text:
Yong Xiao
Institute of Urban Environment, Chinese Academy of Sciences (CAS), China
Luciano T. Kishi
National Laboratory for Scientific Computing (LNCC), Brazil
Zheng Chen
Xi'an Jiaotong-Liverpool University, China
Front. Microbiol., 12 July 2016
Sec. Microbiotechnology
A Novel Electrophototrophic Bacterium Rhodopseudomonas palustris Strain RP2, Exhibits Hydrocarbonoclastic Potential in Anaerobic Environments
Krishnaveni Venkidusamy1,2* and Mallavarapu Megharaj1,2,3
1Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA, Australia
2CRC for Contamination Assessment and Remediation of the Environment, Mawson Lakes, SA, Australia
3Global Centre for Environmental Risk Assessment and Remediation, The University of Newcastle, Callaghan, NSW, Australia
An electrophototrophic, hydrocarbonoclastic bacterium Rhodopseudomonas palustris stain RP2 was isolated from the anodic biofilms of hydrocarbon fed microbial electrochemical remediation systems (MERS). Salient properties of the strain RP2 were direct electrode respiration, dissimilatory metal oxide reduction, spore formation, anaerobic nitrate reduction, free living diazotrophy and the ability to degrade n-alkane components of petroleum hydrocarbons (PH) in anoxic, photic environments. In acetate fed microbial electrochemical cells, a maximum current density of 305 ± 10 mA/m2 (1000Ω) was generated (power density 131.65 ± 10 mW/m2) by strain RP2 with a coulombic efficiency of 46.7 ± 1.3%. Cyclic voltammetry studies showed that anaerobically grown cells of strain RP2 is electrochemically active and likely to transfer electrons extracellularly to solid electron acceptors through membrane bound compounds, however, aerobically grown cells lacked the electrochemical activity. The ability of strain RP2 to produce current (maximum current density 21 ± 3 mA/m2; power density 720 ± 7 μW/m2, 1000 Ω) using PH as a sole energy source was also examined using an initial concentration of 800 mg l-1 of diesel range hydrocarbons (C9-C36) with a concomitant removal of 47.4 ± 2.7% hydrocarbons in MERS. Here, we also report the first study that shows an initial evidence for the existence of a hydrocarbonoclastic behavior in the strain RP2 when grown in different electron accepting and illuminated conditions (anaerobic and MERS degradation). Such observations reveal the importance of photoorganotrophic growth in the utilization of hydrocarbons from contaminated environments. Identification of such novel petrochemical hydrocarbon degrading electricigens, not only expands the knowledge on the range of bacteria known for the hydrocarbon bioremediation but also shows a biotechnological potential that goes well beyond its applications to MERS.
Soil and groundwater petroleum hydrocarbon contamination has long been a serious concern to environmental and public health. Of these petroleum hydrocarbon (PH) contaminants, diesel range hydrocarbons (DRH) have been documented as one of the most abundant pollutants; they can be biodegradable in both oxic and anoxic conditions (Huang et al., 2011). Microbial remediation of these petrochemical compounds is claimed to be an efficient, economic, versatile alternative to the physicochemical methods. However, the rate of microbial utilization of these PH compounds is very slow especially in anaerobic environments where the availability of relevant electron acceptors is limited. The recent research on such recalcitrant contaminant removal using bioelectrochemical systems is leading to a new interest in its practical applications. An emerging microbial electrochemical remediation systems (MERS) shows potential as an effective approach that can exploit microorganisms for treating contaminants while generating electricity in the process. This has recently been proposed for the remediation of PH contaminants by capitalizing on the bio-catalytic potential of electrode respiring bacteria (ERB; Morris et al., 2009; Venkidusamy et al., 2016).
Electrode respiring bacteria are a group that has received much attention in the field of electromicrobiology because of their exoelectrogenic capabilities to degrade substrates that range from easily degradable natural organic compounds to xenobiotic compounds such as PH contaminants. Many studies have shown the presence of diverse, electro active-microbial communities on fuel cell electrodes including members of the Alphaproteobacteria (Zuo et al., 2008), Betaproteobacteria (Chaudhuri and Lovley, 2003), Gammaproteobacteria (Kim et al., 2002), Deltaproteobacteria (Holmes et al., 2006), and Firmicutes (Wrighton et al., 2008). Of these, Gammaproteobacteria was the dominant class found on the anodes of organic contaminants such as PH fed MERS (Morris et al., 2009; Venkidusamy et al., 2016). Several bacterial strains from this class have been isolated either from electrochemical systems fed with wastewater or defined carbons sources and their physiological roles have been studied. However, the microbial community composition is divergent in MERS (Morris et al., 2009; Venkidusamy et al., 2016), and the physiology of such populations remains to be investigated. Therefore, identification of such ERB which can be used in MERS systems for the enhanced removal of PH contaminants is of current importance.
In the present study, such a novel electrode respiring, hydrocarbonoclastic bacterial strain was isolated from the biofilms of PH fed MERS anodes and analyzed its physiology and activities in pure culture studies. This strain was found to be a Fe (III) respiring bacterium, phylogenetically related to Rhodopseudomonas palustris and designated as R. palustris strain RP2. The electrochemical activity was determined by using cyclic voltammetry and fuel cell techniques. Here, we show the existence of hydrocarbonoclastic behavior by the strain RP2, first such organism from MERS environments with several novel features.
Bacterial Strain
The bacterium used in this study was isolated from the anodic biofilm of a MERS through serial dilution techniques. The initial source of inoculum for the PH fed MERS was a PH contaminated ground water inoculated with activated sludge. Bacterial cells of the anodic biofilm were extracted into a sterile phosphate buffer and shaken vigorously to separate cells from the electrode. The extracted cell suspensions were serially diluted and plated on modified Hungate's medium (Hungate, 1950) and incubated anaerobically in a glove box (Don Whitley Scientific, MG500, Australia) for a period of two weeks. Single colonies were randomly selected and transferred to anaerobic agar plates filled with Luria Bertani (LB) medium (Sambrook et al., 1989). Cultures were routinely cultivated using anoxic rich medium (LB) under illuminated conditions. A chemically defined medium supplemented with Wolfe's trace elements and vitamins was used in the fuel cell experiments as previously described (Oh et al., 2004).
Culture Conditions and Biodegradation Experiments
Bacterial cells were grown under anoxic photosynthetic conditions with acetate (20 mM) as an electron donor in phosphate buffer saline supplemented with Wolfe's trace elements and vitamins and sealed with aluminum crimps. For experiments with different electron acceptors 10 mM nitrate, 10 mM sulfate, 10 mM iron (III) citrate, and 10 mM iron (III) oxide were used. The cells were cultured under different physiological conditions, including phototrophic and chemotrophic, oxic and anoxic conditions. The photosynthetic pigments were analyzed by measuring the whole cell absorption spectra in the UV-visible ranges from 300 to 1100 nm as described by Mehrabi et al. (2001). Biolog-GN2 plates were used to determine the carbon source utilization using the strain RP2 (Biolog., USA) according to the manufacturer's instructions under anoxic, illuminated conditions. The cells were also tested for their growth with nitrogen-deficient solid medium lacking a carbon source according to Kranz and Haselkorn (1986). DRHs was also used as the electron donor in biodegradation experiments at a concentration of 4000 mg l-1. Hydrocarbonoclastic potential of the strain RP2 was monitored under different electron accepting, growth environments (nitrate, sulfate, iron (III) as terminal electron acceptors) including phototrophic and chemotrophic conditions. All cell cultures were maintained in triplicates for each experiment. All procedures for anoxic growth experiments, from medium preparation to manipulating the strain were performed using standard anoxic conditions. All culturing was prepared in sealed serum vials with nitrogen/carbon dioxide (80:20, v/v) in the headspace.
Fe(III) Oxide Reduction
For investigating Fe(III) respiration process, cells were grown in two different environments: anoxic photoheterotrophic and chemoheterotrophic culture conditions supplemented with crystalline Fe(III) oxide (10 mM) as the terminal electron acceptor. The cells were grown in Wolfe's medium using acetate (20 mM) supplemented with trace elements and vitamins (Lovley and Phillips, 1988a). Fe(III) reduction was determined using the ferrozine assay (Lovley and Phillips, 1988b). The bacterial suspension was added to a pre-weighed vial containing 0.5 M HCL. HCL extracted samples were added to 5 ml of ferrozine (1 g l-1) in 50 mM HEPS buffer. The filtered samples were then analyzed in a UV-Vis spectrophotometer (maxima@λ 562 nm) to quantify the Fe(II) formation as previously described (Lovley and Phillips, 1988b).
Samples for transmission electron microscopy were fixed in electron microscopy fixative (4% paraformaldehyde/1.25% glutaraldehyde in PBS, + 4% sucrose, pH-7.2) and washed with buffer. Samples were postfixed in 2% aqueous osmium tetroxide. They were dehydrated in a graded series of ethanol, and then infiltrated with procure/araldite epoxy resin. Blocks were polymerized overnight at 70°C. Sections were cut on a Leica UC6 Ultramicrotome using a diamond knife, stained with uranyl acetate and lead citrate, and examined in a FEI Tecnai G2 Spirit Transmission Electron Microscope. The electrode samples were fixed and prepared as described earlier (Venkidusamy et al., 2016). The dried brush samples were examined using a scanning electron microscope (Quanta FEG 450, FEI) at an accelerating voltage of 20 kV.
