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Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell
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DOI: 10.1007/s00253-012-3894-6
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- Timmers, R.A., Rothballer, M., Strik, D.P.B.T.B. et al. Appl Microbiol Biotechnol (2012) 94: 537. doi:10.1007/s00253-012-3894-6
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The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors.
Keywords454 amplicon sequencing Geobacter Microbial community Plant microbial fuel cell Renewable energy Rhizosphere
The plant microbial fuel cell (PMFC) is a new technology that can potentially provide renewable and sustainable energy. The PMFC transforms solar energy into electricity through the oxidation of organic compounds originating from photosynthesis (De Schamphelaire et al. 2008; Strik et al. 2008). Electricity generation in the PMFC is based on the loss of organic compounds by plant roots (rhizodeposition) (Pinton and Varanini 2007) and oxidation of these organic compounds by electrochemically active bacteria (EAB) (Potter 1911). In the PMFC, electrons, proton and carbon dioxide are produced by oxidation of organic compounds lost by plant roots in the anode. The electrons that are transferred to the anode electrode are consumed in the cathode compartment by typically reduction of oxygen to water (Strik et al. 2011).
Several bacterial species are known to produce current in microbial fuel cells (MFC): Shewanella putrefaciens using lactate, pyruvate and formate as electron donor (Kim et al. 1999; Park and Kim 2001); Clostridium butyricum and Clostridium beijerinckii, using glucose, starch, lactate and molasses (Niessen et al. 2004; Park et al. 2001); Geobacter sulfurreducens, using acetate and hydrogen (Bond and Lovley 2002); Rhodoferax ferrireducens, using glucose (Chaudhuri and Lovley 2003); Geobacter metallireducens, using acetate (Min et al. 2005) and Rhodopseudomonas palustris, using acetate, lactate, valerate, fumarate, ethanol, glycerol and yeast extract (Xing et al. 2008). Alcaligenes faecalis, Enterococcus gallinarum and Pseudomonas aeruginosa are known to generate electricity by producing their own mediators, using glucose as an electron donor (Rabaey et al. 2004). To date, Enterobacter cloacae is the only pure culture that is known to generate electricity directly from more complex electron donors, such as cellulose (Rezaei et al. 2009).
Rhizodeposition is the loss and release of organic and inorganic compounds by plant roots into the rhizosphere (the soil volume affected by the presence of plant roots). Rhizodeposition provides a carbon and energy source, thus stimulating the development of bacterial communities in the rhizosphere. In anaerobic environments, rhizodeposition can also provide oxygen as an electron acceptor and may stimulate the development of facultative anaerobic bacterial communities (Hartmann et al. 2009). In addition to prokaryotes (bacteria and archaea), eukaryotes (such as fungi, protozoa, nematodes and meso- and macro-fauna) are also found in the rhizosphere (Phillips et al. 2003).
The genera Geobacter and Desulfobulbus have been found in PMFCs (De Schamphelaire et al. 2010). As both genera contain electrochemically active species that are enriched at the anode of MFCs (Holmes et al. 2004; Jung and Regan 2007), it can be assumed that EAB may have been present in the PMFC. However, no studies have yet confirmed the presence of active EAB in PMFCs. The presence of active EAB is required to prove their role in electricity production by PMFCs.
In a PMFC, the plant roots are located at the anode of the MFC, where rhizodeposition provides EAB with electron donor. Together the MFC anode and the rhizosphere make up a complex system. In this complex system, it is likely that different microorganisms compete for electron donor. Competition for electron donor among the different microorganisms results in a decrease of electron donor available for EAB and thus lower current generation. Analysis and localisation of the microbial community is needed to gain insight into the competition for electron donor in a PMFC.
In this study, we determined the microbial communities present in a PMFC rhizosphere and localised the active EAB in the rhizosphere and on the electrode of the PMFC. Additionally, we investigated the electron donor and electron pathways in the PMFC. We used 454 technology to sequence 16S rRNA gene amplicon libraries from bacteria and archaea. To prove the relevance of dominant pylogenetic groups and to localise those bacteria and archaea, fluorescent in situ hybridisation (FISH) in combination with confocal laser scanning microscopy (CLSM) was performed in samples of high-current-generating and low-current-generating Glyceria maxima (reed mannagrass) PMFC.
Materials and methods
Preparation of PMFC
To close the electrical circuit, two 4-cm golden wires glued to Teflon coated copper wire were connected over a resistance of 900 Ω. To create contact between the anode and the cathode, one of the gold wires was placed in between the graphite granules and the other was woven through the graphite felt.
Preparation of graphite granules
Eight setups were operated without plants to develop electrochemically active biofilms on the graphite granules. The anode was inoculated with 5 ml of anolyte from an MFC running on acetate (Dekker et al. 2009), and the anolyte was extracted with a syringe. The setups were operated in batch mode. As nutrient solution 1/2 Hoagland Medium (Taiz and Zeiger 2006) buffered with an 8-mM potassium phosphate buffer solution (pH 6.8) was used. The electron donor was supplied by adding 10 mM potassium acetate to the nutrient solution of setups 1 through 4 and 10 mM glucose to the nutrient solution of 5 through 8. After 40 days, the acetate-fed setups generated a current density of 0.248 ± 0.004 A m−2, and the glucose fed setups generated a current density of 0.151 ± 0.02 A m−2. Current densities are expressed per square meter of cross-sectional area of the PMFC (0.00096 m2) throughout the document.
Before the graphite granules were used for the six PMFCs, they were thoroughly mixed to ensure the presence of microorganisms able to generate electricity from acetate as well as glucose. After the mixing, the graphite granules were rinsed with tap water to remove a large part of the residual electron donor. One stem of G. maxima (D'n Bart Waterplanten, Handel, the Netherlands) with a weight between 4.4 and 7.8 g was planted in each PMFC.
Operation of PMFCs
The medium used to feed the PMFCs was 1/2 Hoagland buffered with 0.008 mol l−1 potassium phosphate buffer solution as described by Timmers et al. (2010) (pH 6.8, conductivity between 1.5 and 1.7 mS m−1). The medium was fed through a sample point located 0.07 m above the graphite granules. The applied flow rate was 0.17 ml s−1 throughout the experimental period. The feeding frequency of the PMFC with buffered Hoagland medium was 5 min every 12 h.
Both MFC and PMFC setups were placed in a climate control cabinet (Microclima 1750 Snijders, Tilburg, the Netherlands). In this manner, environmental conditions were fixed at illumination period of 14 h day−1, average light density in the photo active region of 596 ± 161 μmol m−2 s−1, temperature of 25°C and humidity of 75%.
Sample selection, preparation and DNA extraction
Samples for analysis were taken from PMFC generating the highest current (PMFC6) and the PMFC generating the lowest current (PMFC3). Both PMFCs were dismantled 225 days after the start of the experiment. The anode compartments of both PMFCs were divided into bottom (0 to 0.15 m) and top (0.15 to 0.3) sections. Figure 1 shows a schematic representation of the PMFC and the sampling points. The root samples were taken from all of the sampling points in both PMFCs and stored at −80°C. DNA was extracted from a total of 500 mg of root material per sample following the protocol of Griffiths et al. (2000) and using a BIO101 lysing matrix (MP Biomedicals, Illkirch, France) and a final purification step with the AllPrep Mini Kit by Qiagen (Munich, Germany). The amount of DNA in solution was quantified using a bioanalyser from Agilent (Böblingen, Germany).
