However, the distribution of extracellular electron transfer (EET) functionality in fresh natural organic materials has not yet been explored. In the present study, we reveal the wide distribution of EET functionality in proteinaceous materials for the first time using an anaerobic pentachlorophenol dechlorinating consortium, whose activity depends on EETM. Out of 11 natural organic materials and 13 reference compounds, seven proteinaceous organic materials (albumin, beef, milk, pork, soybean, yolk, and bovine serum albumin) functioned as EETMs. Carbohydrates and lipids did not function as EETMs. Comparative spectroscopic analyses suggested that a ß-sheet secondary structure was essential for proteins to function as EETMs, regardless of water solubility. A high content of reduced sulfur was potentially involved in EET functionality. Although proteinaceous materials have thus far been considered simply as nutrients, the wide distribution of EET functionality in these materials provides new insights into their impact on biogeochemical cycles. In addition, structural information on EET functionality can provide a scientific basis for the development of eco-friendly EETMs.

The Emergence of Extracellular Electron Mediating Functionality in Rice Straw-Artificial Soil Mixture during Humification.

This study aimed to elucidate the origin of extracellular electron mediating (EEM) functionality and redox-active center(s) in humic substances, where they are ubiquitously distributed. Here, we show the emergence of EEM functionality during the humification of rice straw in artificial soil (kaolin and sand) with a matric potential of -100 cm at 20 °C for one year. We used the dechlorination activity of an EEM material-dependent pentachlorophenol-dechlorinating anaerobic microbial consortium as an index of the EEM functionality. Although rice straw and its mixture with artificial soil did not initially have EEM functionality, it emerged after one month of humification and increased until six months after which the functionality was maintained for one year. Chemical and electrochemical characterizations demonstrated that the emergence and increase in EEM functionality were correlated with the degradation of rice straw, formation of quinone structures, a decrease in aromatic structures, an increase in nitrogenous and aliphatic structures, and specific electric capacitance during humification. The newly formed quinone structure was suggested as a potential redox-active center for the EEM functionality. These findings provide novel insights into the dynamic changes in EEM functionality during the humification of organic materials.

Humin-promoted microbial electrosynthesis of acetate from CO2 by Moorella thermoacetica.

Humin, an insoluble fraction of humic substances at any pH, has been reported to be an extracellular electron mediator (EEM) that functions in carbon dioxide (CO2 )-fixing acetogenesis. Here, we show that humin promotes the microbial electrosynthesis (MES) of acetate from CO2 using Moorella thermoacetica. Yeast extract, essential for the reaction of M. thermoacetica, resulted in the heterotrophic production of organic acids including acetate, hydrogen, and methane. Excluding the effect of yeast extract, MES with 13 g/L of suspended humin poised at -510 mV (vs. Ag/AgCl) achieved a CO2 -fixing acetate production of 24.2 mg-acetate/L/day (1.9 mg-acetate/day/g-humin); this is 10-folds higher than the humin-free MES, with 90.3% of the coulombic efficiency. Although M. thermoacetica is an electroactive bacterium, it obtains electrons for acetogenesis mostly via humin. The suspended humin-assisted MES poised at -810 mV (vs. Ag/AgCl) increased the acetate production rate to 39.3 mg-acetate/L/day using electrons mainly from electrolyzed hydrogen and humin. Immobilization increased the humin's EEM efficiency, as indicated by the acetate production rate of 20.8 mg-acetate/L/day (6.9 mg-acetate/day/g-humin) with a 98.7% coulombic efficiency in MES with 3 g/L of immobilized humin poised at -510 mV (vs. Ag/AgCl). These results suggest that humin-assisted MES has high potential for microbial CO2 fixation.

Promotion of Nitrogen Fixation of Diverse Heterotrophs by Solid-Phase Humin.

