BACKGROUND AND AIMS:Lignin and other phenolics are commonly observed at the interfaces between the haustoria of parasitic plants and tissues of their hosts. As known plant defence compounds, their accumulation at haustorial interfaces has been ascribed to mechanical and chemical resistance of host tissues. Although the possibility that the interfacial lignin deposits may have a parasitic origin has not previously been addressed, the fact that certain parasitic plants, including Rhinanthus and Odontites, can form haustoria in the absence of hosts gives us a tool that can be used to help answer this question. METHODS:We compared the interfaces of haustoria of root hemiparasites yellow rattle Rhinanthus minor and red bartsia Odontites vernus (Orobanchaceae) attached to hosts bulbous oat-grass Arrhenatherum elatius ssp. bulbosum and perennial rye grass Lolium perenne (Poaceae) with the contact surfaces of non-infective prehaustoria attached to a pot surface. We performed histochemistry, immunocytochemistry and Raman spectroscopy to characterize the architecture of contact deposits formed by both. KEY RESULTS:Lignolic deposits, which we will term lignin-rich interfacial deposits (LIDs), were found at the interfaces between haustoria and compatible hosts as well as at the pot-appressed facets of prehaustoria. In both cases the deposits were determined as lignin by histology and Raman spectroscopy. Xyloglucan and arabinogalactan protein glycan epitopes were also detected while mixed-linkage glucan, xylans and pectin were not. We demonstrate that prehaustoria can produce lignolic interfacial deposits of high structural similarity to those of haustorium-host interfaces. CONCLUSIONS:LIDs at haustorium-host interfaces may at least partly be attributed to the parasite and benefit the establishment and functioning of the haustorium. A reinterpretation of the origin and role of interfacial lignin in parasitic plant-host interactions may therefore be necessary.

AIMS:To identify novel brown coal-degrading bacteria, and elucidate the biochemical pathways involved in brown coal degradation. METHODS AND RESULTS:Four bacterial isolates were identified from the surface of Indonesian brown coal, which can utilize naphthalene and 1-methylnaphthalene as carbon sources for growth. The genome sequence of the best-performing Micrococcus luteus K1 strain was determined. A novel aromatic degradation gene cluster was identified, containing several paa genes normally involved in phenylacetic acid degradation, and also containing genes found on aromatic meta-cleavage pathways. 1-Naphthoic acid was generated from 1-methylnaphthalene by M. luteus K1 whole cell biotransformation, and was also utilized as a growth substrate by M. luteus K1. Recombinant ligase PaaK from the new gene cluster was shown to activate either phenylacetic acid or 1-naphthoic acid to their respective CoA esters, consistent with 1-naphthoyl CoA being an intermediate on the pathway. From metabolite analysis and annotation of the gene cluster, a new 1-methylnaphthalene degradation pathway was proposed, via a benzene oxide-oxepin ring opening. Recombinant mono-oxygenase and extradiol catechol dioxygenase enzymes from the gene cluster were expressed, showing activities consistent with the later steps of the proposed pathway. CONCLUSIONS:A new M. luteus K1 isolate was identified as a brown coal degrader, whose genome contains an unusual aromatic degradation cluster containing paa genes. This cluster is hypothesized to be responsible for 1-methylnaphthalene degradation.

Miscanthus is a promising crop for bioenergy and biorefining in Europe. The improvement of Miscanthus as a crop relies on the creation of new varieties through the hybridization of germplasm collected in the wild with genetic variation and suitable characteristics in terms of resilience, yield and quality of the biomass. Local adaptation has likely shaped genetic variation for these characteristics and is therefore important to quantify. A key biomass quality parameter for biorefining is the ease of conversion of cell wall polysaccharides to monomeric sugars. Thus far, the variability of cell wall related traits in Miscanthus has mostly been explored in accessions from limited genetic backgrounds. Here we analysed the soil and climatic conditions of the original collection sites of 592 Miscanthus genotypes, which form eight distinct genetic groups based on discriminant analysis of principal components of 25,014 single-nucleotide polymorphisms. Our results show that species of the genus Miscanthus grow naturally across a range of soil and climate conditions. Based on a detailed analysis of 49 representative genotypes, we report generally minor differences in cell wall characteristics between different genetic groups and high levels of genetic variation within groups, with less investigated species like M. floridulus showing lower recalcitrance compared to the other genetic groups. The results emphasize that both inter- and intra- specific variation in cell wall characteristics and biomass recalcitrance can be used effectively in Miscanthus breeding programmes, while also reinforcing the importance of considering biomass yield when quantifying overall conversion efficiency. Thus, in addition to reflecting the complexity of the interactions between compositional and structural cell wall features and cell wall recalcitrance to sugar release, our results point to traits that could potentially require attention in breeding programmes targeted at improving the Miscanthus biomass crop.