PCR, 454 sequencing and data processing
If not otherwise mentioned, all the steps were performed according to the Roche 454 sequencing protocol for amplicons. To generate the amplicon library for bacteria and archaea, specific primers were selected according to two criteria: (a) the fragment should span 600 bases of the 16S rRNA gene to receive sufficient phylogenetic information and (b) the primers should bind to as many bacterial/archaeal sequences as possible without detecting non-target groups. To verify these criteria, the ARB probe match tool was used with the latest SILVA database (containing over 400,000 sequences), resulting in the following bacterial 16S primers: 926-F (5′-AAACTYAAAKGAATTGACGG-3′, Escherichia coli position 907–926 (Lane 1991)) and 630-R (5′-CAKAAAGGAGGTGATCC-3′, E. coli position 1528–1544 (Juretschko et al. 1998)). The archaeal primers were rSAf(i) (5′-CCTAYGGGGCGCAGCAG-3′, E. coli position 341–357 (Nicol et al. 2003)) and 958r (5′-YCCGGCGTTGAMTCCAATT-3′, E. coli position 940–958 (Bano et al. 2004)). These primers were extended as amplicon fusion primers with respective A and B adapters, key sequence and multiplex identifiers (MID) as recommended and tested in initial PCR reactions to determine their optimal annealing temperatures (50°C, 54°C, 58°C), cycle numbers (20, 22, 25, 30) and amounts of template DNA (50, 100, 200 ng). The conditions under which a sufficient amount (approximately 1012 molecules) of specific amplicons of the right size was generated using the lowest number of cycles in all the samples were determined (50°C, 22 cycles, 50 ng) for bacterial 16S rRNA genes. For archaeal 16S rRNA genes, 30 cycles and the addition of 8% DMSO were necessary to obtain sufficient PCR product for archaea. These conditions were then used to produce four amplicon libraries each (top and middle/bottom part of the high- and low-current-producing PMFC). The PCR products were purified with AMPure Beads (Agencourt, Beckman Coulter, Krefeld, Germany) and pooled in equimolar amounts.
Emulsion PCR, emulsion breaking of DNA-enriched beads and sequencing run of the amplicon pools were performed on a second-generation pyrosequencer (454 GS FLX Titanium, Roche) using titanium reagents and titanium procedures as recommended by the developer following protocols for bidirectional amplicon sequencing. Quality filtering of the pyrosequencing reads was performed using the automatic amplicon pipeline of the GS Run Processor (Roche) to remove failed and low-quality reads from raw data and to remove adaptor sequences.
Newbler assembler v 2.3 (Roche) was used to batch sequences based on MID-identifiers and to combine corresponding sequences derived from forward and reverse reads with a similarity of 99% and an overlap of 400 bases for the bacterial sequences. Due to the low sequence diversity, a similarity value of 98% and an overlap of 200 bases were sufficient for archaeal sequences. The consensus sequences were inspected for chimera with the help of the Bellerophon software (http://foo.maths.uq.edu.au/_huber/bellerophon.pl) and putative chimera were removed from the dataset.
The alignment and phylogenetic analyses were performed using the ARB 5.1 software package (Strunk and Ludwig 1997). After phylogenetic allocation of the sequences down to the family or genus level, the sequences belonging to different phylogenetic groups were added together and depicted as a percentage of the total sequencing reads obtained from each sample. Phyla represented by less than 50 sequencing reads in all four libraries totalled were not included. The assembled unique sequences with their phylogenetic allocation were deposited under the numbers JF731380 and JF732737 in GenBank.
Quantitative real-time PCR of archaeal and bacterial 16S rRNA genes
For Sybr®Green-based quantitative real-time PCR (qRT-PCR), the same primers as for the 454 sequencing were used, but without the 454 specific adaptor, the key and the MID sequences. For standard generation, PCRs using these specific primers were performed with the same conditions as for the amplicon library amplification. The resulting products were cloned into a pSC-A-Amp/Kan Vector using the StrataCloneTM PCR Cloning Kit (Agilent, Palo Alto, CA, USA) after manufacturer's instructions. In each case, eight resulting clones were picked, the inserts sequenced and subsequently allocated by the ARB software package. Sequences either related to the genus Methanobacterium (archaea) or to the genus Clostridium (bacteria) were chosen as standards, as both sequences represented genera commonly found in a large proportion in all four amplicon libraries.
Subsequently, abundances of archaeal and bacterial 16S rRNA genes were measured on the ABI Prism 3300 system (Applied Biosystems, Foster City, CA, USA) under comparable conditions (10 min 95°C, 40 cycles of 20 s 95°C, 1 min 50°C and 30 s 72°C) in triplicates. Signal acquisition was done at 78°C to overcome bias due to primer dimers. PCR reaction mixtures (25 μl volume) contained 2 mM MgCl2, 0.1 μM of respective primers (forward and reverse), 1× Power Sybr®Green (Applied Biosystems, Foster City, CA, USA) and 4 ng of template DNA. Curves for both standards were linear (r2 > 0.99) over five orders of magnitude, and amplification efficiencies were comparable at 87%.
Fluorescent in situ hybridisation and confocal laser scanning microscopy
Fixed bacterial samples were stained for 10 min in the dark with the DNA binding dye Syto Orange 80 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) at a concentration of 5 μM in 10 mM Tris, 1 mM EDTA (pH 8.0) and subsequently washed twice with ultrapure water. For the FISH, the washed root samples were fixed for 2 h at room temperature (Amann et al. 1990) with either 50% ethanol in ultrapure water for Gram-positive bacteria or 4% paraformaldehyde for Gram-negative bacteria. Hybridisation with fluorochrome (Cy3, Cy5)-labelled oligonucleotide probes was carried out following the protocols described by Manz et al. (1992) and Amann et al. (1992). For the microscopic observations, the FISH stained root pieces (ca. 5–10 mm long) were embedded in Citifluor (Citifluor Ltd., Canterbury, UK) and observed by CLSM without further cutting.
rRNA-targeted oligonucleotide probes were synthesised and labelled by Sigma-Aldrich (Steinheim, Germany). EUB338-I (Amann et al. 1990) and EUB338-II and EUB338-III (Daims et al. 1999) were used in an equimolar mixture at different formamide concentrations. To detect the genus Geobacter, an equimolar mixture of the 16S rRNA targeted probes Geo1A and Geo1B was used at a 35% formamide concentration. Geo1A is specific for G. sulfurreducens, Geobacter hydrogenophilus, Geobacter grbiciae and G. metallireducens; Geo1B detects most other Geobacter species (Demaneche et al. 2008). To detect the Ruminococcaceae group members Ruminococcus bromii, Clostridium sporosphaeroides and Clostridium leptum, the Rbro730 probe (Harmsen et al. 2002) was applied in the presence of 20% formamide. To detect most members of Clostridium clusters I and II, the Chis150 probe (Collins et al. 1994; Franks et al. 1998) was used with 35% formamide.