Although biological nitrogen fixation (BNF) proceeds under mild conditions compared to the energy-intensive Haber-Bosch process, the slow kinetics of BNF necessitate the promotion of BNF activity in its practical application. The BNF promotion using purified nitrogenases and using genetically modified microorganisms has been studied, but these enzymes are unstable and expensive; moreover, designing genetically modified microorganisms is also a difficult task. Alternatively, the BNF promotion in non-modified (wild-type) microorganisms (enriched consortia) with humin has been shown, which is a humic substance insoluble at any pH and functions as an extracellular electron mediator. However, the taxonomic distribution of the diazotrophs promoted by humin, the levels of BNF promotion, and the underlying mechanism in BNF promotion with humin remain unknown. In this study, we show that taxonomically diverse heterotrophic diazotrophs, harboring nifH clusters I, II, and III, promoted their BNF by accepting extracellular electrons from humin, based on the characterization of the individual responses of isolated diazotrophs to humin. The reduced humin increased the acetylene reduction activity of the diazotrophs by 194-916% compared to the level achieved by the organic carbon source, causing adenosine triphosphate (ATP) synthesis in the diazotroph cells without increase in the CO2 production and direct electron donation to the MoFe protein of the nitrogenase in the cells without relying on the biological electron transfer system. These would result in BNF promotion in the wild-type diazotroph cells beyond their biochemical capacity. This significant promotion of BNF with humin would serve as a potential basis for sustainable technology for greener nitrogen fixation.

Promotion of biological H2 (Bio-H2) production by the nitrogen-fixing anaerobic microbial consortia using humin, a solid-phase humic substance.

Dark fermentative biological hydrogen (Bio-H2) production is expected to be a clean and sustainable H2 production technology, and the technologies have been studied to increase in the product yield as index. This study achieved high product yields of Bio-H2 using nitrogen-fixing consortia under nitrogen-deficient conditions with glucose or mannitol as substrate and humin as the extracellular electron mediator: 4.12 mol-H2/mol-glucose and 3.12 mol-H2/mol-mannitol. The high Bio-H2 production was observed under the conditions where both nitrogenase and hydrogenase were active in the presence of humin. Nitrogenase activity was confirmed by acetylene reduction activity and hydrogenase activity by Bio-H2 production under nitrogenase-inhibiting conditions with NH4NO3. [Fe-Fe] hydrogenase detected by a specific PCR and acetate, butyrate, formate, lactate, and pyruvate produced as by-products suggested the involvement of both pyruvate-ferredoxin-oxidoreductase and pyruvate formate lyase pathways in Bio-H2 production. Humin promoted the Bio-H2 production beyond the capacity of the consortium, which had reached saturation with the optimum concentrations of glucose and mannitol. Carbon balance suggested the concurrent H2 consumption by hydrogenotrophic methanogenesis and acetogenesis. Bio-H2 production of the washed and starved consortium with reduced humin under conditions with or without NH4NO3 suggests that humin promoted hydrogenase and nitrogenase activity by donating extracellular electrons. Clostridium and Ruminococcus in the consortia were considered major hydrogen producers. Thus, this study demonstrated the outstanding potential of nitrogen-fixing consortia under nitrogen-deficient conditions with humin as an extracellular electron mediator for dark fermentative Bio-H2 production with high yields.

Effect of Humin and Chemical Factors on CO2-Fixing Acetogenesis and Methanogenesis.

Acetogenesis and methanogenesis have attracted attention as CO2-fixing reactions. Humin, a humic substance insoluble at any pH, has been found to assist CO2-fixing acetogenesis as the sole electron donor. Here, using two CO2-fixing consortia with acetogenic and methanogenic activities, the effect of various parameters on these activities was examined. One consortium utilized humin and hydrogen (H2) as electron donors for acetogenesis, either separately or simultaneously, but with a preference for the electron use from humin. The acetogenic activity was accelerated 14 times by FeS at 0.2 g/L as the optimal concentration, while being inhibited by MgSO4 at concentration above 0.02 g/L and by NaCl at concentrations higher than 6 g/L. Another consortium did not utilize humin but H2 as electron donor, suggesting that humin was not a universal electron donor for acetogenesis. For methanogenesis, both consortia did not utilize extracellular electrons from humin unless H2 was present. The methanogenesis was promoted by FeS at 0.2 g/L or higher concentrations, especially without humin, and with NaCl at 2 g/L or higher concentrations regardless of the presence of humin, while no significant effect was observed with MgSO4. Comparative sequence analysis of partial 16S rRNA genes suggested that minor groups were the humin-utilizing acetogens in the consortium dominated by Clostridia, while Methanobacterium was the methanogen utilizing humin with H2.

