Grape and wine analysis: Oenologists to exploit advanced test kits.
Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.
It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.
Megazyme “advanced” wine test kits general characteristics and validation.
Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.
Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.
Microbial bioanodes with high salinity tolerance for microbial fuel cells and microbial electrolysis cells.
Rousseau, R., Dominguez-Benetton, X., Délia, M. L. & Bergel, A. (2013). Electrochemistry Communications, 33, 1-4.
Increasing the conductivity of the electrolytes used in microbial electrochemical systems is an essential prerequisite to the large-scale success of these technologies. Microbial bioanodes formed from a salt marsh inoculum under constant acetate feeding generated up to 85 A•m-2 in media containing 776 mM NaCl (45 g•L-1, 1.5 times the salinity of seawater). These values were the highest salinities accepted by a microbial anode so far and the highest current densities reported with felt graphite electrodes.
The effect of pathogen reduction technology (Mirasol) on platelet quality when treated in additive solution with low plasma carryover.
Johnson, L., Winter, K. M., Reid, S., Hartkopf‐Theis, T., Marschner, S., Goodrich, R. P. & Marks, D. C. (2011). Vox sanguinis, 101(3), 208-214.
Background and Objectives: Pathogen reduction technologies (PRT) for platelets are now compatible with both plasma and platelet additive solutions (PAS). The aim of this study was to examine the effect of PRT on the platelet storage lesion, in the presence of PAS with low plasma carryover. Materials and Methods: PRT-treated (Mirasol) and untreated buffy coat-derived platelet concentrates prepared in 28% plasma/PAS-IIIM were evaluated using in vitro cell quality parameters on days 1, 2, 5, and 7 post-collection. Results: At day 5, there were no significant differences between control and PRT treated platelets for swirl, viability, pO2, pCO2, mean platelet volume and adenosine diphosphate-induced aggregation. PRT treatment did not affect the functional integrity of the mitochondria. However, PRT resulted in a decrease in pH and enhancement of platelet glycolysis and activation, evidenced by increased glucose consumption and lactate production rates, increased expression of CD62P, CD63, annexin V staining and increased secretion of cytokines (P < 0•05). Hypotonic shock response and aggregation in response to collagen were also significantly reduced in PRT treated platelets (P < 0•05). Conclusion: Despite the observed differences in platelet metabolism and activation observed following PRT treatment in PAS and low plasma carryover, the results suggest that treatment and storage of platelets in PAS is no more detrimental to platelets than treatment and storage in plasma.
In vitro assessment of buffy‐coat derived platelet components suspended in SSP+ treated with the INTERCEPT Blood system.
Johnson, L., Loh, Y. S., Kwok, M. & Marks, D. C. (2013). Transfusion Medicine, 23(2), 121-129.
Background: The INTERCEPT Blood System uses amotosalen-HCl and UVA light to cross-link DNA and RNA, thereby inhibiting pathogen replication. Although previous studies have shown that this treatment alters in vitro platelet quality, most studies have assessed apheresis platelets or platelets pooled from 5 or 6 donors. In Australia, platelets are prepared using buffy-coats from 4 donors, with SSP+ and have lower plasma carryover than recommended by the manufacturer (32–47%). As such, it is currently unknown whether these platelet concentrates are suitable for INTERCEPT treatment. Materials and methods: Platelet concentrates were prepared by pooling four buffy-coats with SSP+, resulting in 30% plasma carryover. Two platelet units were pooled and split to generate paired units, with one unit treated with the INTERCEPT System (n = 6), whilst the other remained untreated (n = 6). All units were stored for seven days at 22°C with agitation. Results: INTERCEPT treatment resulted in 10•4 ± 4•3% loss of platelets, but did not significantly affect the functional integrity of mitochondria. INTERCEPT-treated platelets demonstrated a decreased pH, accelerated lactate production and glucose consumption, as well as higher surface expression and increased secretion of P-selectin and reduced collagen-induced aggregation. These changes were particularly evident from day 5 of storage. Conclusion: The observed increase in platelet glycolysis following INTERCEPT treatment is consistent with previous literature reports. Importantly, the in vitro changes were less marked than previously reported indicating that the platelets suspended in SSP+ with reduced plasma carryover are of suitable in vitro quality following INTERCEPT treatment and storage.