The fluorescently labelled cells were detected using a Zeiss LSM510 CLSM. An argon ion laser supplied 488-nm illumination to excite the fluorescein, and two helium-neon lasers provided 543 nm for Cy3 and 633 nm for Cy5. For each hybridisation probe, an EUB338 mix labelled with Cy5 (blue) was combined with another group-specific probe labelled with Cy3 (red). The binding of both probes resulted in a magenta staining of the target cells. The green channel (fluorescein) served as a negative control that showed only autofluorescence by the root or other particles. Syto Orange was detected by excitation with the 488- and 543-nm laser wavelengths, resulting in a yellow/orange fluorescence signal in the target cells; the negative control was the blue channel (excitation at 633 nm).
The optical sectioning of the root was achieved by moving the focus position deeper into the sample in 1 μm steps, which produced z-stacks of individual pictures from the same xy-area with a penetration depth of up to 60 μm. The resulting set of pictures was displayed as an orthogonal view using the LSM510 software package that was provided by Zeiss.
Energetic performance of PMFCs
Differences in the 454 sequencing of bacterial and archaeal 16S rRNA gene fragments between PMFC
A bidirectional sequencing approach of the four pooled bacterial 16S rRNA gene amplicon libraries (top section low-current PMFC, middle/bottom section low-current PMFC, top section high-current PMFC, middle/bottom section high-current PMFC) resulted in a total of 140,000 reads with 114,000 key pass reads and 46,000 reads that passed all of the internal quality filters of the 454 software. An average read length of 517 bases and almost no reads shorter than 500 bases were obtained. The sequencing of the bacterial amplicon libraries was repeated once for verification of the results with slightly lower output and read lengths but no major differences in bacterial abundance.
In the top section of the high-current PMFC, the major portion (36%) of the bacterial 16S belonged to Comamonadaceae, and considerable amounts could be assigned to the Rhodocyclaceae (10%) and Clostridiaceae families (13%). The bottom and top sections of the low-current PMFC were dominated (54% and 35%, respectively) by members of the Comamonadaceae family, but also representatives of the Rhodocyclaceae family were frequently detected (13% and 23%).
Figure 3b shows the archaeal genera found in both PMFCs. In the high-current PMFC, the genus Methanobacterium made up over 95% of the archaeal community. In the low-current PMFC, the archaeal community was more diverse and mainly consisted of the genus Methanobacterium and uncultured Methanobacteriaceae. Furthermore, Methanosarcina was found to account for 5% of the archaeal community in the top section and up to 10% in the bottom section of the low-current PMFC.
The quantification of archaeal and bacterial 16S rRNA genes revealed great differences in the abundances of about two to three orders of magnitude. Gene copy numbers of bacterial 16S rRNA genes per nanogram extracted DNA were (top section, low current; middle/bottom section, low current; top section, high current; middle/bottom section, high current) 0.9, 2.4, 0.7 and 1.2 × 106, respectively. In comparison, gene copy numbers of archaeal 16S rRNA were 5.6, 4.9, 1.4 and 0.8 × 103, respectively. While the abundances of bacteria differed by a maximum of factor 2, when comparing the same sections of the high and the low-current PMFCs, there were four to six times more archaeal 16S genes in the low-current than in the high-current PMFC.
Localisation of characteristic PMFC bacteria by FISH and CLSM
A biofilm was detected, particularly in roots from the upper sections of both PMFCs, where the film completely surrounded the root and was 5 to 10 μm thick. No visual differences in the structure or amount of the biofilms were observed between the PMFCs. For the low-current PMFC, however, fungal hyphae were clearly detected in the biofilms of all observed samples.
FISH was used to localise Geobacter, Clostridiaceae and Ruminococcaceae species that were previously detected by 454 sequencing of the 16S rRNA gene. The Geobacter probe set enabled detection of fluorescent Geobacter cells, which appeared more frequently on the graphite granules than on the root surfaces. With probe Geo1A, specific only for G. sulfurreducens, G. metallireducens, G. grbiciae and G. hydrogenophilus, positive signals were identified on both roots and granules from both PMFCs. Furthermore, the positive signal was more frequently observed on samples from the bottom layer of the high-current PMFC. A Clostridia cluster I and II specific probe yielded very few positive signals, and no clear differences were observed between the individual samples. With probe Rbro730, specific for the three Ruminococcaceae species R. bromii, C. sporosphaeroides and C. leptum, small numbers of cells were positively identified on the root surfaces, but fluorescent signals were detected to a much greater extent in the outer cortex layers of roots of the high-current PMFC.
The presence of electrochemically active bacteria in the PMFCs
In the bottom section of the high-current PMFC, G. sulfurreducens or one of its close relatives (G. metallireducens, G. grbiciae and G. hydrogenophilus) were observed frequently on the graphite granules. The roots in the bottom section of the high-current PMFC were also colonised, although less frequently than the graphite granules (Fig. 4c, f). In the low-current PMFC, G. sulfurreducens or one of its close relatives (G. metallireducens, G. grbiciae and G. hydrogenophilus) were observed less frequently in the top section, and no signal at all was detected in the bottom section.
The difference in the presence of the genus Geobacter between the PMFC types was supported by the differences in the presence of the Geobacteraceae family to which the Geobacter genus belongs (Fig. 4a). An average of 0.74% of the obtained 16S rRNA gene fragment sequences belonged to Geobacteraceae in the high-current PMFC, while an average of only 0.13% belonged to Geobacteraceae in the low-current PMFC.
Anaerobic Ruminococcaceae were predominant in the bottom section of the high-current PMFC
Ruminococcaceae accounted for over half of the bacterial rhizosphere sequences in the bottom section of the high-current PMFC. The members of this family have not been reported to be electrochemically active, but they are known to hydrolyse cellulose into hydrogen and low molecular weight organic compounds. In MFCs, the hydrolysis of cellulose generates electron donors that are suitable for G. sulfurreducens (Maki et al. 2009; Ren et al. 2007).
Based on their cellulolytic activity, the Ruminococcaceae species probably played an important role in electricity generation in the PMFC. They probably hydrolysed the cellulose, originating from dead roots, into suitable electron donors for EAB. Typically, 35–50% of dry-plant weight consists of cellulose (Lynd et al. 1999), which stresses the importance of cellulolytic activity in generating a suitable electron donor for EAB.
The main species of the Ruminococcaceae family was probably C. sporosphaeroides, which is able to ferment glutamate to ammonia, CO2, acetate, butyrate and hydrogen via the hydroxyglutamate pathway (Hsiao et al. 2009). Furthermore, this species has been used to improve the hydrolysis rates of cellulose and hemicelluloses in a biomass fermenter (patent no. WO/2010/072219).