16S rRNA Gene and Phylogenetic Analysis
Genomic DNA of R. palustris strain RP2 was extracted from photoheterotrophically grown cells by using the UltraClean microbial DNA isolation kit (MO BIO, Carlsbad, CA, USA) following the manufacturer's instructions. The universal primers were used to amplify16S rRNA gene according to the procedure devised by Weisburg et al. (1991). The PCR products were purified via the UltraClean PCR clean-up kit (Mo Bio, Carlsbad, CA, USA) following the manufacturer's instructions, and sequenced by the Southern Pathology Sequencing Facility at Flinders Medical Centre. The neighbor joining tree was constructed using the molecular evolutionary genetic analysis package version 5.0 based on 1000 bootstrap values (Tamura et al., 2011). The presence of the pufM gene was identified using published pufM primers (Achenbach et al., 2001). The presence of nifH gene was also analyzed using a gene specific primer (Cantera et al., 2004). In silico analysis was done by using the blast programs to search the GenBank and NCBI databases (http://www.ncbi.nlm.nih.gov).
Microbial Fuel Cell (MFC) construction
Single chamber and dual chamber cubic MFC reactors were used to evaluate power generation from strain RP2. Single chamber bottle MFCs were made from laboratory Schott duran bottles with a capacity of 300 ml as suggested by Logan et al. (2007). The liquid volume of the chamber was 280 ml. Dual chamber cubic MFCs were constructed using two lexan glasses separated by a cation exchange membrane (Membrane International Inc., USA). The liquid volume of a dual chamber cubic MFC was 150 ml. Anodes were carbon paper or graphite fiber brushes of 5 cm in diameter and 7 cm in length. The graphite brushes were treated as previously described (Feng et al., 2010). Plain carbon paper was used as the anode material in dual chamber cubic MFC systems. Single chamber air cathodes contained 0.5 mg cm-2 of platinum catalyst on the liquid facing side with diffusional layers of PTFE applied to the air facing side (Cheng et al., 2006). All the reactors were sterilized before use.
MFC Operational Conditions
Strain RP2 was used for fuel cell experiments with acetate (1 g/L) as the electron donor in 50 mM PBS buffer. A higher concentration (200 mM) of buffer was also used in fuel cell experiments to examine the effect on power generation. A stationary phase culture was used in the anode chamber and the cells were operated under illuminated conditions. The anodic chamber was flushed with nitrogen gas and filled with anaerobic growth medium. The anolyte was agitated using a magnetic stirrer operating at 100 rpm. In the cubic MFC, the cathode chamber was provided with air through a 0.45 μ pore sized membrane filter. Solutions were replaced when the voltage dropped <10 mV. Open circuit MFC studies were also carried out and then switched to closed circuit with a selected external load (R-1000 Ω unless stated otherwise). DRH compounds were also used as sole source of energy in degradation experiments using the strain RP2 at a concentration of 800 mg l-1 in MERS studies. All the reactors were maintained in photoheterotrophic and chemoheterotrophic culture conditions at room temperature in triplicates.
Electrochemical Analysis
The electrochemical activity of strain RP2 was examined using cyclic voltammetry with a conventional three electrode electrochemical cell with a 25 ml capacity as described earlier (Kim et al., 1999). Bacterial cells grown (aerobic and anaerobic growth environments) in Fe(III) oxide liquid cultures were harvested and used for testing electrochemical activities. Cyclic voltammograms of the bacterial suspension were obtained using a potentiostat (Electrochemical analyser, BAS 100B, USA) connected to a personal computer with BAS software. A glassy carbon working electrode (3 mm, diameter, MF-2012, BAS) and silver/silver chloride reference electrode (MW-4130, BAS) and platinum counter electrode (MW-4130, BAS) were used in a conventional three electrode system. The working electrodes were polished with alumina slurry on cotton wool before each measurement. The electrochemical cells were purged with nitrogen gas for 15 minutes before each measurement. Different scan rates were used from 5 to 100 mV/sec with a potential range from -800 to 800 mV.
Analytical Methods and Calculations
Fe(III) reduction was monitored by measuring Fe(II) production by the ferrozine method (Lovley and Phillips, 1988b). The DRH was extracted in acetone-methylene chloride (1:1) mixture, dewatered and concentrated by evaporator, and then concentrations were measured by GC-FID using a HP-5 capillary column (15 m length, 0.32 mm thickness, 0.1 μm internal diameter) following the USEPA protocol (USEPA, 1996). The resulting chromatograms were analyzed using Agilent software (GC-FID Agilent model 6890) to identify the hydrocarbon degradation products. Chemical oxygen demand was measured by COD analyser (Chemetrics, K-7365). Fuel cell power output was monitored using a DMM (Keithly Model 2701, USA) linked to a multi-channel scanner (Module 7700, Keithly Instruments, USA). Data were recorded digitally on an Intel computer via IEEE 488 input system and Keithly cable. To measure the current under closed circuit conditions, the external load was connected (R-1000Ω unless stated otherwise). Current was calculated by using I = V/R, and power was calculated via P = VI. Power density and current density were normalized to the projected surface area of a cathode. Polarization curves were plotted using various external loads with a range of 10 Ω to open circuit. Coulombic efficiency (CE) was calculated at the end of the cycle from COD removal as previously described by an Logan (2008).
Nucleotide Sequence Numbers
The 16S rRNA, pufM, nifH gene sequences have been deposited in the GenBank database under the accession numbers of KJ460004, J1289478, J223658.
Strain Identification
In our laboratory experiments, a strain of R. palustris strain RP2 emerged as a dominant species was isolated from the anodic biofilm PH fed MERS. R. palustris strain RP2 is a cosmopolitan phototrophic bacterium, contains double membrane bilayers, lamellar thylakoid membrane system, produce chains of magnetosomes (Venkidusamy et al., 2015), and grow as long bacillus-shaped (0.5 to 1 μm wide and 2.0 to 6 μm long cells) with asymmetric cell division. Cell division occurred by budding, with dumbbell shaped daughter cells, forming rosette-like structures. Thin section transmission electron micrographs revealed the presence of a lamellar thylakoid membrane system presumably containing the photosynthetic apparatus in anoxic phototrophically grown cells (Supplementary Figure S1A) whereas cells grown in the dark lacked intra cytoplasmic (ICM) membranes (Supplementary Figure S1B).
Photosynthetic Pigments
Photosynthetically grown anoxic liquid cells were dark red (Supplementary Figure S2A) in color whereas chemosynthetically grown anoxic cells were colorless (Supplementary Figure S2B). Cultures grown under aerobic conditions were faint pink to colorless. Thin section transmission electron micrographs revealed the presence of a lamellar thylakoid membrane system presumably containing the photosynthetic apparatus in anoxic phototrophically grown cells. Absorption maxima of the homogenized photosynthetic cells showed three bacteriochlorophyll peaks at 379, 473, and 503 nm, and peaks at 545, 594, 807, and 875 nm indicated the presence of carotenoid pigments of the spillriloxanthin series. In contrast, no peaks were observed in the chemosynthetically grown cells. The presence of the photosynthetic gene (pufM gene) in the strain RP2 was shown using PCR with primers specific for this gene, resulting in a 230 bp product.
Physiological and Metabolic Characters
The cells of strain RP2 have exceptionally flexible growth based on environmental signals such as photoorganotrophic, photolithotrophic, dark fermentative, and aerobic heterotrophic mechanisms. Optimum bacterial growth was observed at 25 to 30°C at a neutral pH, whilst no growth was detected above 40°C. Experiments to determine the growth factor requirements for the strain RP2 clearly shows the need for p-aminobenzoate, pyridoxine HCl, and folic acid (Supplementary Table S1). Salient properties of the strain RP2 were direct electrode respiration, dissimilatory metal oxide reduction, anaerobic nitrate reduction, carbon dioxide fixation, free living diazotrophy, and the ability to degrade n-alkane components of PH under anoxic environments. Strain RP2 showed growth in minimal medium in the absence of both nitrogen and carbon sources under anoxic, photosynthetic conditions. Nitrogen fixation trait was confirmed by the detection of a nifH gene required for nitrogen fixation in the strain. The strain RP2 differed from a previously reported exoelectrogenic strain of R. palustris DX1 (Xing et al., 2008) with respect to its ability of photoassimilating a range of substrates including gluconate, aspartate, glycerol, and amino-ethanol. However, the strain RP2 was unable to utilize some compounds such as sebacic acid, threonine, L-ornithine, and L-proline. The ability to utilize sodium benzoate distinguishes the strain from other species of purple sulfur bacteria. The strain RP2 can assimilate acetate photosynthetically with nitrate, sulfate, and iron as terminal electron acceptors (Supplementary Figure S3). We cultured RP2 cells under two different environmental conditions: anoxic photoheterotrophic and anoxic chemoheterotrophic with crystalline Fe(III) oxide as a terminal electron acceptor to investigate the dissimilatory metal oxide reduction trait. Fe(III) oxide reduction was monitored by color change and hydroxylamine Fe(II)extraction assay. Fe(III) oxide reduction of 69.5% ± 0.41% was observed only in anoxic photoheterotrophic environments (Venkidusamy et al., 2015) whereas chemosynthetic grown cells showed no reduction. During this process, peculiar extracellular electrically conductive nanofilamentous structures were observed in the phototrophic growth conditions as stated previously (Venkidusamy et al., 2015). When colonies are incubated for longer incubations (for a period of 4-5 weeks) under anoxic chemosynthetic conditions, they develop a complex morphology such as spore formation with a well-defined outer layer (Figure 1).
