Dissimilatory Fe(III) reduction by an electrochemically active lactic acid bacterium phylogenetically related to Enterococcus gallinarum isolated from submerged soil

AIMS: The isolation and identification of a glucose-oxidizing Fe(III)-reducing bacteria (FRB) with electrochemical activity from an anoxic environment, and characterization of the role of Fe(III) in its metabolism. METHODS AND RESULTS: A Gram-positive (Firmicutes), nonmotile, coccoid and facultative anaerobic FRB was isolated based on its ability to reduce Fe(III). Using the Vitek Gram-positive identification card kit and 16S rRNA gene sequence analysis, the isolate was identified as Enterococcus gallinarum, designated strain MG25. On glucose this isolate produced lactate plus small amounts of acetate, formate and CO2 and its growth rates were similar in the presence and absence of Fe(O)OH. These results suggest that MG25 can couple glucose oxidation to Fe(III) reduction, but without conservation of energy to support growth. Cyclic voltammetry showed that strain MG25 was electrochemically active. CONCLUSIONS: An electrochemically active and FRB, E. gallinarum MG25, was isolated from submerged soil. Fe(III) is used in the bacterial metabolism as an electron sink. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report concerning the electrochemical activity of glucose-oxidizing FRB, E. gallinarum. This organism and others like it could be used as new biocatalysts to improve the performance of a mediator-less microbial fuel cell.     

Bacterial community structure, compartmentalization and activity in a microbial fuel cell

AIMS: To characterize bacterial populations and their activities within a microbial fuel cell (MFC), using cultivation-independent and cultivation approaches. METHODS AND RESULTS: Electron microscopic observations showed that the fuel cell electrode had a microbial biofilm attached to its surface with loosely associated microbial clumps. Bacterial 16S rRNA gene libraries were constructed and analysed from each of four compartments within the fuel cell: the planktonic community; the membrane biofilm; bacterial clumps (BC) and the anode biofilm. Results showed that the bacterial community structure varied significantly between these compartments. It was observed that Gammaproteobacteria phylotypes were present at higher numbers within libraries from the BC and electrode biofilm compared with other parts of the fuel cell. Community structure of the MFC determined by analyses of bacterial 16S rRNA gene libraries and anaerobic cultivation showed excellent agreement with community profiles from denaturing gradient gel electrophoresis (DGGE) analysis. CONCLUSIONS: Members of the family Enterobacteriaceae, such as Klebsiella sp. and Enterobacter sp. and other Gammaproteobacteria with Fe(III)-reducing and electrochemical activity had a significant potential for energy generation in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that electrochemically active bacteria can be enriched using an electrochemical fuel cell.     

微生物燃料电池中产电微生物的系统发育分析及筛选

对污水处理厂曝气池的产电微生物进行富集并利用纯培养法筛选,采用基于16S rRNA基因序列的系统发育分析方法研究了产电微生物的生物多样性,并基于三电极体系绘制出的循环伏安曲线鉴别出产电性能较强的纯菌株。结果表明,菌株F003、F042和F050与其系统发育关系最密切的有效发表种的典型菌株的16S rRNA基因序列存在较大差异,分别代表新的分类单元。之后又对所获得的38株菌株进行电化学测试活性,得出4株活性较强的菌株,其中菌株F010和F017的电化学活性比菌株F007和F051更为显著。     

微生物燃料电池中产电微生物的研究进展

产电微生物是微生物燃料电池系统的核心组成, 本文从生物学角度介绍了几种产电微生物的分类学地位、形态特征、生理生化特征及在微生物燃料电池中的产电机理和产电能力, 分析了利用产电微生物进行废水处理同时生物发电的应用前景, 提出产电微生物在MFC系统中的进一步研究方向为微生物的富集、驯化、改造和多种菌种优化组合等。     

Phosphate compounds as iron chelators in animal cell cultures

Summary We have studied the capacity of a number of phosphate compounds to act in the double role as a phosphate source and a detoxifier of ferric chloride hydroxo compounds, i.e. as Fe(III) chelators. The tested compounds were: orthophosphate, trimetaphosphate, α-glycerophosphate, β-glycerophosphate, phytic acid, and phosphorylcholine; the test organism the ciliate protozoonTetrahymena thermophila, an animal cell; and the nutrient medium was synthetic, consisting solely of low-molecular-weight compounds. We assessed growth rates of cells in two experimental series. First, phosphate-starved cells were exposed to the tested phosphate compound as the only phosphate source and the ferric chloride concentrations were varied stepwise from 0 to 1000μM. Second, we offered the cells orthophosphate as a phosphate source and selected phosphate compounds as chelators. The cell growth results allow the following conclusions: orthophosphate, trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent phosphate sources; trimetaphosphate, α-glycerophosphate, β-glycerophosphate, and phytic acid are excellent Fe(III) chelators; of the tested compounds trimetaphosphate, α-glycerophosphate, and β-glycerophosphate are excellent in the double role as a phosphate source and a ferric chloride hydroxo detoxifier, i.e. as a Fe(III) chelator.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Electrochemically active biofilms: facts and fiction. A review