C. sporosphaeroides was mainly found in the root cells of the outer root cortex in the high-current PMFC (see Fig. 4e). Due to the large surface area of the outer root cortex, the presence of C. sporosphaeroides there probably resulted in a high rate of hydrolysis (Lynd et al. 2002). It could not be determined whether C. sporosphaeroides acted as a moderate pathogen and actively killed root cells or was simply colonising dead plant material. E. cloacae, able to generate current from complex electron donors as a pure culture, was not detected in the PMFCs (Rezaei et al. 2009). This is another indication that current was likely generated via the hydrolysis of complex electron donors into suitable electron donor for G. sulfurreducens. The importance of the hydrolysis of cellulose for electricity generation is supported by the results of Ren et al. (2007). They demonstrated that a binary culture of Clostridium cellulolyticum and G. sulfurreducens (with carboxymethyl cellulose and MN301 cellulose as electron donors) produced current, while single cultures of these bacteria did not.
The lack of dominance by obligate anaerobes in the top section of the high-current PMFC and in the top and bottom sections of the low-current PMFC indicates the presence of oxygen. Therefore, hydrolysis in the low-current PMFC was likely performed by fungi and/or facultative anaerobic cellulolytic bacteria, such as the Rhodocyclaceae family (Schellenberger et al. 2010). In contrast, the bottom section of the high-current PMFC was dominated by the obligate anaerobe Ruminococcaceae, indicating that cellulose hydrolysis was predominantly carried out by members of this family.
Short-chain fatty acid-utilising denitrifiers are major competitors for electron donor
Except for the bottom section of the high-current MFC, Rhodocyclaceae and Comamonadaceae were the dominant families observed on the PMFCs. Rhodocyclaceae and Comamonadaceae are known to be facultative anaerobic short-chain fatty acid-utilising denitrifiers (Ginige et al. 2005; Nielsen et al. 2006; Thomsen et al. 2007). Nevertheless, both Comamonadaceae and Rhodocyclaceae have been previously observed in MFCs. Comamonadaceae have been found to dominate the bacterial community in the anode of a cellulose-fed MFC (Rismani-Yazdi et al. 2007). Rhodocyclaceae have been found to dominate the bacterial community in the anode of an acetate-fed MFC with an open air cathode (Borole et al. 2009). Furthermore, Comamonas denitrificans and R. ferrireducens species belonging to the Comamonadaceae family are able to generate electricity in an MFC in the absence of oxygen and nitrate (Xing et al. 2008, 2010).
In our PMFC, both oxygen and nitrate were probably present due to the diffusion of oxygen through the roots and the addition of nitrate through the medium. The nitrate flux into the anode was equivalent to 140 g N m−2 day−1 (2 × 10−3 molnitrate day−1, assuming that there was no medium overflow), which is about 125 times higher than the maximum reported rate of nitrogen uptake by emergent macrophytes in a constructed wetland containing G. maxima (Tanner 1996). Therefore, a large portion of the added nitrate was denitrified by facultative anaerobic activity as long as short-chain fatty acids were available as electron donors. Short-chain fatty acids were probably generated by the aerobic cellulolytic activity of Oxalobacteraceae (Lynd et al. 2002) and the anaerobic cellulolytic activity of Rhodocyclaceae (Schellenberger et al. 2010). Offre et al. (2007) associated the presence of Oxalobacteraceae and Comamonadaceae with mycorrhizal roots and suggested a possible symbiosis between both bacterial families and fungi. The presence of fungi in the outer cortex of the roots in the low-current PMFC was consistent with these findings.
The presence of Rhodocyclaceae and Comamonadaceae probably had a negative effect on current generation, as these families do not contribute to electricity generation in the presence of oxygen and nitrate. Furthermore, these families consume short-chain fatty acids, which decreases the amount of short chain fatty acids available for current generation and thus negatively affect current generation. The difference in current generation between the high- and low-current-generating PMFC could likely be attributed to difference in presence of Rhodocyclaceae and Comamonandaceae in the bottom part of both PMFC. In the bottom part of the high-current PMFC, Rhodocyclaceae and Comamonandaceae were almost absent while they were more abundant in the bottom part of the low-current PMFC. As these families consume short-chain fatty acids, it is likely that their presence resulted in lower current generation because less substrate is available for EAB.
The absence of fungi in the outer cortex of roots in the bottom section of the high-current PMFC, together with possible nitrate depletion may have allowed strictly anaerobic cellulolytic microorganisms (Clostridiaceae and Ruminococcaceae) and EAB, such as G. sulfurreducens and G. metallireducens, to proliferate.
Methanogenesis plays a minor role in competition for electron donor
Methanogens can compete with G. sulfurreducens and G. metallireducens for electron donors, as they are able to use acetate, formate, methanol, methylamine, H2 and CO2 (Ishii et al. 2008; Thauer et al. 2008; Thomsen et al. 2007). Quantitative real-time PCR amplification with bacterial and archaeal primers using the same DNA template indicated that considerably less of the DNA present in the sample was derived from archaea than from bacteria. The higher abundance of bacterial DNA compared to archaeal DNA was probably due to the presence of alternative acceptors in the medium, such as oxygen, nitrate and sulphate. As long as these acceptors are present, methanogenic archaea are outcompeted by aerobic bacteria, denitrifiers and/or sulphate-reducing bacteria (Scheid et al. 2004; Thauer et al. 2008). Timmers et al. (2011) did not measure acetate concentrations above 2 mg l−1 (the limit of detection) in the PMFCs of which the biological community was analysed. This finding indicated that hydrolysis is the rate-limiting step in that type of cell, assuming that electricity in the PMFC is generated via fermentation of hydrolyses products to acetate and the subsequent oxidation of acetate by G. sulfurreducens and G. metallireducens. The majority of the methanogenic archaeal community, Methanobacteriacea, are unable to utilise acetate (Thauer et al. 2008). And therefore do not compete with G. sulfurreducens and G. metallireducens for acetate. This restriction is supported by the finding that of all the archaeal genera found in both PMFCs, only the Methanosarcina genera belong to acetate-utilising methanogens (Thauer et al. 2008). Methanosarcina comprised, on average, less than 10% of the archaeal DNA in the low-current PMFC and were barely observed in the high-current PMFC. The relative higher abundance of Methanosarcina in the low-current PMFC compared to the high-current PMFC likely contributed to competition for acetate. The competition for acetate between G. sulfurreducens and G. metallireducens and Methanosarcina likely contributed to the difference in current generation, however less than Rhodocyclaceae and Comamonandaceae. Furthermore, Methanosarcina is a methanogen with cytochrome, which generally have a threshold partial hydrogen pressure above 10 Pa. This consideration may indicate that products other than acetate were formed by fermentation, leading to less acetate production and thus likely a decrease in availability of acetate for generation of current. In addition, the overall abundance of archaeal 16S rRNA genes was considerably lower in the high-current PMFC than in the low-current PMFC. This result leads to the conclusion that the methanogens likely did not consume a major portion of the acetate available for G. sulfurreducens and G. metallireducens.
