Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1

In this work, we investigated the anaerobic decolorization of methyl orange (MO), a typical azo dye, by Shewanella oneidensis MR-1, which can use various organic and inorganic substances as its electron acceptor in natural and engineered environments. S. oneidensis MR-1 was found to be able to obtain energy for growth through anaerobic respiration accompanied with dissimilatory azo-reduction of MO. Chemical analysis shows that MO reduction occurred via the cleavage of azo bond. Block of Mtr respiratory pathway, a transmembrane electron transport chain, resulted in a reduction of decolorization rate by 80%, compared to the wild type. Knockout of cymA resulted in a substantial loss of its azo-reduction ability, indicating that CymA is a key c-type cytochrome in the electron transfer chain to MO. Thus, the MtrA-MtrB-MtrC respiratory pathway is proposed to be mainly responsible for the anaerobic decolorization of azo dyes such as MO by S. oneidensis.     

奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer,EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力;四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一,能够参与多种氧化还原过程,但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用,补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange,MO）作为电子受体,在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点,并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下,细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时,MO在周质空间中的还原速率与其表达水平呈正相关,更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别,可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为,为进一步推进对细菌周质电子传递机制的理解,以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。     

Biological sulphate reduction and redox mediator effects on azo dye decolourisation in anaerobic–aerobic sequencing batch reactors

In this work, the anaerobic period of an anaerobic–aerobic sequencing batch reactor was found to allow the reductive decolourisation of azo dyes. 1-l reactors were operated in 24-h cycles comprising anaerobic and aerobic reaction phases, fed with a simulated textile effluent including a reactive type (Remazol Brilliant Violet 5R) or an acid type (Acid Orange 7) azo dye. The aim was to assess the role of different redox phenomena in the anaerobic decolourisation process. Selective inhibition of sulphate reducing bacteria was carried out in the sulphate-containing, reactive dye fed reactor, resulting in nearly complete, though reversible and inhibition of decolourisation. The acid dye fed reactor's supplementation with sulphate, though resulting in sulphate reduction, did not improve decolourisation. Other redox mediators, namely quinones, were more effective in promoting electron transfer to the azo bond. Bio-augmentation of the acid dye fed reactor with a pure sulphate reducer strain known to decolourise azo dyes, Desulfovibrio alaskensis, was also carried out. Decolourisation was improved, but apparently as a result of the carbon source change required to support D. alaskensis growth. A chemically mediated reduction of the azo bond coupled to biological sulphate reduction, thus seemed to account for the high decolourisation yields of both dyes.     

Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Potential-dependent extracellular electron transfer pathways of exoelectrogens

Exoelectrogens are distinct from other bacteria owing to their unique extracellular electron transfer (EET) abilities that allow for anaerobic respiration with various external redox-active surfaces, including electrode and metal oxides. Although the EET process is known to trigger diverse extracellular redox reactions, the reverse impact has been long overlooked. Recent evidences show that exoelectrogens can sense the potential changes of external surfaces and alter their EET strategies accordingly, which imparts them remarkable abilities in adapting to diverse and redox-variable environment. This mini-review provides a condensed overview and critical analysis about the recent discoveries on redox-dependent EET pathways of exoelectrogens, with focus on Geobacter sulfurreducens and Shewanella oneidensis. We summarize the detailed responses of various EET components, analyze the drives and mechanisms of such responses, highlight the diversity of EET dynamics among different bacterial species and under integrated effects of redox potential and surface chemistry, and discusses the future research needs.     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

Respiration and Growth of Shewanella decolorationis S12 with an Azo Compound as the Sole Electron Acceptor

The ability of Shewanella decolorationis S12 to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory azoreduction was investigated. This microorganism can reduce a variety of azo dyes by use of formate, lactate, pyruvate, or H₂ as the electron donor. Furthermore, strain S12 grew to a maximal density of 3.0 × 10⁷ cells per ml after compete reduction of 2.0 mM amaranth in a defined medium. This was accompanied by a stoichiometric consumption of 4.0 mM formate over time when amaranth and formate were supplied as the sole electron acceptor and donor, respectively, suggesting that microbial azoreduction is an electron transport process and that this electron transport can yield energy to support growth. Purified membranous, periplasmic, and cytoplasmic fractions from S12 were analyzed, but only the membranous fraction was capable of reducing azo dyes with formate, lactate, pyruvate, or H₂ as the electron donor. The presence of 5 μM Cu²⁺ ions, 200 μM dicumarol, 100 μM stigmatellin, and 100 μM metyrapone inhibited anaerobic azoreduction activity by both whole cells and the purified membrane fraction, showing that dehydrogenases, cytochromes, and menaquinone are essential electron transfer components for azoreduction. These results provide evidence that the microbial anaerobic azoreduction is linked to the electron transport chain and suggest that the dissimilatory azoreduction is a form of microbial anaerobic respiration. These findings not only expand the number of potential electron acceptors known for microbial energy conservation but also elucidate the mechanisms of microbial anaerobic azoreduction.     