Epistasis and frequency dependence influence the fitness of an adaptive mutation in a diversifying lineage.
Le Gac, M. & Doebeli, M. (2010). Molecular Ecology, 19(12), 2430-2438.
The opportunity for a mutation to invade a population can dramatically vary depending on the context in which this mutation occurs. Such context dependence is difficult to document as it requires the ability to measure how a mutation affects phenotypes and fitness and to manipulate the context in which the mutation occurs. We identified a mutation in a gene encoding a global regulator in one of two ecotypes that diverged from a common ancestor during 1200 generations of experimental evolution. We replaced the ancestral allele by the mutant allele, and vice versa, in several clones isolated during the time course of the evolution experiment, and compared the phenotype and fitness of clones isogenic except for the focal mutation. We show that the fitness and phenotype of the mutation are strongly affected by epistatic interactions between genes in the same genome, as well as by frequency dependent selection resulting from biotic interactions between individuals in the same population. We conclude that amongst the replicate population in which it spread, the mutation we identified is only adaptive when occurring in specific genomes and competing with specific individuals. This study thus demonstrates that the opportunity for an adaptive mutation to spread in an evolutionary lineage can only be understood in the light of its genomic and competitive environments.
Production of functionalized biopolyester granules by recombinant Lactococcus lactis.
Mifune, J., Grage, K. & Rehm, B. H. A. (2009). Applied and Environmental Microbiology, 75(14), 4668-4675.
Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction.
Gou, J. Y., Miller, L. M., Hou, G., Yu, X. H., Chen, X. Y. & Liu, C. J. (2012). The Plant Cell, 24(1), 50-65.
Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggests that pectin acetylesterase functions as an important structural regulator in plants by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.
Garden compost inoculum leads to microbial bioanodes with potential-independent characteristics.
Cercado, B., Byrne, N., Bertrand, M., Pocaznoi, D., Rimboud, M., Achouak, W. & Bergel, A. (2013). Bioresource Technology, 134, 276-284.
Garden compost leachate was used to form microbial bioanodes under polarization at −0.4, −0.2 and +0.1 V/SCE. Current densities were 6.3 and 8.9 A m-2 on average at −0.4 and +0.1 V/SCE respectively, with acetate 10 mM. The catalytic cyclic voltammetry (CV) showed similar electrochemical characteristics for all bioanodes and indicated that the lower currents recorded at −0.4 V/SCE were due to the slower interfacial electron transfer rate at this potential, consistently with conventional electrochemical kinetics. RNA- and DNA-based DGGE evidenced that the three dominant bacterial groups Geobacter, Anaerophaga and Pelobacter were identical for all bioanodes and did not depend on the polarization potential. Only non-turnover CVs showed differences in the redox equipment of the biofilms, the highest potential promoting multiple electron transfer pathways. This first description of a potential-independent electroactive microbial community opens up promising prospects for the design of stable bioanodes for microbial fuel cells.
Combined intracellular nitrate and NIT2 effects on storage carbohydrate metabolism in Chlamydomonas.
Remacle, C., Eppe, G., Coosemans, N., Fernandez, E. & Vigeolas, H. (2014). Journal of Experimental Botany, 65(1), 23-33.
Microalgae are receiving increasing attention as alternative production systems for renewable energy such as biofuel. The photosynthetic alga Chlamydomonas reinhardtii is widely recognized as the model system to study all aspects of algal physiology, including the molecular mechanisms underlying the accumulation of starch and triacylglycerol (TAG), which are the precursors of biofuel. All of these pathways not only require a carbon (C) supply but also are strongly dependent on a source of nitrogen (N) to sustain optimal growth rate and biomass production. In order to gain a better understanding of the regulation of C and N metabolisms and the accumulation of storage carbohydrates, the effect of different N sources (NH4NO3 and NH4+) on primary metabolism using various mutants impaired in either NIA1, NIT2 or both loci was performed by metabolic analyses. The data demonstrated that, using NH4NO3, nia1 strain displayed the most striking phenotype, including an inhibition of growth, accumulation of intracellular nitrate, and strong starch and TAG accumulation. The measurements of the different C and N intermediate levels (amino, organic, and fatty acids), together with the determination of acetate and NH4+ remaining in the medium, clearly excluded the hypothesis of a slower NH4+ and acetate assimilation in this mutant in the presence of NH4NO3. The results provide evidence of the implication of intracellular nitrate and NIT2 in the control of C partitioning into different storage carbohydrates under mixotrophic conditions in Chlamydomonas. The underlying mechanisms and implications for strategies to increase biomass yield and storage product composition in oleaginous algae are discussed.