De Schamphelaire et al. (2010) performed a phylogenetic community analysis on archaea and found that 45% of the archaeal clone sequence was not related to any of the known methanogenic lineages. Of the archaeal clone sequences related to methanogenic lineages, 20% belonged to Methanobacteriaceae, 18% to Methanosarcina and 10% to Methanosaetaceae. In this study, the amounts of the acetate-utilising methanogens Methanosarcina and Methanosaetaceae were considerably lower (an average of <10% versus an average of 28%). One possible explanation for this discrepancy is the difference in the availability of graphite as an electron acceptor. De Schamphelaire et al. (2010) used a graphite felt in a sediment as the anode electrode for the PMFC, whereas in our setups we used graphite granules (without sediment) as electrodes. The use of graphite granules may have resulted in the increased availability of graphite as an electron acceptor. The limited availability of the graphite felt as an electron acceptor may have given the acetate-utilising methanogens a competitive advantage over EAB. In case the availability of graphite as an electron acceptor indeed limits the competitive advantage of EAB over acetate-utilising methanogens, the availability of graphite as an electron acceptor will be an important factor in the reduction of methanogenesis by the PMFC (De Schamphelaire et al. 2008; Strik et al. 2008).
Possible electron sinks and electron donor transfer pathways
Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, and more EAB were found on the graphite granules than on the root surface. This result implies that current was generated via the hydrolysis of cellulose and not directly from low molecular weight rhizodeposits. When optimising electricity generation in a PMFC, therefore, the focus should be on root biomass production and effective hydrolysis of the biomass.
This research received funding from the European community's 7th framework programme FP7/2007–2013 under grant agreement no. 226532, as well as from Senternovem, the Dutch governmental agency for sustainability and innovation from the Ministry of Finance (grant no. EOSLT06020), and was supported by Alliander. The authors would like to thank Marjolein Helder and Kirsten Steinbusch for their useful comments.
Open Access
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Dolch Reading: Set 21
Ten Dolch words (these, came, what, now, let, yes, any, can, an). Practice spelling & context reading with a crossword puzzle, cloze sentences, a cloze story, and a "find the words" story.
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Brooklyn's Knitting Factory Announces Lineup Through 5/10
The Knitting Factory Brooklyn, located at 361 Metropolitan Ave. (at Havemeyer St. - Subway: L to Lorimer) has announced its upcoming lineup, which kicks off with Jon Benjamin Has a Tour tonight, May 3 and ends May 10 with Mark Sultan, Habibi and Des Roar. See full details and other shows below.
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Tickets: $15.00
Call 347-529-6696 or go online at http://bk.knittingfactory.com/event/104705/
Actor, writer and producer, H. Jon Benjamin has garnered an extensive list of credits in some of television's most entertaining series. This year, he will play the title character on three series. He is the voice of "Sterling Archer" in the FX animated hit, "Archer," which gave him a 2010 Emmy Nomination for Outstanding Voice-Over Performance. He also is the voice of "Bob Belcher" in Fox's "Bob's Burgers." Benjamin will star in "Jon Benjamin Has A Van" premiering on Wednesday, June 15 at 10:30 p.m. on COMEDY CENTRAL.
Wheatus + MC Frontalot + Jetblack Bullseye + Math The Band + Schaffer The Darklord
Fri, May 4, 2012
Doors: 6:30 / Show 7:00
Tickets: $10.00 - $12.00
Call 347-529-6696 or go online at http://bk.knittingfactory.com/event/96423/
Wheatus' debut single "Teenage Dirtbag" cracked the Top 40 BBC Official Sales Chart for a second time in March of 2011, TEN YEARS after its debut at #2 on the same chart. To say that Wheatus have been through the wringer in the decade between is the original understatement. Lawsuits, lineup changes, interstate art heist investigations (yes, really), betrayals, intellectual property theft, physical violence--post-traumatic front man Brendan B. Brown could write a book. To date, their first record has sold more than 5 million copies world wide. Any manager or record label would be happy to tout those stats to the press but Wheatus have remained unsigned & unmanaged for the last 8 years. Unlike many of their peers from the early oughts, wheatus continue to write, record, and tour 15 years after their inception, having taken control of their revenue stream with the home made, "pick-your-price" download system fans can use at wheatus.com. They have released 5 Studio albums since 2000 and are currently in preproduction for album 6.
MC Frontalot is the stage name of Damian Hess, a hip hop musician and self-proclaimed "world's 579th-greatest rapper." Many of his songs deal with things nerdy in nature, which has led him to be categorized as nerdcore, a word he himself coined in his first single, "Nerdcore Hip Hop". Jetblack Bullseye is an American rock band formed in Brooklyn, NY in the summer of 2010. Their debut album, Jetblack Bullseye (2012) was recorded live in their practice space in Greenpoint, BK. Started by a sixteen year old kid who was kicked out of his high school pop-punk band, Math the Band has been going strong for 5 years, playing well over 500 shows throughout the US, Mexico and Canada, with bands such as Andrew WK, Matt and Kim, Polysics and Bob Doland. Schaffer the Darklord (or STD) is a New York City-based rapper and comedian with subversive material best-suited for brainy and/or decadent audiences.
Cinco de Mayo Fiesta with Live Music & Salsa w/ Grupo Arcano + Maquina Mono + The Peeps + Paracuta + Cupéro
Sat, May 5, 2012
Doors: 7:00 / Show 7:30
Tickets: $8.00 - $12.00
Call 347-529-6696 or go online at http://bk.knittingfactory.com/event/110427/
Grupo Arcano is a Salsa band that brings the best from a rich tradition, as well as the hottest trends in salsa music. To packed dance floors, Grupo Arcano plays the hits from decades past as well as explosive original compositions.
Maquina Mono is a bilingual latin rock group with horns from New York City making music to listen to & dance all night. Half-American, half-Colombian, the Peeps bring their own style of latin-influenced punk rock they call "SalsaPunk." Cupéro is an upcoming singer-songwriter artist who has recently completed an EP at the Cutting Room in Greenwich Village, NYC.
Pierced Arrows + Mount Carmel
Sun, May 6, 2012
Doors: 7:30 / Show 8:30
Tickets: $10.00 - $12.00
Call 347-529-6696 or go online at http://bk.knittingfactory.com/event/113337/
After the demise of underground legends DEAD MOON in fall 2006, nobody was surprised to hear that founders Fred and Toody Cole had lost little time in starting a new band. They recruited drummer Kelly Halliburton in April 2007, and soon the PIERCED ARROWS were up and running, playing live shows in the Northwest and recording songs for upcoming releases. PIERCED ARROWS picks up, musically, where DEAD MOON left off - a lo-fi assault on the senses played with the sincerity and feeling that made DM such a special band for all of their fans around the world. Stay tuned to this site for information about upcoming shows, releases, and tours.


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|Nualsri, Charassri
|Beuselinck, Paul
Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/1998
Publication Date: N/A
Citation: N/A Interpretive Summary: Lotus corniculatus L. (broadleafed birdsfoot trefoil) is a forage legume grown in the United States, Canada, South America and Europe. Long-lived and productive stands of L. corniculatus depend on natural reseeding and individual plant persistence. Wild plants of L. corniculatus from Morocco are unique in that they produce rhizomes (stems that spread underground) which may prove useful in increasing the longevity of our cultivated varieties. The objective of this study was to determine how rhizomes are inherited in L. corniculatus. To study the inheritance of rhizomes we developed self-pollinating hybrids and their offspring to use as research tools. Our observations from other studies suggested that rhizomes were inherited as a dominant trait and this research confirmed our observations. Our examination of the hybrids and their offspring indicates that rhizome presence is controlled by a single dominant gene and conforms to simple Mendelian genetics principles. We propose to name the gene for rhizomes in L. corniculatus and identify it by the symbol R. The findings of this research will result in the faster development of new L. corniculatus cultivars with rhizomes, and may lead to the isolation of the rhizome gene. Once the gene is isolated, it can be transferred to different species of plants that could benefit from rhizomes.