FIGURE 1. Micrographs of spore formation in chemosynthetically grown cells of R. palustris strain RP2. (A) SEM micrograph. (B) TEM micrograph.
ClustalW alignment was used to align 1420 bp of the 16S ribosomal RNA gene sequence of strain RP2 with the same region of several related non-sulfur alphaproteobacteria groups. Using this multiple alignment, the neighborhood phylogenetic tree was constructed (Figure 2). Phylogenetic analysis of 16S rRNA gene sequences of strain RP2 demonstrated that the strain was closely linked to the genera of Rhodopseudomonas with 98% identity to sequences from R. palustris ATCC (17005) and Rhodopseudomonas sp. DPT4 (2001).
FIGURE 2. Phylogenetic tree based on 16S rRNA sequences showing the positions of the isolate, R. palustris strain RP2 and representatives of other closely related species. The tree was constructed form 1,420 aligned bases; Scale bar represents 0.002 substitution per nucleotide.
Current Generation by R. palustris Strain RP2 in Acetate MFCs
Current was generated in all the MFCs inoculated with strain RP2 using acetate as an energy source. After three days, voltage started to follow a constant pattern and then stabilized. The fuel cell electrodes were discharged through a 1000 Ω external resistance once it reached the plateau voltage generation stage. The voltage fell quickly from 690 ± 10 mV to 446 ± 5 mV after a 1000 Ω fixed resistor was connected. The maximum output range of voltage and current density were 446 ± 7 mV, 305 ± 10 mA/m2 (R = 1000 Ω) after four cycles of operation. Few representative initial cycles (average current density from triplicates) of current density are shown in (Figure 3A). After six refilling batches with a fresh substrate, the maximum current output of each batch became stable (300 ± 7 mA/m2). The maximum open circuit voltage was 700 ± 7 mV and attainable power density was 131.65 ± 10 mW/m2 (R = 1000 Ω) (Figure 3B). The CEs showed the increased trend with the increased current densities as shown in the Figure 3C. The maximum CE was 46.7% which corresponded to the maximum current density of 259.90 mA/m2. The higher buffer (200 mM) concentration were also examined at the same resistance which produced a maximum power density of 300 ± 20 mW/m2.
FIGURE 3. (A) A few representative initial cycles of current density (average from triplicates) generated by strain RP2 fed with acetate. (B) Power density and voltage generation as a function of current density from strain RP2 inoculated acetate-fed bottle MFCs. (C) Coulombic efficiency (CE) of a few representative batches of acetate-fed bottle MFCs.
Experiments were also conducted using dual chamber cubic MFCs (CMFC) containing carbon flat paper anodes at a fixed resistance of 1000 Ω. The voltage profile of the cubic MFC revealed that the voltage produced was less than that for the air cathode MFC. A long lag time was observed with the carbon paper anode whereas a treated brush anode reduced the strain acclimation period and resulted in an increased power generation. The maximum open circuit potential and power output of CMFC were 600 ± 7 mV and 70 mW/m2, respectively. Constant power and voltage output indicated the formation of a stable biofilm around the electrode surface. Scanning electron micrographs revealed that the bacterial population from the graphite brush anode was homogenous in shape (Figure 4).
FIGURE 4. Anode micrographs of R. palustris strain RP2 inoculated acetate-fed MFCs. (A) Treated control anode, (B) Strain RP2- biofilm on graphite surface and (C) Cells with high magnification.
Electrochemical Activity of R. palustris Strain RP2
Cell suspensions were prepared from the bacterial cells grown under anaerobic and aerobic environments to determine the electrochemical activities of the strain RP2 using cyclic voltammetry (CV) studies. Oxidation and reduction peaks of CV were observed in anaerobic grown bacterial cells (Figure 5A) whereas aerobically grown cells lacked the electrochemical activity (Figure 5B). Reduction peaks ranging from -394 mV to -399 mV and oxidation peaks ranging from -200 mV to +100 mV were observed at the electrode interface from washed cells suspensions. The calculated mid-point potential was about -278 mV. This asymmetric CV peak shows that the redox reaction is a quasi-reversible reaction. The amplitude of the peaks increased according to the growth stage of the culture in batch mode. The highest peaks were present during the exponential growth stage which indicated that the development of biofilm was the main factor for the electron transfer. One redox couple was observed from the CV peak and number of electrons transferred was calculated based on the Nernst equation (Logan, 2008). The CV peaks were not observed from the suspension of aerobically grown cells or autoclaved controls.
FIGURE 5. Cyclic Voltammetry studies of R. palustris strain RP2. (A) Anoxic grown cells; (B) Aerobically grown cells; Scan rate: 5 mV s-1.
Hydrocarbonosclastic Potential of R. palustris Strain RP2 in Anoxic Environments
Degradation of Hydrocarbons in Anoxic, Photosynthetic Incubations
To study the hydrocarbon degradation potential of the strain RP2, experiments were performed under four different environments: oxic, anoxic, photosynthetic, and chemosynthetic. Cultures were inoculated into Wolfe's media with DRHs as a sole carbon source with different electron accepting conditions. These incubation experiments indicate that photosynthetic anaerobic degradation of hydrocarbons occurred, however, no increase in biomass or hydrocarbon degradation was observed in chemosynthetically grown anaerobic or aerobic samples. The rate and extent of biodegradation was interpreted from GC chromatograms of the residual hydrocarbons. Figure 6A shows the possible anoxic photoheterotrophic degradation of DRH compounds under different electron accepting conditions [Photosynthetic only (PS); Photosynthetic + Nitrate (PS+N); Photosynthetic + Sulfate (PS+S); Photosynthetic + Iron (PS+Fe)]. For a substrate concentration of 4000 mg/l, cells showed ~10 to 30% of DRH removal by the end of the experiment. A higher percentage of DRH removal (30.6 ± 0.68%, 90th day) was noticed in the triplicates of sulfate containing anoxic, photoheterotrophic samples. Abiotic loss of DRH was measured under each condition was less than 5%.
FIGURE 6. (A) Photoorganotrophic hydrocarbon degradation under different electron accepting conditions (PS-photosynthetic; PS+N- Photosynthetic + Nitrate; PS+S; Photosynthetic + Sulfate; PS+Fe- Photosynthetic + Iron). (B) A representative current density cycle of diesel fed microbial fuel cell system using strain RP2. (C) Hydrocarbon degradation under different reactor operating conditions (AC, Abiotic controls; OC, Open circuit controls; CC- Closed Circuit).
Degradation of Hydrocarbons in MERS Environments
The ability of the strain RP2 to produce electricity using DRH as a sole carbon source was also examined using an initial concentration of 800 mg l-1 of DRHs for three complete cycles. A lag time of 90 h was observed in DRH fed MERS before a constant current was established. A maximum current and power density generated at this concentration were 21 ± 3 mA/m2 (Figure 6B), 720 ± 7 μW/m2. An average of 47.4 ± 2.7% decrease in DRH was observed in closed circuit MERS inoculated with the strain RP2 by the end of experiment (30days). In the case of the abiotic (AC) and open circuit (OC) controls, DRH removal rates were 6.7 ± 0.43% and 10.1 ± 1.7% by the end of experiment. The current density and degradation profiles suggest that MERS could utilize the DRH as a sole source of energy generation which indicated the acclimation of strain RP2 biofilm (Figure 6C). However, no increase in biomass or hydrocarbon degradation or current generation was observed in chemosynthetically incubated MERS samples.
Rhodopseudomonas species are associated with a variety of environments such as limnetic zones (Eckersley and Dow, 1980), marine environments (Larimer et al., 2004), sewage sludges (Hiraishi and Ueda, 1995), euxinic lagoons (Whittenbury and McLee, 1967), and poorly drained soils. The strain RP2 is a facultatively anaerobic, Gram negative purple non-sulfur photosynthetic rod shaped bacterium, containing lamellar ICMs as previously described (Mehrabi et al., 2001). It is a magnetotactic bacterium (Vainshtein et al., 1997), capable of nitrogen (Cantera et al., 2004), and carbon dioxide fixation (Larimer et al., 2004) as reported earlier. The detection of the nif H and pufM genes confirmed the presence of diazotrophic and photosynthetic traits as stated previously (Achenbach et al., 2001; Oda et al., 2005). With respect to this, it shares general characteristics with the Rhodopseudomonas genus of Rhodopspirillaceae. However, a few distinctive features make this strain different from the existing members of the family, these include photoheterotrophic anoxic Fe(III) oxide reduction, electrically conductive filaments (Venkidusamy et al., 2015), spore formation, (Figure 1) growth factor requirements and the ability to degrade n-alkane components of DRHs in anoxic and MERS environments.