Myxobacteria produce a variety of bioactive secondary metabolites, and with a wealth of under-researched species they hold vast potential for undiscovered compounds. With the ever-increasing need for new antibiotics, the development of novel therapeutics is vitally important. Therefore, this study aimed to extract and elucidate antimicrobial metabolites from the following myxobacteria: Myxococcus xanthus CA010 and AB022; Corallococcus exiguus DSM14696T; Myxococcus stipitatus DSM14675T; Corallococcus aberystwythensis AB050AT. Metabolite mixtures were extracted in acetone from XAD-16 resin incubated in liquid cultures and analysed using GC-MS. Bioactivity was identified using a growth inhibition assay against a panel of clinically relevant prey species including Gram-positive and Gram-negative bacteria and a fungus. The growth of Klebsiella pneumoniae and Enterococcus faecalis was most affected by the metabolite mixtures and the mixtures from AB022 and AB050AT were effective against the most prey. GC-MS analysis revealed metabolites with roles in synthesis and degradation of amino acids and fatty acids, but also identified compounds A and B with a diketopiperazine (DKP) core. With previously confirmed bioactivity of compound A, it is suggested that these DKP compounds are contributing to the antimicrobial activity observed. Furthermore, many compounds could not be identified and so these unknowns present further potential for novel bioactive compounds.

Attenuated total reflectance Fourier transform mid-infrared (ATR-FTIR) spectroscopy is widely applicable for the chemical analysis of biological materials, relatively inexpensive, requires only simple sample preparation, and is of comparatively high-throughput compared to traditional wet chemical or chromatographic methods. It is particularly well suited for the nondestructive analysis of dried and finely ground plant samples for the subsequent prediction of cell wall and other compositional or processing parameters using chemometric regression models. Furthermore, analysis of mid IR spectra by nonregression methods (e.g., principal component analysis) provides a straightforward approach for multivariate comparison of the effects of experimental, processing, and environmental treatments, and genotypic and temporal differences on chemical composition including changes in cell wall composition. There is thus great potential for using ATR-FTIR in the lignocellulosic biomass industry at a number of levels. Here we describe methods for cell wall sample preparation and generation of ATR-FTIR spectra, and suggest techniques for the statistical analysis and/or chemometric pattern recognition between the analyzed samples.

Invasive plants depositing recalcitrant, polyphenol-rich leaf litter may alter decomposition dynamics, leading to an accumulation of soil organic matter. Removing invasives is critical in restoring native habitats, but our understanding of its impacts upon soil processes remains limited. Here, we test the hypothesis that clearing of Rhododendron ponticum leads to increased soil respiration, at a site within Snowdonia National Park, Wales, UK. Soil samples were collected, and soil respiration was monitored over 32 weeks on plots cleared of R. ponticum, plots infested with R. ponticum which were left uncleared, and uninvaded plots of native vegetation. Soil respiration was significantly higher in native vegetation plots, relative to uncleared plots. Clearing R. ponticum led to a short-term (< four weeks) increase in soil respiration relative to uncleared plots and was related to elevated soil temperature post-clearance. However, this respiration response was transient, with no significant difference between cleared and uncleared plots over the whole growing season (32 weeks). Declining soil respiration responses to soil warming have been attributed to altered soil microbial communities and substrate limitation. Analysis of microbial phospholipid fatty acids (PLFAs) detected no differences among cleared, native and uncleared plots post-clearance. However, Fourier-transform mid-infrared spectroscopy detected a decline in organic matter aromaticity over the growing season in the native and uncleared plots, whilst there was no change in the cleared plots. The findings show that despite a pulse in soil respiration during the initial four weeks post-clearance, R. ponticum litter and associated soil organic matter in cleared plots continued to decompose at a similar rate to uncleared plots over the whole growing season. This was likely a result of substrate limitation and altered soil organic matter composition following R. ponticum clearing, with labile carbon becoming depleted and an enrichment of more recalcitrant aromatic structures.

The study reports on the effect of anaerobic digestate derived composts on the metabolite composition and thermal behaviour of rosemary (Rosmarinus officinalis L.). Plants were cultivated in semiarid soil under four different fertiliser treatments (composts of anaerobic digested cattle (C) or pig slurry (P) at 30t/ha and 60 t/ha, and two control treatments (inorganic fertiliser and no fertiliser application). Samples of leaves and stems were analysed to investigate the effect of treatment on chemical composition and thermochemical properties. Three orthogonal analytical approaches were used, namely: Fourier transform mid infrared spectroscopy (FTIR), gas chromatography/mass spectrometry (GC/MS) and thermochemical gravimetric analysis (TGA). FTIR and GC/MS showed fertiliser treatment resulted in tissue specific changes in sample metabolite composition. Fertiliser treatment was detected to change the thermogravimetric properties of the leaf samples and from inorganic and composted pig slurry digestate treatments had greater ash content and lower proportions of fixed carbon compared with samples from the unfertilised control treatment. This study provides information on how the composition of rosemary might be altered by fertiliser application in regions of poor soil, and has implications for biomass quality when rosemary is grown on semi-wild sites for the purpose of soil improvement.