Technical Abstract: Introduction of rhizomes from wild Lotus corniculatus L. (broadleafed birdsfoot trefoil) germplasm from Morocco is considered an important step towards improving persistence of cultivated L. corniculatus, a cross-pollinated herbage crop. Rhizome-production has been successfully transferred from wild germplasm of broadleafed birdsfoot trefoil into domesticated cultivars and autogamous germplasm. The objective of this study was to determine the inheritance of rhizomes in L. corniculatus. Crosses were made between AG-S4, an autogamous, non-rhizomatous germplasm and two rhizomatous accessions from Morocco, G31276 and G31317. Two autogamous progeny from each cross (verified as F1 hybrids by morphological and random amplified polymorphic DNA markers) produced F2 seed and were reciprocally backcrossed (BC). After 10 mo growth in the greenhouse, F2 and BC progeny were scored for rhizome production. Plants failing to express rhizomes in greenhouse conditions were transplanted to the field and reevaluated for presence or absence of rhizomes at the end of the growing season. Segregation ratios for rhizome expression were tested for goodness-of-fit by the Chi-square test. The ratios of rhizome production in F2 and BC's to AG-S4 fit 3:1 and 1:1 dominant:recessive ratios indicating that rhizome presence in L. corniculatus is controlled by a single dominant gene. The gene symbol R is proposed for rhizome expression.


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Comic convention coming to Rochester's ' Underground city'
Local comics and fantasy fans have long been envious of the attention that larger cities in warmer places get from pop culture conventions, like the recent San Diego Comic Con.
Now it looks like they will have their one piece of the action right here in the Med City.
Bill Willingham, the creator of the wildly popular comic Fables as well as being a southern Minnesota transplant, recently told the Comic Con crowd that this was his last appearance at the San Diego event.
Most importantly for local comic and fantasy fanatics like myself, Willingham announced that he is organizing his own three day convention to be called, "Fabletown and Beyond."
It will focus on "mythic fiction" like his dark retelling of fairy tales and Neil Gaiman's "Sandman" series.
Here's the happy ending to this tale. This young Midwestern sibling of Comic Con will be held in downtown Rochester in March.
I got a kick out of how the comics site, bleedingcool.com, described the choice of Rochester as a venue.
"… The show will be held in Rochester, Minnesota. The facilities that have been chosen have been chosen specifically for the expansive indoor space, an underground city built for the wealthy visitors of the Mayo Clinic. This space will enable convention goers to remain indoors in the bitter Minnesota March."
The Post-Bulletin will have more on this as it gets nearer. For more information, check out fablescon.com.


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Bertha Blanche Jackson
Bertha Blanche Jackson, 94, of Uvalde died on Jan. 13, 2014, at her residence. A memorial service will be held Saturday at noon at the Uvalde Church of the Nazarene. Everyone is invited and encouraged to attend. She was born June 15, 1919, in San Francisco, Calif. to Mary Blanche Owen and William Edward Griffith. She later moved to Uvalde. She met her husband, Arthur Jackson, in Wichita Falls. According to the family, Jackson loved her Lord, and served him vigorously. She attended Uvalde Church of the Nazarene, where she rarely missed a service. She loved her family and prayed for them by name every day. Besides her family, her passion was missions. She loved everyone, whether they lived in Uvalde, Zimbabwe, India or anywhere else in the world. The family said Jackson need not know you to love you. When she came to church, she wore the most beautiful and contagious smile anyone has ever owned. She gave tirelessly of herself to her Lord, her family, and to her church family. She was preceded in death by her parents; husband; one daughter, Judy Snider; and one great-grandson, Andrew George Snider. She is survived by two sons David Jackson of California and Arthur Jackson and wife, Lisa, of Florida; son-in-law, George Snider; seven grandchildren, Sam Jackson, Chris Jackson and wife, Suzanne, Steve Jackson and wife, Gracie, Tishri Dobson and husband, Patrick, Laura Snider Wiemelt and husband, Ronald, Jeanette Hull and husband, David, and George Snider III and wife, Michele; 13 great-grandchildren, Asaiah Wiemelt, Benjamin Wiemelt, Aaron Wiemelt, Crystal Wiemelt Green, Sarah Snider Wiemelt, Jon Hull, Sydney Doykin, Jossiah Dobson, Ezekiel Dobson, Christyn Jackson, Kelly Jackson, Ryan Jackson and Nathan Jackson; three great-great-grandchildren, Jay Green, Serinity Green and Dominic Andrew Snider; and numerous nieces and nephews.
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- Sold for: Start Free Trial or Sign In to see what it's worth.
- Item Category: Transportation and Vehicles
- Source: eBay
- Sold Date: May 15, 2011
- Channel: Auction House
*I ship fast > > CHECK OUT MY OTHER AUCTIONS, A VINTAGE *AMERICAN FLYER S GAUGE* , CABLE CAR CONDITION IS AS YOU SEE IN THE PICS > THIS IS A BIG LONG HEAVY CAST CABLE CAR WITH KNUCKLE COUPLERS * IN VERY GOOD CONDITION , > **ALL SALES ARE FINAL, **SOLD IN ASIS CONDITION**NO RETURNS NO REFUNDS . amp; **** ** Payments accepted: ** PAYPAL, MAJOR CREDIT CARDS, **. ** **!. I ship anywhere in the USA . I ship fast, NEXT day after payment arrives, * I SHIP FAST!. **Check my feedback**, & bid with confidance**. Payment expected within 2 days of auction end. ****Please feel free to ask questions, don"t wait to the last minute, I'll try to answer them. ***! Please bid responsibly. Thanks for looking & have a Great day. ****CHECK OUT MY OTHER AUCTIONS****. ** > ATT: > *****THIS IS A USA AUCTION ONLY > *STRICTLY ENFORCED!! > I SHIP FAST ****. > !!****


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Find Hotels in Norsborg
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Lacrosse news straight to your inbox.
Tuesday August 6th, 2013 1:07am
Fallball will soon be coming to a close. Fallball weekends will give way to recruiting weekends for coaches and IL staffers.
As we have all fall, check out our Winners & Losers for the week, and be sure to follow us each individually on Twitter to get the scoop over the weekend while we're in the field.
UMBC — I caught this past weekend's fall tournament at Drexel, featuring the Dragons, Retrievers, Navy and Penn. I came away impressed with the Retrievers. They have balance all around the field, a solid goalie in Adam Cohen, and a player who can take over with his goal-scoring ability in Scott Jones. UMBC showed us what it can do with its surprise win over Maryland a year ago, though that doesn't soften the blow of a 5-8 season. But the team has been young for a few years now, and coach Don Zimmerman knows how to work with young talent and maximize potential. One loss though: Freshman Nate Lewnes broke his elbow at the tournament; he looked solid in the short time he had on the field before getting hurt.