Activation of extracellular electron network in non-electroactive bacteria by Bombyx mori silk.

Extracellular electron transfer material (EETM) has increasingly attracted attentions for the enhancing effect on multiple microbial reactions. Especially, EETM is known to be essential to activate the energy network in non-electroactive bacteria. It is motivated to find out an EETM which is natural-based, environmentally friendly, and easily produced at large-scale. In this study, Bombyx mori silk is found, for the first time, to function as an EETM by using an EETM-dependent pentachlorophenol (PCP) dechlorinating anaerobic microbial culture. Subsequently, by dividing fibroin fiber into different soluble/insoluble fractions and correlating their EET functions with their structural properties based on various spectroscopic analyses, the ß-sheet configuration is suggested as an essential structure supporting the EET function of silk materials. The analyses also suggested the involvement of sulfur-containing amino acids in this function. The EET function is not degraded by boiling or acid/alkaline treatments and the material can be utilized multiple times, although it is susceptible to UV irradiation. Bombyx mori silk also enhance other microbial reactions, including Fe(III)OOH reduction, CO2 reduction to acetate, and nitrogen fixation. This discovery provides a basis for developing biotechnology for environmental remediation, global warming reduction, and biofertilizer production using Bombyx mori silk and its wastes.

Dehalococcoides mccartyi NIT01, a novel isolate, dechlorinates high concentrations of chloroethenes by expressing at least six different reductive dehalogenases.

This study presents the isolation of a novel strain of Dehalococcoides mccartyi, NIT01, which can completely dechlorinate up to 4.0 mM of trichloroethene to ethene via 1,2-cis-dichroroethene and vinyl chloride within 25 days. Strain NIT01 dechlorinated chloroethenes (CEs) at a temperature range of 25-32 °C and pH range of 6.5-7.8. The activity of the strain was inhibited by salt at more than 1.3% and inactivated by 1 h exposure to 2.0% air or 0.5 ppm hypochlorous acid. The genome of NIT01 was highly similar to that of the Dehalococcoides strains DCMB5, GT, 11a5, CBDB1, and CG5, and all included identical 16S rRNA genes. Moreover, NIT01 had 19 rdhA genes including NIT01-rdhA7 and rdhA13, which are almost identical to vcrA and pceA that encode known dehalogenases for tetrachloroethene and vinyl chloride, respectively. We also extracted RdhAs from the membrane fraction of NIT01 using 0.5% n-dodecyl-ß-d-maltoside and separated them by anion exchange chromatography to identify those involved in CE dechlorination. LC/MS identification of the LDS-PAGE bands and RdhA activities in the fractions indicated cellular expression of six RdhAs. NIT01-RdhA7 (VcrA) and NIT01-RdhA15 were highly detected and NIT01-RdhA6 was the third-most detected. Among these three RdhAs, NIT01-RdhA15 and NIT01-RdhA6 had no biochemically identified relatives and were suggested to be novel functional dehalogenases for CEs. The expression of multiple dehalogenases may support bacterial tolerance to high concentrations of CEs.

Promotion of biological nitrogen fixation activity of an anaerobic consortium using humin as an extracellular electron mediator.

Nitrogen fertiliser is manufactured using the industrial Haber-Bosch process, although it is extremely energy-consuming. One sustainable alternative technology is the electrochemical promotion of biological nitrogen fixation (BNF). This study reports the promotion of BNF activity of anaerobic microbial consortia by humin, a solid-phase humic substance, at any pH, functioning as an extracellular electron mediator, to levels of 5.7-11.8 times under nitrogen-deficient conditions. This was evidenced by increased acetylene reduction activity and total nitrogen content of the consortia. Various humins from different origins promoted anaerobic BNF activity, although the degree of promotion differed. The promotion effected by humin differed from the effects of chemical reducing agents and the effects of supplemental micronutrients and vitamins. The promotion of anaerobic BNF activity by only reduced humin without any other electron donor suggested that humin did not serve as organic carbon source but as extracellular electron mediator, for electron donation to the nitrogen-fixing microorganisms. The next generation sequencing (NGS) of partial 16S rRNA genes showed the predominance of Clostridiales (Firmicutes) in the consortia. These findings suggest the effectiveness of humin as a solid-phase extracellular electron mediator for the promotion of anaerobic BNF activity, potentially to serve for the basis for a sustainable technology.