The normal growth mode of photosynthetic purple non-sulfur bacteria is photoheterotrophic; however, strain RP2 is able to switch to other metabolic modes such as photoorganoheterotrophic, phtotoorganoautotrophic, chemorg- anoheterotrophic, photolithoautotrophic, and chemoautotrophic growth. The strain RP2 also showed photolithotrophic growth with thiosulphate as an electron donor whereas pure cultures of R. blastica cannot utilize thiosulphate as an inorganic electron donor (Eckersley and Dow, 1980). Aerobic growth of the strain RP2 was also possible under both illuminated and non-illuminated conditions; however, strains of Rhodopsedumonas such as R. faecalis (Zhang et al., 2002), and R. palustris-17 (Dönmez et al., 1999) are unable to grow in oxygen rich, illuminated environments. The strain RP2 has a quite different pattern of carbon source utilization compared to other members of the genus Rhodopseudomonas (Supplementary Table S1). For example, strain RP2 was able to use gluconate and aspartate whereas R. palustris DX1 and R. palustris TIE-1 were unable to utilize those compounds (Xing et al., 2008). Under anaerobic conditions, the strain RP2 can utilize sodium benzoate as a carbon and energy source whereas the strain Rhodopseudomons DCP-3 can oxidize benzoate only under aerobic conditions (Krooneman et al., 1999). The same pattern of glucose utilization under photoheterotrophic conditions has been reported (Imhoff and Bias-lmhoff, 1995), however few strains of Rhodopseudomonas are unable to grow when the medium is enriched with glucose as a sole substrate (Dönmez et al., 1999). Different patterns of vitamin requirements including p-amino benzoic acid, folic acid and pyridoxine HCl highlighted differences in the requirements of photosynthetic non-sulfur bacteria (Dönmez et al., 1999). The most interesting feature of the strain is photoheterotrophic ferric oxide reduction which required both light and an organic carbon source. This also provided evidence of a dissimilatory metal oxide reduction pathway (DMRB) similar to that seen in other electrochemically active bacterial strains (Lovley et al., 1993; Bretschger et al., 2007). On the other hand, photoferrotrophic growth of R. palustris strain TIE-1 has been demonstrated in the studies of anaerobic oxidation of Fe(II) (Bose et al., 2014; Byrne et al., 2015). Considering these features, this strain RP2 appears to be a novel member of the purple non-sulfur photosynthetic bacterial genus of Rhodopseudomonas.
The strain RP2 can transfer the electrons to extracellular, insoluble electron acceptors as reported earlier (Xing et al., 2008; Venkidusamy et al., 2015). Recent investigations have revealed the potential of using such phototrophic biofilms in a mediator free MFC systems (Park et al., 2014; Li et al., 2015). For instance, Xing et al. (2008), documented the highest power density of 2.72 ± 0.06 W/m2 by R. palustris DX1 using dual chamber MFCs which is higher than that produced by mixed biofilms. R. palustris micro MFCs fed with renewable substrates such as blue green algae produced more power output of 10.4 mW/m3 than other chemical substrates used (Inglesby et al., 2012). The present study showed a maximum power density of 131.65 ± 10 mW/m2 (acetate fed MFC, 50 mM buffer concentration) with an electron recovery of more than 40% as current using the strain RP2. However, the maximum power densities by such pure exoelectrogens are considerably influenced by a number of reactor parameters, operating conditions such as electrodes and its distance, electrode surface area to the volume ratio, pH, dissolved oxygen, resistance, electrolytes, etc. as reported earlier (Min et al., 2005; Logan, 2008). Biofilms and supernatant of the strain RP2 was used to determine the electrochemical activity through the release of redox proteins using CV analysis (Figures 5A,B). These results suggest that the presence of redox compounds in strain RP2 may be involved in extracellular electron transfer. The CV of R. palustris strain RP2 biofilm with Fe(III) oxide revealed the distinct redox peaks at -398 mV, +150 mV. The mid-potential of the CV peaks obtained matched the outer membrane cytochromes (OmcA) that have redox potential around -240 to -320 mV as reported in Shewanella oniendensis MR (Kim et al., 2002), Geobacter, Desulfuromonas acetoxidans (Lojou and Bianco, 2004). Further, CV curves from a bacterial cell suspension showed a smaller cell potential value than the theoretical electrode potential which was responsible for poor performance and operating conditions of MFCs (Kim et al., 1999). The results also suggest that oxygenated liquid cultures prevent the synthesis of the outer membrane cytochromes which plays important role in extracellular electron transfer to insoluble metal oxides.
Metabolic Specialization and Biotechnological Potential of R. palustris Strain RP2
The genus Rhodopseudomonas has been studied as a versatile bioremediation candidate because of its exceptional growth flexibility involving the metabolism of diverse compounds. These include utilization of N-aromatic rings (Dutton and Evans, 1969), heterocyclic compounds, chlorinated compounds (McGrath and Harfoot, 1997), heavy metals and other groups of xenobiotic pollutants, for instance, phenolic compounds (Mehrabi et al., 2001), and other dihydroxylated aromatic aldehydes (Harwood and Gibson, 1986). However, the degradation of aliphatic hydrocarbons compounds by the genus Rhodopseudomonas is previously unknown. It was demonstrated here for the first time that the strain RP2 is capable of mineralizing DRHs in anaerobic environments.
In order to assess the hydrocarbon degradation potential of the strain RP2, GC scan was performed using the photoheterotrophicaly grown samples grown under different electron accepting conditions. We showed evidence for the existence of anaerobic hydrocarbon degrading capability in the strain RP2 when grown under illuminated conditions in the presence of thiosulfate. To obtain deeper insights into hydrocarbon degradation mechanism by the strain RP2, further research is required in terms of catabolic genes encoding alkane degrading enzymes such as Cytochrome 450, alk genes including alkB, alkM, alkA etc., The presence of such specific catabolic genes was investigated using the PCR mediated amplification method with various oligonucleotide primers (Supplementary Table S3). This strain, however, showed negative and non-specific results for CYP153A, alkB and its related, alkM and alkA genes. Efforts are being made using R. palustris strain RP2 genome sequence analysis to improve our understanding of anoxic photoorganotrophic hydrocarbon degradation mechanism in strain RP2. Metabolic prediction analysis using KEGG (Kyoto encyclopedia of Genes and Genome) reveals the presence of key enzymes involved in biodegradation of hydrocarbons. Supplementary Table S2 shows the comparison of metabolic genes present in the genome of R. palustris strain RP2 with other genomes obtained from database. Results of this study shows that the strain RP2 possesses alkane sulfonate monooxygenase and catechol 1,2-dioxygenase for hydrocarbon degradation as recently mentioned in Pseudomonas aeruginosa strain N0002 (Roy et al., 2013).
Previously, the remediation of hydrocarbons in MERS systems required mixed biofilms or enriched biofilms (Morris et al., 2009; Venkidusamy et al., 2016). The present study also demonstrated the potential of generating current in the presence of hydrocarbon compounds using the strain RP2 in MERS for the first time. However, the maximum current and power densities of hydrocarbons fed MERS were much lesser than the mixed culture MERS studies using freshly inoculated anodes (70.57 mA/m2 at a concentration of 800 mg L-1) (Venkidusamy et al., 2016), suggesting that the lower rate of hydrocarbons assimilation (47.4 ± 2.7% by 30th day) limited the power generation. Whereas mixed culture MERS system showed nearly 84% of DRH removal at this concentration by the end of batch experiment (Venkidusamy et al., 2016). This perhaps indicates the presence of microbial interactions and its synergistic effects between different species in the mixed culture MERS systems. Interestingly, the strain RP2 also showed the physiological induction of electrically conductive nanofilaments in energetically engineered environments as recently reported (Venkidusamy et al., 2015). It will be interesting to examine whether the induction of these nanofilaments are involved in enhanced bioremediation of hydrocarbon contaminants in energetically engineered environments.
The members of Rhodopseudomonas species appear to have a cosmopolitan distribution as their presence has been detected in marine environments. The present study demonstrated for the first time, the hydrocarbon bioremediation potential of R. palustris strain RP2 in anaerobic and MERS environments. The findings of this study not only expand our knowledge of the range of bacteria known to degrade hydrocarbon contaminants but also provide further research opportunities in the field of sustainable remediation (MERS), molecular biology of hydrocarbon bio-degradative genes in EABs and their interactions in oil contaminated sites. Thus, this study will undoubtedly contribute to the biotechnological applications involved in advanced bioremediation techniques (MERS) and bioremedial process of hydrocarbons at photic, marine environments.
KV and MM proposed the study. KV conducted the experiments under the supervision of MM. KV prepared the draft with contributions from MM.
The authors thank R. Lockington for comments and suggestions on previous versions of this manuscript. KV thanks Australian Federal Government, University of South Australia for International Postgraduate scholarship award (IPRS) and CRC CARE for the research top-up award.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2016.01071
Achenbach, L. A., Carey, J., and Madigan, M. T. (2001). Photosynthetic and phylogenetic primers for detection of anoxygenic phototrophs in natural environments. App. Environ. Microbiol. 67, 2922–2926. doi: 10.1128/AEM.67.7.2922-2926.2001
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Keywords: microbial electrochemical remediation, hydrocarbonoclastic bacterium, photoorganotrophic hydrocarbon degradation, electrode respiring bacteria, Rhodopseudomonas palustris strain RP2
Citation: Venkidusamy K and Megharaj M (2016) A Novel Electrophototrophic Bacterium Rhodopseudomonas palustris Strain RP2, Exhibits Hydrocarbonoclastic Potential in Anaerobic Environments. Front. Microbiol. 7:1071. doi: 10.3389/fmicb.2016.01071
Received: 19 April 2016; Accepted: 27 June 2016;
Published: 12 July 2016.
Yong Xiao, The Chinese Academy of Sciences, China
Luciano Takeshi Kishi, Universidade Estadual Paulista, Brazil
Zheng Chen, Xi'an Jiaotong-Liverpool University, China
Copyright © 2016 Venkidusamy and Megharaj. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Krishnaveni Venkidusamy, krishnaveni.venkidusamy@mymail.unisa.edu.au
Electrochemically active microorganisms