Azo Reductase Activity of Intact Saccharomyces cerevisiae Cells Is Dependent on the Fre1p Component of Plasma Membrane Ferric Reductase

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae Δfre1 and Δfre1 Δfre2 mutant strains, but not the Δfre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae Δfre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.     

Biogenic Formation of As-S Nanotubes by Diverse Shewanella Strains

Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S₂O₃²⁻ under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.A number of bacterial strains have been shown to contribute to the formation of diverse arsenic minerals (4). If sulfide is present as a ligand for immobilization of arsenic, As-S precipitates often form. Desulfosporosinus auripigmentum, which can be isolated from lake sediments, reduces As(V) to As(III) and S(VI) to S(−II) during anaerobic respiration and forms a yellow arsenic sulfide precipitate (7). While Desulfovibrio strain Ben-RB also produces precipitated arsenic sulfide in culture media, As reduction was not correlated with energy conservation (6). Other taxonomically divergent microorganisms isolated from various arsenic-rich sites have also been shown to reduce As(V) to As(III) and form arsenic sulfide precipitates (1, 2).We previously reported that Shewanella sp. strain HN-41 produces an extensive extracellular network of filamentous arsenic-sulfide (As-S) nanotubes via its dissimilatory metal-reducing activity (4). The As-S nanotubes, which formed via the reduction of As(V) and S₂O₃²⁻, were initially amorphous As₂S₃ but evolved with increasing incubation time toward polycrystalline phases of the chalcogenide minerals realgar (AsS) and duranusite (As₄S). Because the Shewanella As-S nanotubes behaved both as metals and as semiconductors, in terms of their electrical and photoconductive properties, respectively, it was postulated that they may provide useful materials for novel nano- and optoelectronic devices (4).While several bacterial species have been shown to produce amorphous and particulate As-S precipitates (1, 2, 4, 7), the formation of the As-S nanotubes by other bacteria has not yet been described, suggesting that this may be a unique property of Shewanella strains. To test this hypothesis, 10 different Shewanella strains, including Shewanella sp. strains PV-4 and HN-41, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were inoculated into HEPES-buffered basal medium (3, 5) containing 10 mM sodium dl-lactate as the electron donor and 5 mM arsenate (Na₂HAsO₄·7H₂O) and 5 mM thiosulfate (Na₂S₂O₃·5H₂O) as the electron acceptors. All chemicals and methods for sample preparation and characterization used in this study were previously described (4).Of the 10 different Shewanella strains examined, only four strains, Shewanella sp. strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1, produced As-S yellow precipitates in culture medium following incubation in the presence of arsenate and thiosulfate. Shewanella sp. strain HN-41 and S. putrefaciens CN-32 produced yellow precipitates of As-S after 7 days of incubation, whereas S. alga BrY and S. oneidensis MR-1 produced only a small amount of visible precipitate after 30 days of incubation. The remainder of the tested Shewanella strains failed to produce yellow precipitates, regardless of incubation time.The culture medium of the strains tested was periodically sampled during the bacterial incubation period to determine the concentrations of lactate, acetate, arsenic, and sulfide in the aqueous solution. Among the 10 strains examined, Shewanella strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 metabolized lactate in growth medium containing arsenate and thiosulfate (Table ​(Table1).1). Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly consumed lactate both as an electron donor and as a carbon source (see Fig. S1 in the supplemental material). Cultures of S. alga BrY and S. oneidensis MR-1 consumed ∼1.4 mM lactate after 7 days, while Shewanella sp. strain HN-41 and S. putrefaciens CN-32 consumed 1.7 mM and 2.3 mM lactate, respectively. Although S. putrefaciens CN-32 reduced As(V) in the culture medium supplemented with 5 mM As(V) as the sole electron acceptor, Shewanella sp. strain HN-41, S. alga BrY, and S. oneidensis MR-1 did not reduce As(V) and did not oxidize lactate to acetate (data not shown). Consequently, the latter three strains could not utilize As(V) as an electron acceptor for respiratory metabolism.

TABLE 1.

Influence of thiosulfate on the consumption of lactate, reduction of As(V), and formation of As-S nanotubes by Shewanella strains in medium containing lactate and 5 mM As(V)

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K _m and V _max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.     