Humin: No longer inactive natural organic matter.

The discovery of the function of humin (HM), an insoluble fraction of humic substances (HSs), as an extracellular electron mediator (EEM) in 2012 has provided insight into the role of HM in nature and its potential for in situ bioremediation of pollutants. The EEM function is thought to enable the energy network of various microorganisms using HM. Recently, a number of studies on the application of HM as EEM in anaerobic microbial cultures have been conducted. Even so, there is a need for developing a holistic view of HM EEM function. In this paper, we summarize all the available information on the properties of HM EEM function, its applications, possible redox-active structures, and the interaction between HM and microbial cells. We also suggest scopes for future HM research.

Humin Assists Reductive Acetogenesis in Absence of Other External Electron Donor.

The utilization of extracellular electron transfer by microorganism is highly engaging for remediation of toxic pollutants under "energy-starved" conditions. Humin, an organo-mineral complex of soil, has been instrumental as an external electron mediator for suitable electron donors in the remediative works of reductive dehalogenation, denitrification, and so forth. Here, we report, for the first time, that humin assists microbial acetogenesis as the extracellular electron donor using the electron acceptor CO 2 . Humin was obtained from Kamajima paddy soil, Japan. The anaerobic acetogenic consortium in mineral medium containing CO 2 / HCO 3 - as the inorganic carbon source used suspended humin as the energy source under mesophilic dark conditions. Retardation of acetogenesis under the CO 2 -deficient conditions demonstrated that humin did not function as the organic carbon source but as electron donor in the CO 2 -reducing acetogenesis. The consortium with humin also achieved anaerobic dechlorination with limited methanogenic activity. Total electron-donating capacity of humin was estimated at about 87 µeeq/g-humin. The metagenomic sequencing of 16S rRNA genes showed the predominance of Firmicutes (71.8 ± 2.5%) in the consortium, and Lachnospiraceae and Ruminococcaceae were considered as the CO 2 -reducing acetogens in the consortium. Thus, microbial fixation of CO 2 using humin introduces new insight to the holistic approach for sustainable treatment of contaminants in environment.

Anaerobic Dechlorination by a Humin-Dependent Pentachlorophenol-Dechlorinating Consortium under Autotrophic Conditions Induced by Homoacetogenesis.

Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H2) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H2 directly, but rather through a reduced state of humin-a solid-phase humic substance-as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H2 was used for carbon-dioxide fixation to acetate; the acetate produced was used for the reduction of humin; and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H2 dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination.

Aggregatilinea lenta gen. nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated from subseafloor sediment, and proposal of the new order Aggregatilineales ord. nov. within the class Anaerolineae of the phylum Chloroflexi.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5-0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20-37 °C (optimum, 30 °C), pH 5.5-8.0 (pH 6.5-7.0), and 0-30 g l-1 NaCl (5 g l-1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml-1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

Humin as an External Electron Mediator for Microbial Pentachlorophenol Dechlorination: Exploration of Redox Active Structures Influenced by Isolation Methods.

Humin (HM) has been reported to function as an external electron mediator (EEM) in various microbial reducing reactions. In this study, the effect of isolation methods on EEM functionality and the chemical/electrochemical structures of HM were examined based on the correlation between dechlorination rates in the anaerobic HM-dependent pentachlorophenol (PCP)-dechlorinating consortium and the chemical/electrochemical structures of HM. A lack of PCP dechlorination activity suggested no EEM function in the HM samples prepared as a soluble fraction in dimethyl sulfoxide and sulfuric acid (which did not contain any electric capacitance). Other HM samples exhibited EEM functionality as shown by the dechlorination activity ranging from 0.55 to 3.48 (µmol Cl-) L-1d-1. The comparison of dechlorination activity with chemical structural characteristics suggested that HM with EEM functionalities had predominantly aliphatic and carbohydrate carbons with the partial structures C=O, O=C⁻N, and O=C⁻O. EEM functionality positively correlated with the proportion of O=C⁻N and O=C⁻O, suggesting an association between peptidoglycan structure and EEM functionality. The lack of detection of a quinone structure in one HM sample with EEM functionality and a negative correlation with aromatic or C=C carbon suggested that the mechanism containing quinone structures is a minor component for the functionality of EEM.