Bonus: Lots of parents came up to me this weekend while I was at Drexel and said hello. Thanks to the Navy parents who showed appreciation for the story I wrote this weekend, and thanks to the other parents and fans who came up just to talk lacrosse and say hello. This business is a two-way street, and we always welcome conversation and feedback — and we love talking lacrosse.
Penn — As Kinnear said in his write up from Saturday, Penn looked good during the Drexel Fall Play Day. They have been defensively sound the past few seasons, but last year their offense lacked punch. That identity might flip a little this year as Penn looked fast and potent on offense. true it's just a one game sample, but their production on Saturday has to be encouraging for the Quakers.
Face-Off Yearbook 2013 — Though the season is just a glint in the eyes of a baby spring, and my erstwhile compatriots at IL are attacking the autumnal tundra to provide depth of coverage from lacrosse fallball, myself and assistant art director Keely Knopp are working feverishly in the deep bowels of our Baltimore headquarters, plowing through production for this, our 20th edition of the F/O YB. Oh what nuggets of information we'll have for you, dear reader! And, though it is early in this endeavor, our eyes, in fact, have not popped out of the comfort of their sockets yet. Still, deadline day approaches swiftly, and anything can happen in the interim...
Vintage lacrosse gear collectors — It was a great weekend on eBay if you're a vintage stick collector. I scored a few items I'd been after for a long time, some of them at great prices. ILGear.com wins there too, as we'll get a chance to share some of those cool old items with you in the near future. And if you still don't believe that there's a growing market for vintage lacrosse gear on eBay, you should. Monday night, an Amisco Warrior was sold for $405 dollars.
Here's a link to my original Amisco Warrior post (the text of which was pulled and used for the description on the listing for the Amisco Warrior).
The family of John Schimoler — It was a pleasure to be part of a Simmons Lacrosse Foundation Fundraiser for former Syracuse national champion John Schimoler at the Powderly household Friday night. Schimoler passed away at age 50 in August, and members of his family, including son Gunnar, a promising sophomore attackman at South River (Md) High, were present Friday night. So were Brad Kotz, Todd Curry, Tim Nelson, Roy Simmons III, Roy Simmons Jr and a host of other supporters. Click this link to donate.
Under Armour Lacrosse — Under Armour was able to sign Steele Stanwick as an endorsed athlete this week. Both Under Armour and Stanwick have become Baltimore staples so it makes sense for both to sign the deal. Myself, John Jiloty, Terry Foy and Ryan Rabidou were able to stop by UA's first video/photo shoot with Steele yesterday at the Stanwick compound and from the looks of the crew and equipment involved in the shoot, Under Armour is taking this relationship very seriously. It looked like a blockbuster film was being shot in the famous Stanwick garage. I'm excited to check out the results.
Hopefully Kim Roper — The match4kim campaign kicked off last week with a website and social media effort meant to raise awareness and drive registration for bone marrow donor registrations. The hoped-for beneficiary is Kim Roper, whose son Drew is a senior midfielder at Penn State. Men's Lacrosse Mustache Madness also started last Friday, and a fall season during which numerous charities benefited from a handful of events is winding down, so the evidence of the lacrosse community's desire and ability to help is all around.
Fall Scoreboard Watchers — We're all starved for lacrosse and hungry for any information about what are teams are doing. But please, don't let scores mislead you because they do not tell even half the story in the fall. The 9-6 score would tell you Penn struggled offensively in its loss to UMBC, but it doesn't tell you that second- and third-stringers were going against UMBC's first line. And, it doesn't tell you how good Penn's freshmen offensive players were in the earlier game vs. Drexel. Navy's 14-3 loss against Notre Dame certainly is eye-opening, but it doesn't tell you that Navy had players missing because of military commitments and injuries, and the game wasn't nearly as bad as the scrimmage a year ago. A lot of people were hounding me on Twitter for score updates during fallball weekends, but let's treat fallball as it is: A time for coaches to work out some kinks, see where they are, and see what they need to do leading up to February. Bring on spring.
The Rules AGAIN — Saturday's double-header in Drexel probably puts me at about a dozen or more games I've seen this fall and there was one steadfast trend in all of them: the inconsistency and nuance of the application of the new rules. Some games I saw felt much faster because of the quicker whistles and no horns, while in others teams were able to play at their old pace without issue. To that end, different refs had different ideas about just how fast a whistle should be, how much lead time a player coming on the field could get, and how much they let opposing defenses get set before restarting play. And in the dozen or more games I saw, I have no idea how many 30-second shot clocks were put on, a bad sign for the coming season if coaches, fans or players have similar confusion. The lack of a visible clock and a discernible qualification for when it clicks on are going to be points of consternation come the spring.
The fall is the perfect time to experiment and explore rule changes like this. Unfortunately early results seem inconclusive. Rules proposals that may have seemed well thought, helpful and limited may prove too be tepid, confusing half measures.
@ILPreps Twitter Feed – Not for any negative reason, only in that it's about to get heated on the @ILPreps feed, thanks to a ton of recruiting events kicking off starting in two weeks with the first turn of November. IL will be at various recruiting events across the country, from Florida to Philadelphia and beyond. Make sure to follow our poor, overworked @ILPreps as we get going for recruiting second season.
Me — See above. This past weekend, my vintage love affair hit my wallet. Hard.
Athletic Goalies — As Mark Dixon pointed out in his story on IL.com this week, since goalies no longer have five seconds to get back to their crease before a restart, they're likely going to be less inclined to leave the goal area to chase balls to the endline or help with clears. And while quicker restarts have been pretty positively received this fall, I hadn't thought of the impact on crazy, athletic goalies like Brett Queener, who are quick enough and have good-enough sticks to serve as an extra player on clears. That was an exciting part of the game, albeit one I'm sure coaches aren't sad to see going away.
Nobody — That's the way I feel about Nicky Galasso's transfer to Syracuse — there were no losers between Nicky, 'Cuse and UNC. Over the last two weeks, there were five stories published about the news on InsideLacrosse.com that garnered a total of 103 comments — many of those casting blame or condemning one or more of the parties involved in the decision. I don't see it that way. Galasso had some frustrations at Carolina and wanted a change. He got it. Syracuse had room and likely a need for a offensive player — the Orange got one. Carolina was clearly set on its offensive personnel moving forward and likely avoided some difficult situations with Galasso's departure. I wouldn't argue it's an ideal situation, but I do think there were only winners in how this played out.


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1 Recommendation
debbyNY's recommendation
If youâre bored with your Rubikâs Cube you can combine it with the challenge of Sudoku with the Sudoku Puzzle Cube. It isnât exactly a Sudoku puzzle, as the challenge is to get the numbers 1 through 9 on each of the six sides of the cube.
My Collections:


text:
Pasadena, CA, USA < change city >
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OUR WEDDINGJune, 2008 in Pasadena
"Our wedding was wonderful, but the time flew by so quickly. Thank goodness many people had cameras to catch the event on film.
My wife and I worked on the details months in advance. She did the majority of the work and it was done the way she wanted it. I provided help with the logistics, reservations, and printing of the various invitations and programs.
For any young couple, I would suggest having the groom involved in the planning process. This way you both can work together as a team to plan the event. It is a wedding for both of you."