Hydrogen and Formate Oxidation Coupled to Dissimilatory Reduction of Iron or Manganese by Alteromonas putrefaciens

The ability of Alteromonas putrefaciens to obtain energy for growth by coupling the oxidation of various electron donors to dissimilatory Fe(III) or Mn(IV) reduction was investigated. A. putrefaciens grew with hydrogen, formate, lactate, or pyruvate as the sole electron donor and Fe(III) as the sole electron acceptor. Lactate and pyruvate were oxidized to acetate, which was not metabolized further. With Fe(III) as the electron acceptor, A. putrefaciens had a high affinity for hydrogen and formate and metabolized hydrogen at partial pressures that were 25-fold lower than those of hydrogen that can be metabolized by pure cultures of sulfate reducers or methanogens. The electron donors for Fe(III) reduction also supported Mn(IV) reduction. The electron donors for Fe(III) and Mn(IV) reduction and the inability of A. putrefaciens to completely oxidize multicarbon substrates to carbon dioxide distinguish A. putrefaciens from GS-15, the only other organism that is known to obtain energy for growth by coupling the oxidation of organic compounds to the reduction of Fe(III) or Mn(IV). The ability of A. putrefaciens to reduce large quantities of Fe(III) and to grow in a defined medium distinguishes it from a Pseudomonas sp., which is the only other known hydrogen-oxidizing, Fe(III)-reducing microorganism. Furthermore, A. putrefaciens is the first organism that is known to grow with hydrogen as the electron donor and Mn(IV) as the electron acceptor and is the first organism that is known to couple the oxidation of formate to the reduction of Fe(III) or Mn(IV). Thus, A. putrefaciens provides a much needed microbial model for key reactions in the oxidation of sediment organic matter coupled to Fe(III) and Mn(IV) reduction.     

Microbial fuel cell with an azo-dye-feeding cathode

Microbial fuel cells (MFCs) were constructed using azo dyes as the cathode oxidants to accept the electrons produced from the respiration of Klebsiella pneumoniae strain L17 in the anode. Experimental results showed that a methyl orange (MO)-feeding MFC produced a comparable performance against that of an air-based one at pH 3.0 and that azo dyes including MO, Orange I, and Orange II could be successfully degraded in such cathodes. The reaction rate constant (k) of azo dye reduction was positively correlated with the power output which was highly dependent on the catholyte pH and the dye molecular structure. When pH was varied from 3.0 to 9.0, the k value in relation to MO degradation decreased from 0.298 to 0.016 μmol min⁻¹, and the maximum power density decreased from 34.77 to 1.51 mW m⁻². The performances of the MFC fed with different azo dyes can be ranked from good to poor as MO > Orange I > Orange II. Furthermore, the cyclic voltammograms of azo dyes disclosed that the pH and the dye structure determined their redox potentials. A higher redox potential corresponded to a higher reaction rate.     

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.     

Two different electron transfer pathways may involve in azoreduction in Shewanella decolorationis S12

Electron transfer pathways for azoreduction by S. decolorationis S12 were studied using a mutant S12-22 which had a transposon insertion in ccmA. The results imply that there are two different pathways for electron transport to azo bonds. The colony of S12-22 was whitish and incapable of producing mature c-type cytochromes whose α-peak was at 553 nm in the wild type S12. The mutant S12-22 could not use formate as the sole electron donor for azoreduction either in vivo or in vitro, but intact cells of S12-22 were able to reduce azo dyes of low polarity, such as methyl red, when NADH was served as the sole electron donor. Although the highly polar-sulfonated amaranth could not be reduced by intact cells of S12-22, it could be efficiently reduced by cell extracts of the mutant when NADH was provided as the sole electron donor. These results suggest that the mature c-type cytochromes are essential electron mediators for the extracellular azoreduction of intact cells, while the other pathway without the involvement of mature c-type cytochromes, NADH-dependent oxidoreductase-mediated electron transfer pathway can reduce lowly polar sulfonated azo dyes inside the whole cells or highly polar sulfonated azo dyes in the cell extracts without bacterial membrane barriers.     

Adsorption and decolorization kinetics of methyl orange by anaerobic sludge

Adsorption and decolorization kinetics of methyl orange (MO) by anaerobic sludge in anaerobic sequencing batch reactors were investigated. The anaerobic sludge was found to have a saturated adsorption capacity of 36 ± 1 mg g MLSS⁻¹ to MO. UV/visible spectrophotometer and high-performance liquid chromatography analytical results indicated that the MO adsorption and decolorization occurred simultaneously in this system. This process at various substrate concentrations could be well simulated using a modified two-stage model with apparent pseudo first-order kinetics. Furthermore, a noncompetitive inhibition kinetic model was also developed to describe the MO decolorization process at high NaCl concentrations, and an inhibition constant of 3.67 g NaCl l⁻¹ was estimated. This study offers an insight into the adsorption and decolorization processes of azo dyes by anaerobic sludge and provides a better understanding of the anaerobic dye decolorization mechanisms.     