Bacteroides sedimenti sp. nov., isolated from a chloroethenes-dechlorinating consortium enriched from river sediment.

A Gram-negative, anaerobic, non-motile, non-spore-forming bacterial strain, designated YN3PY1T, was isolated from a chloroethene-dechlorinating consortium originally enriched from river sediment. The strain enhanced the dechlorination of cis-dichloroethene to ethene by Dehalococcoides, especially at the early stages of cultivation. Strain YN3PY1T was the first isolate of the genus Bacteroides, obtained from animal-independent environments, and its 16S rRNA gene had the highest sequence similarity (97.1%) with Bacteroides luti JCM 19020T in the 'Coprosuis' clade of the genus Bacteroides. Strain YN3PY1T formed a phylogenetic cluster with other phylotypes detected from sediments and paddy soil, and the cluster was affiliated with a linage of so-called free-living Bacteroides detected from animal-independent environments, suggesting specific adaptations to sediment-like environments. The strain showed typical phenotypes of Bacteroides, i.e., polysaccharolytic anaerobe having anteiso-C15:0 as the most abundant fatty acid and MK-11 as one of the major respiratory quinones. Additionally, the strain uniquely transforms glucose to lactate and malate, has MK-12 as another major respiratory quinone, and grows at comparatively low temperatures, i.e. 10-40°C, with an optimum at 28°C. Based on the presented data, strain YN3PY1T (= KCTC 15656T = NBRC 113168T) can be proposed as a novel species of the genus Bacteroides and named as Bacteroides sedimenti sp. nov.

Identification of Multiple Dehalogenase Genes Involved in Tetrachloroethene-to-Ethene Dechlorination in a Dehalococcoides-Dominated Enrichment Culture.

Chloroethenes (CEs) are widespread groundwater toxicants that are reductively dechlorinated to nontoxic ethene (ETH) by members of Dehalococcoides. This study established a Dehalococcoides-dominated enrichment culture (designated "YN3") that dechlorinates tetrachloroethene (PCE) to ETH with high dechlorination activity, that is, complete dechlorination of 800 µM PCE to ETH within 14 days in the presence of Dehalococcoides species at 5.7 ± 1.9 × 107 copies of 16S rRNA gene/mL. The metagenome of YN3 harbored 18 rdhA genes (designated YN3rdhA1-18) encoding the catalytic subunit of reductive dehalogenase (RdhA), four of which were suggested to be involved in PCE-to-ETH dechlorination based on significant increases in their transcription in response to CE addition. The predicted proteins for two of these four genes, YN3RdhA8 and YN3RdhA16, showed 94% and 97% of amino acid similarity with PceA and VcrA, which are well known to dechlorinate PCE to trichloroethene (TCE) and TCE to ETH, respectively. The other two rdhAs, YN3rdhA6 and YN3rdhA12, which were never proved as rdhA for CEs, showed particularly high transcription upon addition of vinyl chloride (VC), with 75 ± 38 and 16 ± 8.6 mRNA copies per gene, respectively, suggesting their possible functions as novel VC-reductive dehalogenases. Moreover, metagenome data indicated the presence of three coexisting bacterial species, including novel species of the genus Bacteroides, which might promote CE dechlorination by Dehalococcoides.

Phenol-degrading anode biofilm with high coulombic efficiency in graphite electrodes microbial fuel cell.

A microbial fuel cell (MFC), with graphite electrodes as both the anode and cathode, was operated with a soil-free anaerobic consortium for phenol degradation. This phenol-degrading MFC showed high efficiency with a current density of 120 mA/m2 and a coulombic efficiency of 22.7%, despite the lack of a platinum catalyst cathode and inoculation of sediment/soil. Removal of planktonic bacteria by renewing the anaerobic medium did not decrease the performance, suggesting that the phenol-degrading MFC was not maintained by the planktonic bacteria but by the microorganisms in the anode biofilm. Cyclic voltammetry analysis of the anode biofilm showed distinct oxidation and reduction peaks. Analysis of the microbial community structure of the anode biofilm and the planktonic bacteria based on 16S rRNA gene sequences suggested that Geobacter sp. was the phenol degrader in the anode biofilm and was responsible for current generation.