Serene one level living w/ exquisite island, mountain & bay views. Home features include open concept kitchen w/ eating nook, a family room w/exposed wood beam ceiling & cozy fireplace, a formal dining area & living room w/ Nano doors that open to an entertainment deck, providing great indoor/outdoor flow. The master is equipped with an ensuite, walk-in closet & fireplace. The daylight lower level is ideal for guest quarters with gas fireplace plus large storage area, wine cellar, utility room & separate entry. This secluded yet accessible property is located in the gated Chuckanut Crest community and minutes from beaches & trails.


text:
package de.hshannover.f4.trust.irongpm.rest;

import java.io.InputStreamReader;
import java.net.URL;
import java.net.URLConnection;

import org.apache.log4j.Logger;

import com.google.gson.JsonElement;
import com.google.gson.JsonParser;
import com.google.gson.stream.JsonReader;

import de.hshannover.f4.trust.irongpm.Pulldozer;

/**
 *
 * @author Leonard Renners
 *
 */
public class VisitmetaResource {
	private boolean mRawXml;
	private String mBaseUrl;
	private static final Logger LOGGER = Logger.getLogger(Pulldozer.class);

	/**
	 * Constructor with default url: <b>http://localhost:8000/default/graph/</b>
	 */
	public VisitmetaResource() {
		this("http://localhost:8000/default/graph/");
	}

	/**
	 * Constructor.
	 *
	 * @param resource
	 *            URL of the base REST interface of the visitmeta dataservice
	 * @param rawXml
	 *            Whether rawXML should be requested or not
	 */
	public VisitmetaResource(String resource, boolean rawXml) {
		mBaseUrl = resource;
		mRawXml = rawXml;

	}

	/**
	 * Constructor.
	 *
	 * @param resource
	 *            URL of the base REST interface of the visitmeta dataservice
	 */
	public VisitmetaResource(String resource) {
		this(resource, true);
	}

	/**
	 * Requests the REST interface for the given path, e.g. "current" or "changes"
	 *
	 * @param path
	 *            The path for the HTTP request
	 * @return The JSON response string
	 */
	public String get(String path) {
		String json = null;
		boolean connected = true;
		do {
			try {
				URLConnection conn = new URL(mBaseUrl + path + "?rawData=" + mRawXml).openConnection();
				conn.setConnectTimeout(2000);
				conn.connect();
				JsonReader reader = new JsonReader(new InputStreamReader(conn.getInputStream()));
				JsonParser parser = new JsonParser();
				JsonElement rootElement = parser.parse(reader);
				json = rootElement.toString();
				if (!connected) {
					LOGGER.info("VisitmetaResource connection (re-)established");
				}
				connected = true;
			} catch (Exception e) {
				try {
					LOGGER.warn("VisitmetaResource connection failed on " + mBaseUrl
							+ ". Trying again in 5 seconds. Reason: [" + e + "]");
					Thread.sleep(5000);
				} catch (InterruptedException e1) {
					// TODO Auto-generated catch block
					e1.printStackTrace();
				}
				connected = false;
			}
		} while (!connected);
		return json;
	}
}

redpajama_set_name: RedPajamaGithub


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The components are all completely interchangeable and compatible from all of our modular systems, so you can always add to your stand whenever it suits you.
The only tools required to assemble the system is a 4mm hex allen key, supplied with every set, the larger components simply twist together for ease of assembly.
We can supply the printed panels and all of the components required to make a complete exhibition stand.
Two panels can either create a larger (1500mm) single expanse, or split with poles and/or spacers to create a two piece unit of 2 x 750mm panels, as the images to the right.


The following text is an unofficial, lightly edited transcript of testimony delivered by Dr. Ahmed Kutty to a subcommittee of New Hampshire's House Health, Human Services and Elderly Affairs Committee on Sept. 14, 2016. The session was co-chaired by state Reps. Thomas Sherman, M.D., and John Fothergill, M.D.; and included subcommittee member Rep. Susan Emerson.
Complexity and opaqueness are built into the private system, in contrast to the simplicity and transparency of a public system.
3. Economic benefits. Health care expenses borne by business do not enhance value and add to the cost of goods and services, passed on to the consumer. Removing the cost burden of employee health insurance from businesses, large, medium and small, could increase the profit margin and potentially lead to job growth via reinvestment in expansion and innovation, thus facilitating rejuvenation of the local and regional economy.
Workers aged 50 to 64 are often expensive to insure and less likely to find new employment opportunities. Delinking health insurance and employment will increase work-related mobility in this demographic segment. Job loss from illness will not result in loss of coverage, frequently when most needed, as under the current system.
A public system will remove an onerous obligation currently heaped on local entities such as municipalities, school boards and county governments.
4. Cost control by means of price negotiations for drugs, durable equipment, devices, hospital stays and fees for professional services are integral to a publicly funded system. Nationally, savings of $650 billion annually is projected from these measures and from reduction of wasteful administrative spending that prevails in our current, fragmented, multi-payer system, with about 6,000 plans operating in the country, offering 28,000 "products"! This is money siphoned off from funding for direct patient care in all settings.
5. Societal health. The U.S. ranks 19th in the world, in measures of population health as life expectancy, infant mortality and other preventable causes of death, mostly due to lack of access to affordable and timely health care.
6. Administrative advantages. Medicare overhead is 3 percent vs. private insurers' average of 18 percent – and in some plans up to 28 percent – generated by the need to advertise, distribute dividends to shareholders, and pay exorbitant CEO salaries.
7. Single Payer vs. Socialized Medicine. Single payer is system of privately delivered and publicly funded care, like our Medicare and the Canadian Medicare. The Veterans Health Administration, the military medical system, the Indian Health Service, and the National Health Service in UK are examples of "socialized" systems.
8. Electronic medical records (EMR). A streamlined, interoperable, nationwide information system, similar to the VISTA in use by the Veterans Administration for over a decade, will help reduce waste and redundancy and improve efficiency, if it is designed with clinical utility and service benefit, rather than the bottom line of the vendors, as the focus. At the state level, such a system will be organic to a publicly funded program.
Society in 2007. In several surveys, a tax-funded financing system has enjoyed public support at 60 percent to 70 percent level nationally, over the last two decades.
10. Public health issues. A publicly funded system is significantly better designed to deal with epidemics like opioid addiction (prevention, treatment and rehabilitation), a major public health crisis in our state and in the New England region.
11. Activity in other states. California, New York, Vermont, Oregon, Hawaii and others have bills to legislate or for study commissions to get "a single pipeline mechanism" established for public funding of ALL medically necessary, comprehensive health care services, including drugs and devices. Colorado has a ballot initiative (Amendment 69), with enormous potential to engender systemic change, if it is approved by voters this November.
Therefore a Study Commission to consider all aspects of a "Medicare for All" system, providing universal, medically appropriate care through public funding, is strongly warranted and eminently worthy of bipartisan support in the General Court.
As members of the Granite State chapter of the Physicians for a National Health Program, we request your careful consideration of the above bullet points and to take action leading us towards creation of a health care system that supplies truly universal, affordable, high-quality comprehensive care, in a fiscally sustainable milieu here in New Hampshire. Your public voice and your vote for the Study Commission bill will be a major contribution to the sustained multipronged effort needed for resolving the looming crisis in health care facing our state and very likely for the nation as well.
Thank you, Dr. Sherman, for giving me the opportunity to speak before your esteemed subcommittee today on this critical issue that has been concerning our society for over a century. Having heard us, can we hope our words will be heeded?