Effects of electron donors and acceptors on anaerobic reduction of azo dyes by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12 was able to reduce various azo dyes in a defined medium with formate, lactate, and pyruvate or H₂ as electron donors under anaerobic conditions. Purified membranous, periplasmic, and cytoplasmic fractions from strain S12 analyzed, respectively, only membranous fraction was capable of reducing azo dye in the presence of electron donor, indicating that the enzyme system for anaerobic azoreduction was located on cellular membrane. Respiratory inhibitor Cu²⁺, dicumarol, stigmatellin, and metyrapone inhibited anaerobic azoreduction by purified membrane fraction, suggesting that the bacterial anaerobic azoreduction by strain S12 was a biochemical process that oxidizes the electron donors and transfers the electrons to the acceptors through a multicompound system related to electron transport chain. Dehydrogenases, cytochromes, and menaquinones were essential electron transport components for the azoreduction. The electron transport process for azoreduction was almost fully inhibited by O₂, 6 mM of , and 0.9 mM of , but not by 10 mM of Fe³⁺. The inhibition may be a result from the competition for electrons from electron donors. These findings impact on the understanding of the mechanism of bacterial anaerobic azoreduction and have implication for improving treatment methods of wastewater contaminated by azo dyes.     

Reduction and partial degradation mechanisms of naphthylaminesulfonic azo dye amaranth by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Reduction and biodegradation mechanisms of naphthylaminesulfonic azo dye amaranth using a newly isolated Shewanella decolorationis strain S12 were investigated. Under anaerobic conditions, amaranth was reduced by strain S12, and a stoichiometric amount of two reduction products RP-1 and RP-2 were generated. UV/visible spectrophotometric and high performance liquid chromatography (HPLC) analysis indicated that RP-1 and RP-2 were 1-aminenaphthylene -4-sulfonic acid and 1-aminenaphthylene-2-hydroxy-3, 6-disulfonic acid. The result strongly supports a mechanism of azo dye reduction by the process via the reductive cleavage of the azo bond to form corresponding aromatic amines. The result of HPLC analyses revealed that these aromatic amines were not able to be mineralized by strain S12 under anaerobic conditions. But after re-aeration of the decolorized culture, RP-2 was mineralized completely by this microorganism, but the consumption of RP-1 was not observed. Ames test showed that amaranth had mutagenic but no cytotoxic potential. The mutagenic potential was relieved after the anaerobic treatment with strain S12 as the mutagenic effect of the two reduction products from amaranth was not detected by Ames test. Thus, the ability of strain S12 to reduce and partially mineralize the naphthylaminesulfonic azo dye efficiently was demonstrated, which can potentially be used to biodegrade and detoxify wastewater containing azo dyes using an alternating anaerobic/aerobic treatment procedure.     

Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems

Biocathodes in bioelectrochemical systems (BESs) can be used to convert CO₂ into diverse organic compounds through a process called microbial electrosynthesis. Unfortunately, start-up of anaerobic biocathodes in BESs is a difficult and time consuming process. Here, a pre-enrichment method was developed to improve start-up of anaerobic facultatively autotrophic biocathodes capable of using cathodes as the electron donor (electrotrophs) and CO₂ as the electron acceptor. Anaerobic enrichment of bacteria from freshwater bog sediment samples was first performed in batch cultures fed with glucose and then used to inoculate BES cathode chambers set at −0.4 V (versus a standard hydrogen electrode; SHE). After two weeks of heterotrophic operation of BESs, CO₂ was provided as the sole electron acceptor and carbon source. Consumption of electrons from cathodes increased gradually and was sustained for about two months in concert with a significant decrease in cathode chamber headspace CO₂. The maximum current density consumed was −34 ± 4 mA/m². Biosynthesis resulted in organic compounds that included butanol, ethanol, acetate, propionate, butyrate, and hydrogen gas. Bacterial community analyses based on 16S rRNA gene clone libraries revealed Trichococcus palustris DSM 9172 (99% sequence identity) as the prevailing species in biocathode communities, followed by Oscillibacter sp. and Clostridium sp. Isolates from autotrophic cultivation were most closely related to Clostridium propionicum (99% sequence identity; ZZ16), Clostridium celerecrescens (98–99%; ZZ22, ZZ23), Desulfotomaculum sp. (97%; ZZ21), and Tissierella sp. (98%; ZZ25). This pre-enrichment procedure enables simplified start-up of anaerobic biocathodes for applications such as electrofuel production by facultatively autotrophic electrotrophs.