Denitrification by Pseudomonas stutzeri coupled with CO2 reduction by Sporomusa ovata with hydrogen as an electron donor assisted by solid-phase humin.

A co-culture system comprising an acetogenic bacterium, Sporomusa ovata DSMZ2662, and a denitrifying bacterium, Pseudomonas stutzeri JCM20778, enabled denitrification using H2 as the sole external electron donor and CO2 as the sole external carbon source. Acetate produced by S. ovata supported the heterotrophic denitrification of P. stutzeri. A nitrogen balance study showed the reduction of nitrate to nitrogen gas without the accumulation of nitrite and nitrous oxide in the co-culture system. S. ovata did not show nitrate reduction to ammonium in the co-culture system. Significant proportions of the consumed H2 were utilized for denitrification: 79.9 ± 4.6% in the co-culture system containing solid-phase humin and 62.9±11.1% in the humin-free co-culture system. The higher utilization efficiency of hydrogen in the humin-containing system was attributed to the higher denitrification activity of P. stutzeri under the acetate deficient conditions. The nitrogen removal rate of the humin-containing co-culture system reached 0.19 kg NO3(-)-N·m(-3)·d(-1). Stable denitrification activity for 61 days of successive sub-culturing suggested the robustness of this co-culture system. This study provides a novel strategy for the in situ enhancement of microbial denitrification.

Enhanced denitrification of Pseudomonas stutzeri by a bioelectrochemical system assisted with solid-phase humin.

The denitrification reactions performed by Pseudomonas stutzeri JCM20778 were enhanced electrochemically with the use of solid-phase humin, although P. stutzeri itself was incapable of receiving electrons directly from the graphite electrode. Electrochemically reduced humin enhanced the microbial, but not abiotic, denitrification reactions. Electric current and cyclic voltammetry analyses suggested that the solid-phase humin functioned as an electron donor for the denitrification reactions of P. stutzeri. Nitrogen balance study and the estimation of the first-order rate constants of the consecutive denitrification reactions suggested that the solid-phase humin enhanced all reducing reactions from nitrate to nitrogen gas. Considering the wide distribution of humin in the environment, the findings that solid-phase humin can assist in electron transfer, from the electrode to a denitrifying bacterium that has little ability to directly utilize external electrons, has important implications for the widespread application of bioelectrochemical systems assisted by solid-phase humin for enhancing microbial denitrification.

Spatial Abundance and Distribution of Potential Microbes and Functional Genes Associated with Anaerobic Mineralization of Pentachlorophenol in a Cylindrical Reactor.

Functional interplays of microbial activity, genetic diversity and contaminant transformation are poorly understood in reactors for mineralizing halogenated aromatics anaerobically. Here, we investigated abundance and distribution of potential microbes and functional genes associated with pentachlorophenol (PCP) anaerobic mineralization in a continuous-flow cylindrical reactor (15 cm in length). PCP dechlorination and the metabolite (phenol) were observed at segments 0-8 cm from inlet, where key microbes, including potential reductive dechlorinators (Dehalobacter, Sulfurospirillum, Desulfitobacterium and Desulfovibrio spp.) and phenol degraders (Cryptanaerobacter and Syntrophus spp.), as well as putative functional genes, including putative chlorophenol reductive dehalogenase (cprA) and benzoyl-CoA reductase (bamB), were highly enriched simultaneously. Five types of putative cprAs, three types of putative bamBs and seven types of putative nitrogenase reductase (nifHs) were determined, with their copy numbers decreased gradually from inlet to outlet. Distribution of chemicals, bacteria and putative genes confirmed PCP dechlorination and phenol degradation accomplished in segments 0-5 cm and 0-8 cm, respectively, contributing to a high PCP mineralization rate of 3.86 µM d(-1). Through long-term incubation, dechlorination, phenol degradation and nitrogen fixation bacteria coexisted and functioned simultaneously near inlet (0-8 cm), verified the feasibility of anaerobic mineralization of halogenated aromatics in the compact reactor containing multiple functional microbes.