This review examines the electrochemical techniques used to study extracellular electron transfer in the electrochemically active biofilms that are used in microbial fuel cells and other bioelectrochemical systems. Electrochemically active biofilms are defined as biofilms that exchange electrons with conductive surfaces: electrodes. Following the electrochemical conventions, and recognizing that electrodes can be considered reactants in these bioelectrochemical processes, biofilms that deliver electrons to the biofilm electrode are called anodic, ie electrode-reducing, biofilms, while biofilms that accept electrons from the biofilm electrode are called cathodic, ie electrode-oxidizing, biofilms. How to grow these electrochemically active biofilms in bioelectrochemical systems is discussed and also the critical choices made in the experimental setup that affect the experimental results. The reactor configurations used in bioelectrochemical systems research are also described and the authors demonstrate how to use selected voltammetric techniques to study extracellular electron transfer in bioelectrochemical systems. Finally, some critical concerns with the proposed electron transfer mechanisms in bioelectrochemical systems are addressed together with the prospects of bioelectrochemical systems as energy-converting and energy-harvesting devices.     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

Secondary Mineral Formation During Ferrihydrite Reduction by Shewanella oneidensis MR-1 Depends on Incubation Vessel Orientation and Resulting Gradients of Cells,Fe2+ and Fe Minerals

In previous studies on microbial ferric iron (Fe(III)) reduction varying results regarding reduction rates and secondary mineral formation have been reported for almost identical conditions regarding temperature, pH, medium composition, Fe(III) mineral identity and bulk iron concentration. Here we show that in addition to physico-chemical parameters also geometric aspects, i.e., incubation orientation and dimension of cultivation vessels, influence the reduction rates and mineralogy. We incubated the Fe(III)-reducer Shewanella oneidensis MR-1 in test tubes at ferrihydrite (FH) concentrations of 1.3–50 mM either in vertical or horizontal orientation. Cells and minerals formed a pellet at the bottom of the tubes with different thicknesses at the same initial FH concentration depending on the incubation orientation. In vertically incubated tubes thick FH pellets were present at the bottom of the tubes and magnetite was formed in all setups with ≥2.5 mM initial FH. In tubes that were incubated horizontally no magnetite was formed in presence of <5 mM initial FH. Spatially resolved analysis of the supernatant and mineral sediment including voltammetric microelectrodes, Xray diffraction and Mössbauer spectroscopy revealed strong gradients of Fe²⁺ in both the aqueous supernatant and mineral pellets, whereas a heterogeneous distribution of cells and minerals in the sediment pellet was detected. The highest cell density and, consequently, the initiation of FH reduction was found at the mineral-supernatant interface. This study demonstrates that small changes in incubation conditions can significantly influence and even change the experimental results of geomicrobiological experiments.     

异化金属还原菌的研究进展

微生物利用金属氧化物作呼吸作用的最终电子受体是一种新的代谢途径。该过程微生物利用有机底物异化还原金属氧化物进行生长代谢。异化金属还原菌对于研究探索古生物呼吸形式、界定生命的上限温度等生命科学问题具有重要研究价值,同时在生物整治、微生物燃料电池等方面具有广阔的应用前景。对异化金属还原菌进行了综述,并对这类菌的研究应用给了评述和展望。     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putrefaciens. Part 2. Oxo-and hydroxo-bridged polynuclear Fe(III) complexes

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes byShewanella putrefaciens intact cells and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H₃heidi =N-(2-hydroxyethyl)iminodiacetic acid) or nta (H₃nta = nitrilotriacetic acid), or the potential tridentate ida (H₂ida = iminodiacetic acid). A number of defined small complexes with varied nuclearity and solubility properties were employed, as well as undefined species prepared by mixing different molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(III) reduction determined by an assay for Fe(II) formation with ferrozine were validated by monitoringc-type cytochrome oxidation and re-reduction associated with electron transport. For the undefined Fe(III) polymeric species, reduction rates in whole cells and membranes were considerably faster in the presence of heidi compared to ida. This is believed to result from generally smaller and more reactive clusters forming with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importance. The increases in reduction rates of the undefined ida species with Fe(III) using membranes relative to whole cells reinforce such a view. Using soluble synthetic Fe(III) clusters, slow reduction was noted for an oxo-bridged dimer coordinatively saturated with ida and featuring unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were found to be reduced by whole cells, but dissimilation rates increased markedly using membranes. These data suggest that Fe(III) reductase activity may be located at both the outer membrane and the cytoplasmic membrane ofS. putrefaciens. Slower reduction of the heidi multimer relative to the heidi dimer reflects the presence of a central hydroxo(oxo)-bridged core containing nine Fe(III) nuclei within the former cluster. This unit is a poor substrate for dissimilation, owing to the fact that the Fe(III) is not ligated by aminocarboxylate. The faster reduction noted for the heidi dimer in membranes than for a soluble ida monomer suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insoluble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively saturated by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the bacterium.Previous paper in this series: Dobbin PS, Powell AK, McEwan AG, Richardson DJ. 1995 The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putefraciens.BioMetals 8, 163–173.     