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Ceremony: Holliston Church
Reception: Holliston Church
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- DJ: Tom Hickey
- Photographer: Ogden Photography
- Ceremony and Reception: Holliston Church
- Restaurant: Hamburger Hamlet
- Hotel: Ramada Inn Pasadena / Kingston
- Restaurant: Islands
- Hotel: The Saga Motor Hotel
- Restaurant: Rubio's Baja Grill
- Hotel: Westway Inn
- Restaurant: The Kitchen
- Restaurant: Brits Restaurant
- Grocery store: Vons
- Restaurant: El Torito Restaurant
Tom HickeyPasdena, CA, USA
"We have known Tom for many years. He plays and has a wide variety of music. He can read the crowd and knows what to play to keep the guests dancing."
Ogden Photography901 Hermosa Ave
Hermosa Beach, CA, United States
"We have known Cheryl for years. She does fantastic work. Her photographs capture the event. She does the typical required wedding photos, photos using the avaliable background to make a special enjoyable photo, as well as the candid shots of the guests."
Ceremony and Reception:
Holliston Church1305 E. Colorado Blvd
Pasadena, CA 91106
"What a wonderful church to have a wedding. Beautiful architecture inside the church. Plenty of seating for a large crowd. The backgrounds are great for group photos."
Ramada Inn Pasadena / Kingston2156 E Colorado Blvd
Pasadena, CA 91107, US
"We made reservations with them months in advance. They were quite helpful in reserving a particular set of rooms to accomodate our group. The rates were very reasonable. We would definitely stay here again."
The Saga Motor Hotel1633 E Colorado Blvd
Pasadena, CA 91106, US
Westway Inn1599 E Colorado Blvd
Pasadena, CA 91106, US
Brits Restaurant1770 E Colorado Blvd
Pasadena, CA 91106, US
(626) 578-1301
El Torito Restaurant3333 E Foothill Blvd
Pasadena, CA 91107, US
(626) 351-8995
Hamburger Hamlet214 S Lake Ave
Pasadena, CA 91101, US
(626) 449-8520
Islands3533 E Foothill Blvd
Pasadena, CA 91107, US
(626) 351-6543
Rubio's Baja Grill330 E Colorado Blvd
Pasadena, CA 91101, US
(626) 535-9383
The Kitchen78 W Union St
Pasadena, CA 91103-3627, US
Grocery store:
Vons2355 E Colorado Blvd
Pasadena, CA, United States


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LAW Writer® Ohio Laws and Rules
No unauthorized persons are allowed in the dressing rooms other than contestants scheduled for the event and the contestant's manager, trainer, second, and the promoter or the promoter's representative. The promoter shall provide separate dressing rooms for male and female contestants, the referees and for each corner as well as the main event contestants and shall provide security in the dressing room areas.
119.032 review dates:
05/21/2010 and
Promulgated Under: 119.03
Statutory Authority: 3773.34
Rule Amplifies: 3773.34
Prior Effective Dates: 9/12/86, 7/31/99, 9/20/05


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April 14, 2010
Box Score | Quotes | Notes | Video Highlights
Site: Ann Arbor, Mich. (Wilpon Softball Complex)
Score: #2 Michigan 8, Central Michigan 0 (5 inn.)
Records: U-M (31-5), Central Michigan (17-13)
Next U-M Event: Saturday, April 17 -- vs. Northwestern (Wilpon Softball Complex), 6 p.m. (TV: Big Ten Network)
ANN ARBOR, Mich. -- Exploding for six runs in the bottom of the fifth inning, the No. 2-ranked University of Michigan softball team rolled to an 8-0, five-inning decision against intrastate rival Central Michigan on Wednesday evening (April 14) in front of 1,309 fans at the Wilpon Softball Complex, home of Alumni Field. The run-rule decision was the Wolverines' fourth in a row and sixth in the last seven games.
Freshman RHP Stephanie Speierman (Columbia, Md./Hammond HS) went the distance in her first collegiate start, striking out three Chippewa batters while allowing just one hit and a walk. She improved to 2-0 on the season.
Michigan produced just four hits -- its lowest tally in 10 games -- but accepted a season-high nine walks to key its offensive surges. Junior first baseman Dorian Shaw (Burke, Va./Robinson SS) extended her career-long hitting streak to 10 games with her 14th home run of the season, headlining the Wolverines' big fifth inning with a three-run bomb. Senior shortstop Molly Bausher (Las Vegas, Nev./Spring Valley HS) capped the frame with a game-ending, two-run double.
After CMU pitcher Kari Seddon walked the bases loaded in the first inning, sophomore second baseman Amanda Chidester (Allen Park, Mich./Cabrini HS) provided Michigan the only advantage it needed, shrugging off a two-game hitless streak with a two-run liner back up the middle.
A one-out walk spoiled Speierman's perfect-game bid in the fourth and the Wolverine rookie lost her no-hitter in the top of the fifth when second baseman Brittani Merchant sent a one-hopper down the third baseline. Senior third baseman Maggie Viefhaus (Eureka, Mo./Eureka HS) stretched to reel it in but could not make the throw to first in time.
Senior catcher Roya St. Clair (Livonia, Mich./Stevenson HS) initiated the Wolverines' fifth-inning attack with one out and runners on first and second, roping a line-drive RBI double to leftfield. The CMU outfielder misjudged the ball off the bat, taking several steps forward as the ball zipped past her to the wall and allowed sophomore centerfielder Bree Evans (Bloomington, Calif./Rialto HS) to score from second base.
Shaw connected with the 2-1 pitch in the next at-bat, clanging her three-run blast off the top of the new scoreboard -- down the leftfield line -- for her second home run in as many games. The Chippewas followed with three consecutive walks to load the bases for Bausher, who doubled the first pitch off the wall in right centerfield to push U-M into run-rule territory and bring an immediate end to the game.
The Wolverines will resume Big Ten Conference play this weekend, hosting rival Northwestern for a two-game series at the Wilpon Softball Complex, home of Alumni Field. U-M will face the Wildcats at 6 p.m. on Saturday (April 17) and 3 p.m. on Sunday (April 18); both games will be aired live on the Big Ten Network.
Contact: Leah Howard (734) 763-4423
Sign up for Michigan Insider to be the first to learn about 2018 Michigan softball ticket opportunities.


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1999 passat gls 1.8t(aeb/k03)
so for some reason my car isnt boosting, peak at like 1-2 in 1st and 2nd and no higher then 3-4psi beyond that.
smoking a bit but not like a blown turbo(prolly due to catless and 1 o2 sensor being run)
It doesnt sound like a blown turbo or act like it, but im curious what the problem is? dv? N75?


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Wrinkle Cream--Is there any brand that actually works?
I'm 25 and I've just started noticing that I have wrinkles! :shock: I never thought it would happen to me. :sad:sad20gif
Is there any product out there that actually reduces wrinkles? I see tons of ads for these "miracle" products but I am very skeptical. I'm not willing to hand over the cash unless I'm guaranteed results!
Also, does anyone know of a good under-eye cream? I have dark circles and my skin there isn't the best.
Thanks girlies!
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Modern bouquet of vanda and cymbidium orchids with ti leaf and lily grass collar
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