Independent Benthic Microbial Fuel Cells Powering Sensors and Acoustic Communications with the MARS Underwater Observatory.
Schrader, P. S., Reimers, C. E., Girguis, P., Delaney, J., Doolan, C., Wolf, M. & Green, D. (2016). Journal of Atmospheric and Oceanic Technology, 33(3), 607-617.
Most oceanographic instruments on the seafloor have no connections with the surface and therefore have to run on batteries and store data until recovery. To demonstrate a developing technology, sensors and acoustic modems were powered with energy harvested from the seafloor, and data were relayed acoustically in near–real time to the Monterey Accelerated Research System (MARS) observatory in Monterey Bay, California, and to surface research vessels. MARS is a cabled observatory in deep water (~890 m) at the edge of Monterey Canyon. An acoustic modem was attached to the MARS node and configured to send out commands to, and relay data received from, remote modems. Two benthic microbial fuel cells (BMFCs) positioned approximately 0.5 km away from MARS supplied power to the remote modems and sensors. At their peak performance, these BMFCs produced continuous power densities of ~35 mW m-2 (footprint area). The modems utilized in this study contained an integrated power management platform (PMP) designed to manage and store the electrical energy generated by each BMFC and to record BMFC performance parameters and sensor data on an hourly basis. Temperature and either oxygen or conductivity sensors were chosen because of their common use and environmental relevance. Acoustically transmitted data records show that the BMFCs renewed energy stores and that the oceanographic sensors measured dissolved oxygen, temperature, and conductivity reliably throughout the operational life of each BMFC system (~6 months). These systems remained in place for more than 12 months.
Halotolerant bioanodes: The applied potential modulates the electrochemical characteristics, the biofilm structure and the ratio of the two dominant genera.
Rousseau, R., Santaella, C., Bonnafous, A., Achouak, W., Godon, J. J., Delia, M. L. & Bergel, A. (2016). Bioelectrochemistry, 112, 24-32.
The development of economically-efficient microbial electrochemical technologies remains hindered by the low ionic conductivity of the culture media used as the electrolyte. To overcome this drawback, halotolerant bioanodes were designed with salt marsh sediment used as the inoculum in electrolytes containing NaCl at 30 or 45 g/L (ionic conductivity 7.0 or 10.4 S·m-1). The bioanodes were formed at four different potentials − 0.4, − 0.2, 0.0 and 0.2 V/SCE to identify the effect on the electrochemical kinetic parameters, the biofilm structures and the composition of the microbial communities. The bioanodes formed at − 0.4 V/SCE were largely dominated by Marinobacter spp. Voltammetry showed that they provided higher currents than the other bioanodes in the range of low potentials, but the maximum currents were limited by the poor surface colonization. The bioanodes formed at − 0.2, 0.0 and 0.2 V/SCE showed similar ratios of Marinobacter and Desulfuromonas spp. and higher values of the maximum current density. The combined analysis of kinetic parameters, biofilm structure and biofilm composition showed that Marinobacter spp., which ensured a higher electron transfer rate, were promising species for the design of halotolerant bioanodes. The challenge is now to overcome its limited surface colonization in the absence of Desulfuromonas spp.
Novel role for carbohydrate responsive element binding protein in the control of ethanol metabolism and susceptibility to binge drinking.
Marmier, S., Dentin, R., Daujat‐Chavanieu, M., Guillou, H., Bertrand‐Michel, J., Gerbal‐Chaloin, S., Girard, J., Lotersztajn, S. & Postic, C. (2015). Hepatology, 62(4), 1086-1100.