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Joanne H. Yi
Western Michigan University Cooley Law SchoolVirginia Commonwealth University and College of William and MaryIllinois and VirginiaVirginia State Bar and Northern Virginia Bankruptcy Bar Association
David Colin Jones
University of Virginia School of LawUniversity of Virginia4th Circuit, Virginia and Eastern District of VirginiaVirginia State Bar and Fairfax Bar Association
The LII Lawyer Directory contains lawyers who have claimed their profiles and are actively seeking clients. Find more Centreville Bankruptcy Lawyers in the Justia Legal Services and Lawyers Directory which includes profiles of more than one million lawyers licensed to practice in the United States, in addition to profiles of legal aid, pro bono and legal service organizations.

redpajama_set_name: RedPajamaCommonCrawl


text:
4 Tracks From Solange's 'When I Get Home' Will Generate No Money Off Spotify Streams
Photo by Natasha Moustache/Getty Images for ABA
by Grant Rindner
@DontGetIt
Referenced Artists
Referenced Albums
A Seat at the Table
Referenced Songs
Interlude: The Glory Is in You
Interlude: I Got So Much Magic, You Can Have It
Stay Flo
Nothing Without Intention (interlude)
Almeda
Not Screwed! (interlude)
S McGregor (interlude)
Can I Hold the Mic (Interlude)
Solange also had two ultra short interludes on 'A Seat at the Table.'
Solange's fourth album When I Get Home contains several short interludes, and per Quartz, she will not be receiving Spotify royalties on four of them. A track must be at least 30 seconds long for its artist to receive royalties the streaming service.
The interludes that don't pass the 30-second mark are "S McGregor," "Can I Hold the Mic," "Nothing Without Intention," and "Not Screwed!" Genius explored the origins of each sample, which include clips of Phylicia Rashad and Debbie Allen, as well as members of Atlanta rap group Crime Mob.
On "Can I Hold the Mic," a clip of Crime Mob's Diamond and Princess leads to Solange discussing the challenge of showing the many different aspects of her being. She says:
I can't be a singular expression of myself, there's too many parts, too many spaces, too many manifestations, too many lines, too many curves, too many troubles, too many journeys, too many mountains, too many rivers, so many
Since its March 1 release, When I Get Home's full-length tracks have performed well on Spotify. "Almeda" leads the way with over 5 million streams, while "Stay Flo" has exceeded more than 3.1 million plays on the service.
Short interludes were also a hallmark of Solange's 2016 album A Seat at the Table, which included two sub-30 second interludes, "The Glory Is in You" with Master P and "I Got So Much Magic, You Can Have It" with Kelly Rowland and Nia Andrews.
Spotify counts something as a song stream when at least 30 seconds of the track has been played. Apple Music does not appear to have a stipulation about minimum track length for payment in the way that Spotify does.
In the episode of Genius' series For The Record, Rob Markman and his guests discussed the way revenue is split in the present day music industry.
Read the full lyrics to "Almeda" and the rest of Solange's discography on Genius now.
A Guide To All The Interlude Samples On Solange's New Album 'When I Get Home'
Solange's Playboi Carti Collaboration "Almeda" Is A Celebration Of Blackness & Houston Culture
Watch Solange's 'When I Get Home' Short Film Here


text:
Q: Versioning system with per file history vs. commit history I'm looking for a versioning system, that can show the version history with comments per file.
I've used SVN (not CVS as stated earlier) for a while and have some projects in git.
Both (svn and git) are "commit oriented" (I use this term for lack of a better one): when I look at the repository git shows all the commits, and when I open a commit, I see, which files and changes it contains.
I want it the other way around: I want to see all the files and folders currently in my repository and for each file I see the history of commits it went through.
For each commit I have to enter a summary comment as well as a per file comment describing the file's changes.
The reason I'm looking for this is my NAV (formerly Navision) designer's licence: I am allowed to change objects in my NAV system, but I can't export the sources. Therefore i only have several versions of compiled ".fob" files without any chance to see code changes, so I have to describe the change in a per file comment.

A:
I have to describe the change in a per file comment

This goal does not seem like a reason which makes git unsuitable. Basically, you only need to:

*

*commit each file separately, not all of them at once

*being able to query only commits which change the selected file. Which git is able to do, and is pretty reliable and fast about it, at least if you don't rename the file.

Also, you still can keep together different changes in same commit, if you find them logically coupled, just write there comments about both.

A: CVS is file-oriented in terms of SCM: for a set of files you can't define single revision and get all files, corresponding to this (revision-slice) state
Use any current SCM, but commit single file at a time

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This is the Official USB Charger for Grasshopper Vape. This is a well-needed accessory in order to use and charger your vaporizer. Even as a spare because you could live one at work and one at home that way you won't be forgetting the charger wherever you go.


Innovative Advocacy Group Professionals are Experienced,Qualified, and well versed in fighting for the rights of People with Disabilities. We are Familiar with the Laws that affect Disability Rights.. Our Advocates are Parents of Special Needs Children and Adults,who have been faced with Major Challenges,Barriers,and have successfully litigated Cases against School Districts,Regional Centers,and Governmental Agencies. Our Advocates Represent Clients in Administrative Hearings,Attend Meetings,Review Documents,Write Letters,and even assist with Document Preparation. We Represent both Children and Adults with Disabilities .We Believe In Equal Rights,and Equal Opportunity!
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Wow!! It's been a very fun five years, but Midnight Cargo is going to end their performances as a band. We would like to thank ALL of our friends and family who have supported us, and hope you come out one last time to hear the band. You will still see us, as individuals, still performing at some of the local venues in other bands. What a blast it's been!!!!!


Opportunity to buy quality garments for your customers, discounts on Tops, blouses and bodys for renovation of our collection.
White mini dress with embroidery detailing. V-neck and tie closure. Wide 3/4 sleeves. Side pockets. Smooth woven fabric. Relaxed fit. Embroidery detail in gold. Pop up your summer tan with this beautiful dress.
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Sleeveless navy striped shirt with printed birds. High neckline with ruffle detail. Button placket. Ruffle detail in the front and back.
Model is: 85-64-90. Height: 1'75m.


text:
Q: Expected Squared Derivative of White Noise? Let $W_t$ denote the continuous time Wiener process at time $t$. Then its time derivative $\xi(t):=\frac{d W_t}{dt}$ is white noise, and may be "seen" in the weak sense via inner products $\langle \xi, \psi\rangle = \langle W', \psi\rangle = \int \psi dW$ where $\psi$ is a test function.
From this SO question, I learned that we can make similar statements about the second derivative of the Wiener process, or the derivative of white noise. Namely,
$$
\langle W'', \psi\rangle = \langle W, \psi''\rangle = \int \psi''dW
$$
I am interested in a related quantity, namely $\langle (W'')^2, \psi\rangle $. Is there some way to express this random variable as an integral of (some function of) $\psi$ with respect to the Wiener process?


text:
General-Articles
__Social Media
__History
__Finance
_Envirinment
_e-Commerce
_Marketing
HomeMoneyThe Great Lakes Economy: The Growth Engine of North America #Infographic
The Great Lakes Economy: The Growth Engine of North America #Infographic
Extremeworldinfo.com June 20, 2020
We don't really think of the states and Canadian provinces that surround the Great Lakes as their own economy-but maybe we should.
The country is tightly interconnected in terms of commerce, after all. It alone accounts for more than 50 percent of all bilateral border trade between the United States and Canada and sees more than 200 million tons of cargo shipped annually. It would have a GDP of US$ 6 trillion if it were its own country-making it the world's third largest economy.
The Great Lakes Region comprises eight states (Minnesota, Wisconsin, Illinois , Indiana, Michigan, New York, Ohio, and Pennsylvania) and two provinces in Canada (Ontario and Quebec) surrounding the five interconnected freshwater bodies known as the Great Lakes. The region is home to 107 million people, 51 million jobs and a US$ 6 trillion GDP – making the Great Lakes Economy a global powerhouse.
The area is especially well-known worldwide for its successful prowess. It is home to giants in the automobile and aerospace sectors such as Ford, GM, Chrysler, Bombardier, GE Aviation and Magna International and many other diverse industries as well. Some of the other major industries that generate business for the region are education and health, shipping and logistics, agriculture , mining and energy, tourism, and finance.
infographic by: councilgreatlakesregion.org
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Interesting infographic
How To Make A DIY Fleshlight Drying Rack In 7 Easy Steps #infographic
A Guide To Homemade Coconut Oil Beauty Products #infographic
Understanding diabetes on Aging Adults #Infographic
What is Extreme World?
We strive to curate the most exciting infographics on the Web at Extreme World. We search the internet to view information with the finest content and design to give our users easy information and numbers.
Extreme World has given the web knowledge and know-how on a large number of subjects for one year. We are proud to help you become smarter every day, from Technology to sporting activities, marketing methods and brilliant news
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text:
World Championship Schedule
The final of Season 4 of Farming Simulator League is imminent
On the weekend of November 19 and 20, 2022, the best eight teams of Farming Simulator League (FSL) face each other. During this thrilling tournament weekend, the title of world championship will be at stake, as well as the prize money of € 40,000! Viewers can follow the matches on both days from 4 am (EDT) in the livestreams on Twitch, YouTube and Facebook Gaming.
A look at the standings
The current title contender by the standings are the teams Trelleborg, New Holland (former Fermiers Flamboyants) and mYinsanity, who earned the most circuit points during the season through playing skill. The midfield of the table was tighter, with one team currently still open.
The eighth, and thus last, rank for the participation in the world championship is currently occupied by the teams of Corteva Agriscience and Astragon, which have the same number of points. The decision game between these two will be broadcasted on Friday, November 18th 2022 at 12 pm (EDT).
Which team will prevail in the end andn go home with the world title?
Exciting season final
The season finale takes into account the standings and diivides the eight participating teams into two groups. Within each of these groups, there is an upper and a lower bracket to be contested. With the double knockout system thus played, the round losers can play their way back to the top. The division:


