A novel electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas hydrophila,isolated from a microbial fuel cell

A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA-DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila.     

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.     

Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment

A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment. The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone). Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation. Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures. Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies. 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii. Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose. Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed. The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM. A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors. Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.     

Carbohydrate oxidation coupled to Fe(III) reduction, a novel form of anaerobic metabolism

An isolate, designated GC-29, that could incompletely oxidize glucose to acetate and carbon dioxide with Fe(III) serving as the electron acceptor was recovered from freshwater sediments of the Potomac River, Maryland. This metabolism yielded energy to support cell growth. Strain GC-29 is a facultatively anaerobic, gram-negative motile rod which, in addition to glucose, also used sucrose, lactate, pyruvate, yeast extract, casamino acids or H2 as alternative electron donors for Fe(III) reduction. Stain GC-29 could reduce NO3(-), Mn(IV), U(VI), fumarate, malate, S2O3(2-), and colloidal S0 as well as the humics analog, 2,6-anthraquinone disulfonate. Analysis of the almost complete 16S rRNA sequence indicated that strain GC-29 belongs in the Shewanella genus in the epsilon subdivision of the Proteobacteria. The name Shewanella saccharophilia is proposed. Shewanella saccharophilia differs from previously described fermentative microorganisms that metabolize glucose with the reduction of Fe(III) because it transfers significantly more electron equivalents to Fe(III); acetate and carbon dioxide are the only products of glucose metabolism; energy is conserved from Fe(III) reduction; and glucose is not metabolized in the absence of Fe(III). The metabolism of organisms like S. saccharophilia may account for the fact that glucose is metabolized primarily to acetate and carbon dioxide in a variety of sediments in which Fe(III) reduction is the terminal electron accepting process.     

Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17

Aims:  To isolate an iron-reducing bacterium and examine its ability of Fe(III) oxide reduction and dechlorination.
Methods and Results:  A fermentative facultative anaerobe, strain L17 isolated from subterranean sediment, can reduce Fe(III) oxides and carbon tetrachloride (CT). It was identified as Klebsiella pneumoniae by 16S rRNA sequence analysis. Strain L17 can metabolize fermentable substrates such as citrate, glycerol, glucose and sucrose coupled with the reduction of hydrous ferric oxide, goethite, lepidocrocite and hematite. Fe(III) reduction was influenced by crystal structure of Fe(III) oxide, type of fermentable substrate, metabolic status of the strain, and significantly enhanced by addition of anthraquinone-2,6-disulfonate (AQDS). Strain L17 could dechlorinate CT to chloroform, and the rate was accelerated in the presence of Fe(III) oxide and AQDS. Biotic dechlorination by strain L17 and abiotic dechlorination by sorbed Fe(II) were proposed as the two main mechanisms. AQDS might accelerate the dechlorination by transferring electrons from strain L17 to Fe(III) oxide and CT.
Conclusions:  K. pneumoniae L17 can reduce Fe(III) oxides and CT. The two reductions can occur simultaneously, and be significantly promoted by AQDS.
Significance and Impact of the Study:  This is the first report of a strain of K. pneumoniae capable of reducing Fe(III) oxides and CT. As a strain of environmental origin, strain L17 may have the potential for bioremediation of chlorinated compound-contaminated sites.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

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.     

异化铁还原细菌Clostridium sp.LQ25分离及其产氢与铁还原特性

[背景] 一些异化铁还原细菌兼具铁还原和发酵产氢能力,可作为发酵型异化铁还原细菌还原机制研究的对象。[目的] 筛选出一株发酵型异化铁还原细菌。在异化铁还原细菌培养体系中,设置不同电子供体并分析电子供体。[方法] 通过三层平板法从海洋沉积物中筛选纯菌株,基于16S rRNA基因序列进行菌株鉴定。通过测定细菌培养液Fe （II）浓度及发酵产氢量分析菌株异化铁还原和产氢性质。[结果] 菌株LQ25与Clostridium butyricum的16S rRNA基因序列相似性达到100%,结合电镜形态观察,菌株命名为Clostridium sp.LQ25。在氢氧化铁为电子受体培养条件下,菌株生长较对照组（未添加氢氧化铁）显著提高。菌株LQ25能够利用丙酮酸钠、葡萄糖和乳酸钠进行生长。丙酮酸钠为电子供体时,菌株LQ25细胞生长和异化铁还原效率最高,菌体蛋白质含量是（78.88±3.40） mg/L,累积产生Fe （II）浓度为（8.27±0.23） mg/L。以葡萄糖为电子供体时,菌株LQ25发酵产氢量最高,达（475.2±14.4） mL/L,相比对照组（未添加氢氧化铁）产氢量提高87.7%。[结论] 筛选到一株具有异化铁还原和发酵产氢能力的菌株Clostridium sp.LQ25,为探究发酵型异化铁还原细菌胞外电子传递机制提供了新的实验材料。     

Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions

AIMS: The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers. METHODS AND RESULTS: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost. The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42. Good growth of the strain was observed at pH 10. The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses. The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B. clausii. The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol%. The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11.0 and 60 degrees C. CONCLUSIONS: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents.     

Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose.

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12 degrees C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H(2) was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4', 6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2. 3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H(2). Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H(2).     

Involvement of thermophilic archaea in the biocorrosion of oil pipelines

Two thermophilic archaea, strain PK and strain MG, were isolated from a culture enriched at 80°C from the inner surface material of a hot oil pipeline. Strain PK could ferment complex organic nitrogen sources (e.g. yeast extract, peptone, tryptone) and was able to reduce elemental sulfur (S°), Fe(3+) and Mn(4+) . Phylogenetic analysis revealed that the organism belonged to the order Thermococcales. Incubations of this strain with elemental iron (Fe°) resulted in the abiotic formation of ferrous iron and the accumulation of volatile fatty acids during yeast extract fermentation. The other isolate, strain MG, was a H(2) :CO(2) -utilizing methanogen, phylogenetically affiliated with the genus Methanothermobacter family. Co-cultures of the strains grew as aggregates that produced CH(4) without exogenous H(2) amendment. The co-culture produced the same suite but greater concentrations of fatty acids from yeast extract than did strain PK alone. Thus, the physiological characteristics of organisms both alone and in combination could conceivably contribute to pipeline corrosion. The Thermococcus strain PK could reduce elemental sulfur to sulfide, produce fatty acids and reduce ferric iron. The hydrogenotrophic methanogen strain MG enhanced fatty acid production by fermentative organisms but could not couple the dissolution Fe° with the consumption of water-derived H(2) like other methanogens.     

Microbial Reduction of Fe(III) in Acidic Sediments: Isolation of Acidiphilium cryptum JF-5 Capable of Coupling the Reduction of Fe(III) to the Oxidation of Glucose

To evaluate the microbial populations involved in the reduction of Fe(III) in an acidic, iron-rich sediment, the anaerobic flow of supplemental carbon and reductant was evaluated in sediment microcosms at the in situ temperature of 12°C. Supplemental glucose and cellobiose stimulated the formation of Fe(II); 42 and 21% of the reducing equivalents that were theoretically obtained from glucose and cellobiose, respectively, were recovered in Fe(II). Likewise, supplemental H₂ was consumed by acidic sediments and yielded additional amounts of Fe(II) in a ratio of approximately 1:2. In contrast, supplemental lactate did not stimulate the formation of Fe(II). Supplemental acetate was not consumed and inhibited the formation of Fe(II). Most-probable-number estimates demonstrated that glucose-utilizing acidophilic Fe(III)-reducing bacteria approximated to 1% of the total direct counts of 4′,6-diamidino-2-phenylindole-stained bacteria. From the highest growth-positive dilution of the most-probable-number series at pH 2.3 supplemented with glucose, an isolate, JF-5, that could dissimilate Fe(III) was obtained. JF-5 was an acidophilic, gram-negative, facultative anaerobe that completely oxidized the following substrates via the dissimilation of Fe(III): glucose, fructose, xylose, ethanol, glycerol, malate, glutamate, fumarate, citrate, succinate, and H₂. Growth and the reduction of Fe(III) did not occur in the presence of acetate. Cells of JF-5 grown under Fe(III)-reducing conditions formed blebs, i.e., protrusions that were still in contact with the cytoplasmic membrane. Analysis of the 16S rRNA gene sequence of JF-5 demonstrated that it was closely related to an Australian isolate of Acidiphilium cryptum (99.6% sequence similarity), an organism not previously shown to couple the complete oxidation of sugars to the reduction of Fe(III). These collective results indicate that the in situ reduction of Fe(III) in acidic sediments can be mediated by heterotrophic Acidiphilium species that are capable of coupling the reduction of Fe(III) to the complete oxidation of a large variety of substrates including glucose and H₂.     