Carbohydrate responsive element binding protein (ChREBP) is central for de novo fatty acid synthesis under physiological conditions and in the context of nonalcoholic fatty liver disease. We explored its contribution to alcohol-induced steatosis in a mouse model of binge drinking as acute ethanol (EtOH) intoxication has become an alarming health problem. Within 6 hours, ChREBP acetylation and its recruitment onto target gene promoters were increased in liver of EtOH-fed mice. Acetylation of ChREBP was dependent on alcohol metabolism because inhibition of alcohol dehydrogenase (ADH) activity blunted ChREBP EtOH-induced acetylation in mouse hepatocytes. Transfection of an acetylation-defective mutant of ChREBP (ChREBPK672A) in HepG2 cells impaired the stimulatory effect of EtOH on ChREBP activity. Importantly, ChREBP silencing in the liver of EtOH-fed mice prevented alcohol-induced triglyceride accumulation through an inhibition of the lipogenic pathway but also led, unexpectedly, to hypothermia, increased blood acetaldehyde concentrations, and enhanced lethality. This phenotype was associated with impaired hepatic EtOH metabolism as a consequence of reduced ADH activity. While the expression and activity of the NAD+ dependent deacetylase sirtuin 1, a ChREBP-negative target, were down-regulated in the liver of alcohol-fed mice, they were restored to control levels upon ChREBP silencing. In turn, ADH acetylation was reduced, suggesting that ChREBP regulates EtOH metabolism and ADH activity through its direct control of sirtuin 1 expression. Indeed, when sirtuin 1 activity was rescued by resveratrol pretreatment in EtOH-treated hepatocytes, a significant decrease in ADH protein content and/or acetylation was observed. Conclusion: our study describes a novel role for ChREBP in EtOH metabolism and unravels its protective effect against severe intoxication in response to binge drinking.
Comparison of synthetic medium and wastewater used as dilution medium to design scalable microbial anodes: application to food waste treatment.
Blanchet, E., Desmond, E., Erable, B., Bridier, A., Bouchez, T. & Bergel, A. (2015). Bioresource Technology, 185, 106-115.
The objective was to replace synthetic medium by wastewater as a strategy to design low-cost scalable bioanodes. The addition of activated sludge was necessary to form primary bioanodes that were then used as the inoculum to form the secondary bioanodes. Bioanodes formed in synthetic medium with acetate 10 mM provided current densities of 21.9 ± 2.1 A/m2, while bioanodes formed in wastewater gave 10.3 ± 0.1 A/m2. The difference was explained in terms of biofilm structure, electrochemical kinetics and redox charge content of the biofilms. In both media, current densities were straightforwardly correlated with the biofilm enrichment in Geobacteraceae but, inside this family, Geobacter sulfurreducens and an uncultured Geobacter sp. were dominant in the synthetic medium, while growth of another Geobacter sp. was favoured in wastewater. Finally, the primary/secondary procedure succeeded in designing bioanodes to treat food wastes by using wastewater as dilution medium, with current densities of 7 ± 1.1 A/m2.
Influence of the electrode size on microbial anode performance.
Oliot, M., Chong, P., Erable, B. & Bergel, A. (2017). Chemical Engineering Journal, In press.
The performance of microbial fuel cells and other related microbial electrochemical processes is seen to deteriorate severely when they are scaled up. This crucial problem is addressed here by comparing the kinetics of microbial anodes with projected surface areas of 9 and 50 cm2 under well-controlled electrochemical conditions. The microbial anode kinetics were characterized by low scan rate voltammetry. The 9-cm2 anodes showed Nernstian behaviour, while the 50-cm2 anodes showed significantly lower performance. The distribution of the electrostatic potential in the experimental set-up was modelled numerically. The model predicted the general trend of the voltammetry curves recorded with the 50-cm2 anodes well, showing that part of the performance deterioration was due to ohmic drop and to non-uniformity of the local potential over the anode surface. Furthermore, the biofilm presented slightly different electrochemical characteristics when grown on the 9-cm2 or 50-cm2 anodes, and the difference in local potential over the 50-cm2 anodes induced spatial heterogeneity in biofilm development. The effect of local potential on biofilm characteristics was an additional cause of the lower performance obtained with the 50-cm2 anodes. In the current state of the art, the soundest way to design large-sized microbial anodes is to adopt the dual main aim of minimizing the ohmic drop while keeping the most uniform possible potential over the electrode surface. Modelling potential distribution inside the reactor should make an essential contribution to this.