text:
In order to Receive Empathy, we must first Teach it - Part 1
I grew up in a household where we were encouraged to keep ourselves to ourselves. If someone asked for help and we could give it, then we would - except if that help was deemed "too much work" or if that person was considered a "crazy person". We would never offer help though - not without being asked because it was the job of the "helpless" to ask for support. "Crazy people" were drunks, "druggies" and older, senile people. We never helped them unless we were cornered into it - and even then, the help that was given was only ever a means of escape. These days of course, I recognize that these are terrible and callous attitudes to have but suppose I hadn't been taught that. Would I be the same today? I remember my mother picking up an old woman in the car. She made it clear to us before picking the woman up that she was a "mad woman" and my sister and I sat in the back seat fearing for our lives. We later asked h
Some insights on Empathy
The concept of empathy is a much discussed topic in Asperger's circles but I'm starting to wonder what all the fuss is about. You see, I feel that empathy has little to do with Asperger's syndrome. So why continue to write about it? I guess I just want the myths understood and "busted". Our family has just been through twenty-one days of hell and while the worst is clearly over, there's still a quite bit to come. It has been a tense emotional and very very painful situation in which we needed all of the help and empathy we could find. It's hard For me to take a step back from the situation and look at things objectively but the situation revealed some surprising insights into empathy and asperger's syndrome which I feel need to be shared. Particular insights include; There is a huge learned component to empathy. In order to receive empathy, we must first teach it. Neurotypcials can be far less empathetic than people with aspergers syndro
In order to Receive Empathy, we must first Teach i...


This is a database front-end for keeping track of chord files (a.k.a. fake sheets). It accepts chord and chordpro files as inputs, and creates output sets in a large variety of formats (pdf, text, chordpro, latex).
perl-based scripts to transform xml'ish files. It has scripts to scan C text,make some xml'ish intermediate form, transform the xml'ish data, and it hasscripts to spit out the xml'ish data as html or docbook.
Explode your large PDF into many PDFs. Extract pages corresponding to any Table of Contents (TOC) entry in your PDF. For example, extract PDF of any or each chapter in a long PDF text or manual.


Games Galore 58 is here and we get started in Segment 1 with Brian breaking down this week's NFL action. In Segment 2, MyBookie.ag's Christopher Smith (Twitter handle: @cfblocksmith) joins the show to give out his two favorite picks of the bowl season. C-Smith is extremely bullish on Vandy and Missouri, and he explains why. Also, he has several selections for Saturday's bowl card that has five games. We had hoped to get with our guy Jay Greeson to get his take on Oscar Liar, in addition to the dysfunction going down at his alma mater (Auburn!). However, technical difficulties prevented us from making this happen We'll get with Jay next week. In the final segment, B.E. hits on a little UFC, the 2019 Heisman Trophy odds posted at MyBookie.ag and more. We hope you enjoyed GG 58!
Episode 57 - OSCAR!!! WTF, Kirby 'Not Very' Smart?
Episode 55. - Holy Zion!
Episode 53 - J-Greeson & B-Powers bring the heat!


text:
Index :: Interesting things on the web. :: Silent Circle: 'surveillance-proof' smartphone app
The technology uses a sophisticated peer-to-peer encryption technique that allows users to send encrypted files of up to 60 megabytes through a Silent Text app. The sender of the file can set it on a timer so that it will automatically burn—deleting it from both devices after a set period of, say, seven minutes. Until now, sending encrypted documents has been frustratingly difficult for anyone who isn't a sophisticated technology user, requiring knowledge of how to use and install various kinds of specialist software.
What Silent Circle has done is to remove these hurdles, essentially democratizing encryption. It's a game-changer that will almost certainly make life easier and safer for journalists, dissidents, diplomats, and companies trying to evade state surveillance or corporate espionage. Governments pushing for more snooping powers, however, will not be pleased.
Silent Circle's revolutionary technology will assist many people in difficult environments, maybe even saving lives, there's also a dark side. Law enforcement agencies will almost certainly be seriously concerned about how it could be used to aid criminals. The FBI, for instance, wants all communications providers to build in backdoors so it can secretly spy on suspects.
Silent Circle is pushing hard in the exact opposite direction—it has an explicit policy that it cannot and will not comply with law enforcement eavesdropping requests. Now, having come up with a way not only to easily communicate encrypted but to send files encrypted and without a trace, the company might be setting itself up for a serious confrontation with the feds. Some governments could even try to ban the technology.
Silent Circle's solution is simple: The team will close up shop and move to a jurisdiction that won't try to force them to comply with surveillance.


Nuance Communications, a speech products vendor, has announced its "Can't Stop the Stupid Calls" contest, described by company officials as "a light-hearted program designed to provide call center agents a forum where they can shed light on the realities of trying to help some of those customers out there who really cannot help themselves."
Through sharing their stories, call center agents will earn a chance to win a number of cash prizes. Nuance is collecting entries now at www.cantstopstupidcalls.com. The first 20 entries automatically win $100. Please see the official rules for details.
Giving an example, the site tells of one call center agent who received a call from a customer who tried to thaw peas in a microwave, which didn't work. Then she placed peas in a clothes dryer, resulting in -- guess what? -- smashed peas on the interior of the machine.
So why did she call customer service? To ask how to get the smashed peas out of the lint capture container. Some calls you just don't get trained for.
"Nuance wants to acknowledge some of that heroism that happens in call centers every day and have some fun while doing it," said Lynda Kate Smith, general manager, of the Care business unit at Nuance. "Sometimes there are problems you just can't resolve."
The Web site is designed to not only capture stories, Nuance officials say, but to "create a sense of community where people can share stories, offer comments and have fun. It will be closely moderated and no actual customer or company names will be referenced in the publicly shared story."
First 20 entries win $100 automatically. Judges will award $1,000 each to the following category winners -- "You've Got to Be Kidding Me!," "Sounds Like Fiction" and "Vacation Day Earned!" Plus $1,000 goes to the entry receiving the most votes online by October 6.
The contest begins today, and winners will be announced the week of October 27.
Aspect Software, a vendor of communications for the contact center, has announced the appointment of Andy Bezaitis as senior vice president of corporate development.
In this role, Bezaitis' responsibilities will include "driving partnerships with strategic players and exploring and evaluating potential acquisition candidates," according to Aspect officials, who say one objective will be "identifying opportunities to further enhance the Aspect Software Unified Communications for the Contact Center strategy."
Bezaitis called the appointment "a great opportunity... our strategic alliance with Microsoft has created tremendous energy and possibilities for Aspect."
Bezaitis has more than 20 years of experience in business development and communications technology. Most recently, he was the senior vice president of business development at Cantata Technology.
He's also served as founder and chief executive officer of Apopleo, as well as founder and chief executive officer of Cambia Networks. He has also held senior management positions at 3COM Corporation, Motorola, Inc., and IIT Research Institute.
Bezaitis has authored several wireless data networking white papers and frequently speaks at industry trade events. Bezaitis has also been awarded nine United States patents for various technologies, including mobile Internet protocol, point-to-point protocol connection, wireless networking authentication, and voice over Internet Protocol.
NetSuite has announced the latest companies to switch from Microsoft Great Plains to NetSuite.
Company officials say the firms switched "largely to take advantage of NetSuite's integration of enterprise resource planning (ERP)/Accounting, customer relationship management and e-commerce capabilities."
These new customers, which include CMC Energy Services, Symbiot, Safir Rosetti and Advantage Sign Supply, also cited "advanced functionality and ease of use" as the other compelling reasons for moving to NetSuite.
Angelica Biehl, IT Support, Home Energy Tune-UP, a new national residential energy audit program of CMC Energy Services, based in Bethesda, Maryland said her company needed "CRM data integrated with accounting and ERP data."
Older generation software applications like Microsoft Great Plains were once very popular within mid-sized companies, NetSuite officials say, adding that "NetSuite is designed as a single system for ERP, CRM and e-commerce operations."
"After a little more than two years of using Great Plains and Salesforce.com, we decided we really needed a completely integrated system to gain better visibility, so we switched to NetSuite," said Ray Jones, Senior VP, Infrastructure/Supply Chain Management, Symbiot, adding that "the ability to access our accounting information along with CRM data from anywhere in the country is a huge benefit for our company.
Ringback tones are on track to become the most attractive mobile content category by 2012, according to new market research from MultiMedia Intelligence.
With worldwide revenue nearly tripling to $4.7 billion, the study finds, ringback tones will fall just short of mobile gambling revenue in capturing the largest share of the mobile premium content market (well, not including mobile video and mobile TV).
Overall, the global retail market for mobile premium is projected to reach $29 billion by 2012, the research firm finds. The research report, "Dough-Ra-Mi: A Look at Mobile Music, Ringtones, Ringbacks, Games, Graphics & Adult Content," examines and forecasts market verticals in the mobile handset market.
"Developed markets, such as Japan and Korea, have seen dramatic growth in premium content, with some operators seeing data revenue exceed 30 percent of total service revenue," the study reports, adding that "as key Asian markets continue to evolve, growth has shifted to Europe and then the Americas."
"Ringback tones are quickly becoming the Golden Child of the mobile music market," said Frank Dickson, Chief Research Officer with MultiMedia Intelligence. Dickson pointed to "a combination of consumer popularity, and minimal impact from DRM or piracy" as reasons for the surge.
"The current leading mobile premium content category of realtones is already facing challenges," in that "consumers increasingly are able to create their own ringtones. The price disparity compared to a full track download is also a growing factor," Dickson noted.
By 2012, the study finds, "the mobile music market in Japan, for example, will be over 7 times the size of the mobile music market in South Korea."
Last September Mark Kirstein, Frank Dickson, and Rick Sizemore formed MultiMedia Intelligence, to provide "actionable intelligence on the markets and technologies for delivering IP video to the nth screen -- the world beyond the classic three screens of TVs, mobile handsets, and computers," according to company officials.
As far as dumb stories to call centers go, First Coffee's favorite is still the one, probably apocryphal, where someone calls to find out how to get back into the Word Perfect program -- "Well, I was just typing along, and all of a sudden the words went away... It's blank; it won't accept anything when I type." -- and after many minutes tells the call center agent no, he can't see if the cable's plugged into the back of his computer because he doesn't have an office light because there's a power failure, and the agent tells him to pack his computer up and ship it back because he's too stupid to own one.