Sinorhodobacter ferrireducens gen. nov., sp. nov., a non-phototrophic iron-reducing bacterium closely related to phototrophic Rhodobacter species

An Fe(III) oxide-reducing bacterium designated as SgZ-3^T, which could couple glucose oxidation to Fe(III) oxide reduction for energy conservation, was isolated from an electrochemical biofilm. The isolate was Gram-negative, catalase-positive and oxidase-negative, and facultative anaerobic. NaCl was not required for growth, but NaCl concentrations up to 5 % (w/v) were tolerated. Growth occurred in TSB (tryptic soy broth) at 20–40 °C (optimum 30 °C) and at pH 6.0–7.5 (optimum 7.0). Phototrophic growth could not be demonstrated. No vesicular photosynthetic membrane was observed. Bacteriochlorophyll α and carotenoids were absent. Biotin and thiamine were required as growth factors for the isolate. Phylogenetic analysis of the 16S rRNA gene sequence placed strain SgZ-3^T within the family Rhodobacteraceae and affiliated with an phototrophic genera Rhodobacter. The G+C content of the genomic DNA was 68.6 mol%. Strain SgZ-3^T contained Q-10 as the predominant quinone. The major cellular fatty acids were C_18:1 ω6c and/or C_18:1 ω7c (66.9 %) and C_16:0 (9.5 %). Based on its phenotypic and phylogenetic properties, chemotaxonomic analysis and the results of physiological and biochemical tests, strain SgZ-3^T (=KACC 16603^T = CCTCC AB2012026^T) was designated as the type strain of a new genus and a novel species of the family Rhodobacteraceae, for which the name Sinorhodobacter ferrireducens sp. nov. was proposed.     

Efficiency of chlorocatechol metabolism in natural and constructed chlorobenzoate and chlorobiphenyl degraders

AIMS: A possibility for the complementation of both ortho- and meta-cleavage pathway for chlorocatechols in one strain and its impact on degradation of chlorobenzoates accumulated during degradation of polychlorinated biphenyls was investigated. METHODS AND RESULTS: Genes responsible for ortho-cleavage of chlorocatechols were subcloned into two biphenyl degraders and the activities of chlorocatechol dioxygenases responsible for ortho- and meta-cleavage in these hybrid strains were monitored spectrophotometrically and also electrochemically by ion-selective electrode. CONCLUSIONS: While strain Pseudomonas fluorescens S12/C apparently gained metabolic advantage from this gene manipulation, strain Burkholderia cepacia P166/C did not express better degradation features in comparison with the parental strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach has the potential to enhance chlorocatechol metabolism in selected biphenyl degraders.     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.     

Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains

Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10^-4 ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population.     

Microbial communities and electrochemical performance of titanium-based anodic electrodes in a microbial fuel cell

Four types of titanium (Ti)-based electrodes were tested in the same microbial fuel cell (MFC) anodic compartment. Their electrochemical performances and the dominant microbial communities of the electrode biofilms were compared. The electrodes were identical in shape, macroscopic surface area, and core material but differed in either surface coating (Pt- or Ta-coated metal composites) or surface texture (smooth or rough). The MFC was inoculated with electrochemically active, neutrophilic microorganisms that had been enriched in the anodic compartments of acetate-fed MFCs over a period of 4 years. The original inoculum consisted of bioreactor sludge samples amended with Geobacter sulfurreducens strain PCA. Overall, the Pt- and Ta-coated Ti bioanodes (electrode-biofilm association) showed higher current production than the uncoated Ti bioanodes. Analyses of extracted DNA of the anodic liquid and the Pt- and Ta-coated Ti electrode biofilms indicated differences in the dominant bacterial communities. Biofilm formation on the uncoated electrodes was poor and insufficient for further analyses. Bioanode samples from the Pt- and Ta-coated Ti electrodes incubated with Fe(III) and acetate showed several Fe(III)-reducing bacteria, of which selected species were dominant, on the surface of the electrodes. In contrast, nitrate-enriched samples showed less diversity, and the enriched strains were not dominant on the electrode surface. Isolated Fe(III)-reducing strains were phylogenetically related, but not all identical, to Geobacter sulfurreducens strain PCA. Other bacterial species were also detected in the system, such as a Propionicimonas-related species that was dominant in the anodic liquid and Pseudomonas-, Clostridium-, Desulfovibrio-, Azospira-, and Aeromonas-related species.     

Novel Pyridinium compound from marine actinomycete, Amycolatopsis alba var. nov. DVR D4 showing antimicrobial and cytotoxic activities in vitro

Marine sediment samples from Visakhapatnam coast of Bay of Bengal, India, were investigated as a source of actinomycetes to screen for the production of antibiotics and cytotoxic compounds. Actinomycete strain DVR D4 with interesting bioactivity profile was isolated during our systematic study of marine actinomycetes. Based on biochemical properties and 16S rDNA analysis the isolate DVR D4 was identified as a strain of Amycolatopsis alba. A solvent extraction followed by a chromatographic purification helped to isolate a cytotoxic compound, which was identified as 1(10-aminodecyl) Pyridinium salt antibiotic, on the basis of spectral data. The compound showed potent cytotoxic activity against cancer cell lines of cervix (HeLa), breast (MCF-7) and brain (U87MG) in vitro and also exhibited antibacterial activities against Gram-positive and Gram-negative bacteria.