Effects of inoculation strategy and cultivation approach on the performance of microbial fuel cell using marine sediment as bio-matrix

Aims: To investigate the effects of inoculation strategy and cultivation approach on the performance of microbial fuel cell (MFC). Methods and Results: A dual‐chamber sediment fuel cell was set up fed with glucose under batch condition. At day 30, the supernatant consortium was partly transferred and used as inoculum for the evaluation of cultivation approach. Power output gradually increased to 9·9 mW m^?2 over 180 days, corresponding to coulombic efficiency (CE) of 29·6%. Separated biofilms attached anode enabled power output and CE dramatically up to 100·9 mW m^?2 and over 50%, respectively, whereas the residual sediment catalysed MFC gave a poor performance. MFC catalysed by in situ supernatant consortium demonstrated more than twice higher power than MFC catalysed by the supernatant consortium after Fe(OH)₃ cultivation. However, the re‐generation of biofilms from the latter largely enhanced the cell performance. Conclusions: MFC exhibited a more efficient inducement of electroactive consortium than Fe(OH)₃ cultivation. MFC performance varied depending on different inoculation strategies. Significance and Impact of the Study: This is the first time to study cultivation approach affecting electricity generation. In addition, anodic limitations of mass and electron transfer were discussed through MFC catalysed by sediment‐based bio‐matrix.     

Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01)

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.     

Intracellular Localization and Characterization of Beef Liver Aldehyde Dehydrogenase Isozymes

Various physiological roles of mammalian aldehyde dehydrogenase had been anticipated because of its broad substrate specificity. In order to clarify roles of the enzyme and the regulation of aldehyde metabolisms in liver, the intracellular distribution and isozyme of beef liver aldehyde dehydrogenase were studied.

The presence of the mitochondrial, the microsomal and the cytoplasmic isozymes were proved by the isoelectric focusing. These isozymes were different from each other in pH-activity curve in the responces for steroid hormones and disulfiram.

It was suggested by comparing the reactivities of these isozymes for various aldehydes that particular aldehyde might be oxidized by a favorite isozyme at particular locality in the liver cells and that a share of physiological role among these isozymes is probable.     

4株高效产电菌的电化学活性及其生物学特征

采用循环伏安法对从湖南省吉首大田湾污水处理厂曝气池活性污泥中富集和筛选的37株产电菌的电化学活性进行考察。结果发现,4株菌株（F012、F015、F021、F026）的电化学活性较为显著,其中F026的电化学活性最好。对4株产电菌的系统发育分析表明,菌株F012属于Dyella属,与Dyella marensis CS5-B2^T系统发育关系最为密切（相似性为97.22%）;菌株F015属于Paludibacterium属,与该属的Paludibacterium yongneupense 5YN8-15^T系统发育关系最为密切（相似性为97.70%）;菌株F021和F026都属于Pseudomonas属,分别与该属的Pseudomonas simiae OLi^T和Pseudomonas otitidis MCC10330^T系统发育关系最为密切（相似性分别为99.60%和98.62%）。生物学特性研究表明,电化学活性最好的产电菌F026的生长温度范围为20~40 ℃,最适宜生长温度为30~35 ℃;生长pH范围为5~9,最适pH生长范围为8~9,适合作为微生物燃料电池的高效产电菌。     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) by Shewanella putrefaciens

The ability of S. putrefaciens to reduce Fe(III) complexed by a variety of ligands has been investigated. All of the ligands tested caused the cation to be more susceptible to reduction by harvested whole cells than when uncomplexed, although some complexes were more readily reduced than others. Monitoring rates of reduction by a ferrozine assay for Fe(II) formation proved inadequate using Fe(III) ligands giving Fe(II) complexes of low kinetic lability (e.g. EDTA). A more suitable assay for Fe(III) reduction in the presence of such ligands proved to be the observation of associated cytochrome oxidation and re-reduction. Where possible, an assay for Fe(III) reduction based upon the disappearance of Fe(III) complex was also employed. Reduction of all Fe(III) complexes tested was totally inhibited by the presence of O₂, partially inhibited by HQNO and slower in the absence of a physiological electron donor. Upon cell fractionation, Fe(III) reductase activity was detected exclusively in the membranes. Using different physiological electron donors in assays on membranes, relative reduction rates of Fe(III) complexes complemented the data from whole cells. The differences in susceptibility to reduction of the various complexes are discussed, as is evidence for the respiratory nature of the reduction.