text:
07779076792 info@martyncroston.com
London Wedding Pianist For Hire
Add a personal touch to your wedding with some live piano music
Contact Martyn
Professional Wedding Pianist For Hire In London
Want to hear your favourite music performed live on your wedding day?
Martyn Croston is an experienced pianist who regularly performs at weddings in London and the home counties, including Kent, Surrey, Hertfordshire, Essex and East/West Sussex.
Martyn can add a touch of class to your wedding and play your favourite music as you walk down the aisle, during the signing and for the exit.
Whether you're a lifelong fan of the classics, or popular songs, Martyn can help you create a song list that speaks to you personally, as well as engaging your guests.
During the wedding ceremony, Martyn willl play your choice of music as you walk down the aisle, during the signing and for the exit.
For the wedding breakfast and drinks reception, Martyn can play a variety of songs on the piano, in different styles, so there'll be something for everyone to enjoy listening to.
Ideal for creating the right atmosphere for you and your guests.
Click the button below and contact Martyn today…
Book Martyn Today
No piano..no problem
If you do not have a piano at your venue, Martyn can bring his portable stage piano which can be quickly set up in any small space. If you have any other questions about booking Martyn for your wedding, just get in touch and he will respond within 24 hours.
You can also visit his FAQs page for some general aspects to consider when hiring a wedding pianist.
Martyn conducted himself at our wedding with full professionalism and his mannerisms were impeccable. I would highly recommend Martyn if you want to have a very special wedding with lovely piano background music like we did.
Andrew Man
51 Buckingham Gate, London
Have a favourite song in mind for your wedding ceremony, but you're unsure how it will sound on the piano?
Give Martyn a call on Skype for FREE and hear a song of your choice played live on the piano
Martyn has played at several venues across London in recent years. Here are a couple of examples.
Wedding @ 51 Buckingham Gate, St James's Park, London
Martyn was asked to play at a wedding ceremony and drinks reception at 51 Buckingham Gate for Andrew Man and his bride-to- be.
Although a digital piano was available at the venue, Andrew was unsure about using it so both he and Martyn met up to check it out a week before the big day.
Martyn reassured Andrew that it would do the job perfectly.
Picture Source: http://yallabook.com/guide/en/show.php?nid=1633&taj-51-buckingham-gate,-suites-and-residences
For the wedding ceremony, Martyn played Pachelbel's Canon in during the bridal entrance. For the signing, Andrew had requested 3 songs; Have I Told You Lately by Van Morrison, Bach's Prelude in C and a famous piece of Chinese music called I Am Willing by the artist Faye Wong. Both Andrew and his bride walked out to Nina Simone's My Baby Just Cares For Me. A great choice!
During the drinks reception, Andrew was happy for Martyn to play songs from his varied repertoire including a mixture of jazz and pop songs. However, he had a few requests which Martyn played on the day including Lionel Richie's Stuck On You, Take That's A Million Love Songs and a couple of David Foster songs.
Everything went according to plan and here's what Andrew had to say about Martyn's playing…
Other London Wedding Venues Where Martyn Has Played
London Marriott Hotel County Hall
Prism Brasserie, 147 Leadenhall Street, London
Pitzhanger Manor, Ealing, London
Trafalgar Tavern, Greenwich
Music Clips
Take That - Greatest Day
Christina Perri - Thousand Years
Bart Howard - Fly Me To The Moon
Billy Joel – She's Always A Woman
https://www.martyncroston.com/wp-content/uploads/2014/06/Shes-Always-A-Woman.mp3
https://www.martyncroston.com/wp-content/uploads/2014/06/Just-the-way-you-are.mp3
J.S Bach – Prelude in C
https://www.martyncroston.com/wp-content/uploads/2014/06/Prelude-in-C.mp3
Here is just a small selection of Martyn's repertoire:
A Thousand Years – Christina Perri
Thinking Out Loud – Ed Sheeran
Rocket Man – Elton John
Here Come's The Sun – The Beatles
We Are The Champions – Queen
Greatest Day – Take That
What A Wonderful World – Louis Armstrong
Canon in D – Pachelbel
I Giorni – Einaudi
See Martyn's full repertoire
Click 'Book Now' and check the availability of your date
To get in touch with Martyn, fill in your details here, or contact him via e-mail or phone:
E-mail: info@martyncroston.com


text:
Kubuntu 15.10 seems fine so far
Written by Mike C. on Oct. 28, 2015 in Tuxedo.
Last update on Oct. 28, 2015 .
So I pulled the upgrade switch on the (Dell Inspiron) laptop on Friday. It trundled for almost the whole day doing the upgrade, then rebooted to a black-and-totally-unresponsive screen, with no way to switch to virtual consoles... I did a hard-reboot, booted to recovery console and reinstalled the fglrx (proprietary AMD) drivers, then rebooted... and everything came up fine. Since then it's basically been working fine (though there seems to be a few graphic hiccups where it takes a heartbeat to refresh the screen).
The suspend/resume still works. Eric got de-configured somehow (I'm guessing because it's now using a packaged release instead of the source-code release). KDE's date-and-time settings seem to have been reworked to the detriment of usability (one of those "I see what you're doing, and I understand that in the code that's how you think about this, but users do *not* think about date-and-time-formats that way). Bug filed on that. Plasma 5 hasn't changed much since 15.04, the "updates available" widget is cleaner/less insistent, which is nice. Other than that (and the clock format settings mentioned above) I can't actually detect any changes that I notice in day-to-day work.
KDE/Plasma 5 seems to have hit the point where the desktop is "finished" and new updates are just tweaks/enhancements. Yay, but... aww, I wanted shiny new toys.
Update: Duh. So I just had a full-session crash, started a new Chromium tab and boom, logged right out of session (kicked back to the DM login page). Also seeing occasional error dialogues from Eric 6 (not crashing, just warning error dialogues). That's obviously a regression from 15.04, where I haven't seen such issues. And another full-system-crash, again while opening a new tab in Chromium... but I can't find any obvious place where the error is being logged... the desktop just crashes in full and dumps me back to the DM. So this is happening about once per day it always seems to happen when opening a new Chromium tab... but what precisely is killing KDE/Plasma is a mystery.
Short url : http://blog.vrplumber.com/b/23Q/
Discussions : 2 comments
KDE Seems to run on fglrx now!
Ubuntu Upgrades are Magic unless they aren't
Black Screen on KDE after disconnecting external monitor
Ubuntu 20.04 unhappy on the old Dell XPS
Has it really been 18 years since I did Java? →
← That point where you wish you'd asked for an hour
Bit of work with TurboGears (Have to work with it more often)
Back to work with me (Gentoo is built, but not yet booting)
The Rain of Tiny Blows
I need to do *something* tonight (Too pitiful to sit here working all weekend)
Voice Dictation Privilege


An online concert at Concert Window, with original and traditional holiday music. Tickets are available at whatever price you wish to spend.
A children's holiday event, The Candy Cane Hunt takes place at the Apollo Center annually in Highland, MI. I provide a musical backdrop while the children make smores, drink hot chocolate, do crafts, face painting, story time and a visit with Santa. There is a small entry fee for this event.
A Goldfish Christmas. This year, I'm joined by special guests, Laura Belliston, Robin Monterosso, Bobby Pennock as we perform holiday favorites, both new and old, in the round and accompanied by Devon Benbow on bass. This is the annual party of shows, and a personal favorite. Goldfish Tea, in Royal Oak, is the place to be. Last year's party was in Wyandotte, and was filled to capacity, with people standing or sitting on the floor and singing along. It's also Devon's birthday, so feel free to give him some birthday cheer amidst the holiday fun.