Joseph Gallagher

Seasonal variation in Laminaria digitata and its impact on biochemical conversion routes to biofuels.

Laminaria digitata is a highly prevalent kelp growing off the coast of the UK but has rarely been considered as a source of biomass to date. This study shows it can be used as a feedstock in both ethanol fermentation and anaerobic digestion for methane production. The study optimised several parameters in the fermentation of L. digitata and investigated the suitability of the macroalgae through the year using samples harvested every month. For both methane and ethanol production, minimum yields were seen in material harvested in March when the carbohydrates laminarin and mannitol were lowest. July material contained the highest combined laminarin and mannitol content and maximum yields of 167 mL ethanol and 0.219 m(3) kg(-1)L. digitata.

Sucrose and the integration of metabolism in vascular plants

We consider the hypothesis that sucrose is a signal as well as a substrate. We suggest that the significance of sugar sensing in plants is the integration of whole-plant carbon flux so that the capacity of sources to produce sucrose matches the capacity of sinks to consume it. We pay particular attention to difficulties with this hypothesis and the areas where further or better evidence is needed. We conclude that there is strong correlative evidence for a link between sucrose metabolism and the level of expression of key genes, but that a number of different mechanisms may be involved.

Association of Candidate Genes With Flowering Time and Water-Soluble Carbohydrate Content in Lolium perenne (L.)

We describe a candidate gene approach for associating SNPs with variation in flowering time and water-soluble carbohydrate (WSC) content and other quality traits in the temperate forage grass species Lolium perenne. Three analysis methods were used, which took the significant population structure into account. First, a linear mixed model was used enabling a structured association analysis to be incorporated with the nine populations identified in the structure analysis as random variables. Second, a within-population analysis of variance was performed. Third, a tree-scanning method was used, in which haplotype trees were associated with phenotypes on the basis of inferred haplotypes. Analysis of variance within populations identified several associations between WSC, nitrogen (N), and dry matter digestibility with allelic variants within an alkaline invertase candidate gene LpcAI. These associations were only detected in material harvested in one of the two years. By contrast, consistent associations between the L. perenne homolog (LpHD1) of the rice photoperiod control gene HD1 and flowering time were identified. One SNP, in the immediate upstream region of the LpHD1 coding sequence (C-4443-A), was significant in the linear mixed model. Within-population analysis of variance and tree-scanning analysis confirmed and extended this result to the 2118 polymorphisms in some of the populations. The merits of the tree-scanning method are compared to the single SNP analysis. The potential usefulness of the 4443 SNP in marker-assisted selection is currently being evaluated in test crosses of genotypes from this work with turf-grass varieties.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6  h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6  hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6  hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6  hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

Rubisco Small Subunit, Chlorophyll a/b-Binding Protein and Sucrose:Fructan-6-Fructosyl Transferase Gene Expression and Sugar Status in Single Barley Leaf Cells in Situ. Cell Type Specificity and Induction by Light

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA.Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab,RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT,Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.

Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli

SummaryA synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n=1) to six (n=6) gene copies. The gene, placed in pλ-ncyt under the control of the λPL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5. The expressed levels of cytochrome b5 in Escherichia coli pλ-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing pλ-6cyt produced 75 μg cytochrome b5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the halocytochrome b5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E. coli.

Ryegrass leaf fructan synthesis is oxygen dependent and abolished by endomembrane inhibitors

Valid models are the foundation of systems biology. However, even well-established models may warrant reassessment. A testable feature of the currently accepted vacuolar model for fructan biosynthesis is its independence from metabolic energy at substrate level. The effects of limiting energy provision on fructan biosynthesis in grass leaves were determined. It was found that, in darkness in air, the rate of fructan accumulation was reduced to half relative to a light control. In darkness under anoxia the process was immediately abolished. In the light, the leaf sucrose concentration remained high, but in darkness +/- O(2), 40% of this sucrose was rapidly degraded. The constant rate of dark-aerobic fructan accumulation was independent of the decrease in sucrose concentration. Constant rates of aerobic fructan synthesis were independent of marked changes in extractable polymerase rates. In the dark under anoxia, fructan accumulation was abolished but leaves maintained > or = 80% of the extractable polymerase. Extractable polymerase rates cannot explain the rates of fructan accumulation observed in vivo, if the process is vacuolar. It was shown that the results were inconsistent with a vacuolar site for fructan synthesis. Six inhibitors of endomembrane function were shown to abolish fructan synthesis in vivo.

Optimisation of slow-pyrolysis process conditions to maximise char yield and heavy metal adsorption of biochar produced from different feedstocks

The objective of this work was to identify biomass feedstocks and optimum pyrolysis process conditions to produce a biochar capable of adsorbing metals from polluted groundwater. Taguchi experimental design was used to determine the effects of slow-pyrolysis process conditions on char yield and zinc adsorption. Treatments were repeated using six candidate feedstocks (Lolium perenne, Lolium perenne fibre, Miscanthus x giganteus, Salix viminalis, Fraxinus excelsior and Picea sitchensis) and the resultant chars were tested for metal adsorption performance. Chars produced from L. perenne and its extracted fibre displayed the greatest zinc adsorption performance and removed 83.27-92.96% respectively. Optimum process conditions in terms of both char yield and zinc adsorption performance were achieved from slow-pyrolysis at 300°C for 2h using a feedstock with a particle size of less than 1mm.

Overview on Commercial Production of Xylitol, Economic Analysis and Market Trends

The interest in xylitol has increased considerably in recent years, due to many commercial applications in different industrial sectors like food, dental related products, and pharmaceuticals. As industrial biotechnological routes to xylitol are costly they currently represents a small fraction of the marketshare. Therefore, over the past few decades much effort has been devoted to the development of cost-effective and environmentally-friendly biotechnological processes by evaluating cheaper lignocellulosic substrates. In this chapter, xylitol commercial processes, cost and market trends are discussed with a special focus on biorefining and biotechnological methods. Increasing commercial and scientific interest in xylitol has led to a strong demand for this product in the global market, of more than 125,000 tons per anum, with a value that is relatively high (4.5–5.5