Non-food biomass crops e.g. switchgrass (Panicum virgatum L.), Miscanthus x giganteus, and short-rotation coppice poplar (Poplus spp.) and willow (Salix spp.) offer a sustainable source of energy and platform chemicals (Sims et al., 2006). The majority of the energy stored in the crop biomass is in the cell wall which constitutes the largest fraction of lignocellulosic biomass. The three polymers that constitute the bulk of plant cell wall (cellulose, hemicellulose and lignin) rank amongst the most abundant biopolymers on the planet and their proportional concentrations range generally between 40 – 50%, 10 – 40% and 5 – 30% of biomass by weight respectively (McKendry, 2002). The absolute and relative concentrations of the components of the cell wall have a great influence on biomass quality i.e. its suitability for conversion to heat, power and chemical products. However, because biomass can be utilised by a number of conversion routes with differing feedstock demands measures of feed-stock quality are often quite specific to how the material is to be used. For example, biomass can be processed thermochemically. These routes include combustion or cocombustion with coal to generate heat and electricity (Allison et al., 2010). Alternatively, biomass can be converted by fast pyrolysis to bio-char, which is receiving much attention as a soil improver and a means of sequestering carbon from the atmosphere into the soil (Laird, 2008; Woolf et al., 2010), and bio-oil, a liquid fuel (Bridgwater, 2003; Mohan et al., 2006). Biomass can also be gasified to produce a combustible gas which has application for the generation of heat and power, and for the chemical synthesis of liquid transport fuels and industrial chemicals (Ptasinski et al., 2007). These thermochemical processes demand feedstocks with low moisture content and high energy density, often equating with high levels of the poly aromatic polymer, lignin. In contrast, non-thermochemical processes e.g. the production of bioethanol and industrial platform chemicals by biological conversion processes are often inhibited by high levels of lignin. High concentrations of lignin in the feedstock necessitate harsh chemical and heat pre-treatments of the biomass prior to enzymic saccharification. This increases energy inputs and often damages the polysachharide components of the cell wall giving rise to inhibitory products (Carroll and Somerville, 2009; Chang, 2007; Fahmi et al., 2007; Fahmi et al., 2008; Grabber, 2005). There is therefore considerable pressure to optimise feedstock composition and at present the most feasible way to achieve this at a commercial scale is by breeding

Understanding rumen plant-microbe interactions is central for development of novel methodologies allowing improvements in ruminant nutrient use efficiency. This study investigated rumen bacterial colonization of fresh plant material and changes in plant chemistry over a period of 24 h period using three different fresh forages: Lolium perenne (perennial ryegrass; PRG), Lotus corniculatus (bird's foot trefoil; BFT) and Trifolium pratense (red clover; RC). We show using 16S rRNA gene ion torrent sequencing that plant epiphytic populations present pre-incubation (0 h) were substantially different to those attached post incubations in the presence of rumen fluid on all forages. Thereafter primary and secondary colonization events were evident as defined by changes in relative abundances of attached bacteria and changes in plant chemistry, as assessed using Fourier transform infrared (FTIR) spectroscopy. For PRG colonization, primary colonization occurred for up to 4 h and secondary colonization from 4 h onward. The changes from primary to secondary colonization occurred significantly later with BFT and RC, with primary colonization being up to 6 h and secondary colonization post 6 h of incubation. Across all 3 forages the main colonizing bacteria present at all time points post-incubation were Prevotella, Pseudobutyrivibrio, Ruminococcus, Olsenella, Butyrivibrio, and Anaeroplasma (14.2, 5.4, 1.9, 2.7, 1.8, and 2.0% on average respectively), with Pseudobutyrivibrio and Anaeroplasma having a higher relative abundance during secondary colonization. Using CowPI, we predict differences between bacterial metabolic function during primary and secondary colonization. Specifically, our results infer an increase in carbohydrate metabolism in the bacteria attached during secondary colonization, irrespective of forage type. The CowPI data coupled with the FTIR plant chemistry data suggest that attached bacterial function is similar irrespective of forage type, with the main changes occurring between primary and secondary colonization. These data suggest that the sward composition of pasture may have major implications for the temporal availability of nutrients for animal.

An alkali lignin preparation from wheat straw was found to be compatible with a previously developed nitrated lignin assay for microbial lignin oxidation, allowing comparison of the susceptibility of different wheat lignins to microbial oxidation. Alkali lignin was prepared from a panel of 18 wheat varieties, and was analysed by FT-IR spectroscopy, revealing minor variations in the lignin structure. Samples of nitrated alkali lignin were assayed against five lignin-degrading bacteria: Streptomyces viridosporus, Rhodococcus jostii RHA1, Pseudomonas putida mt-2, Microbacterium phyllosphaerae, and Sphingobacterium sp. T2. Up to 5-fold variation in rates of lignin oxidation were observed between different wheat varieties, and between different bacterial strain, with Sphingobacterium sp. T2 showing consistently highest absorbance changes. Testing of wheat varieties from two consecutive harvests, and from different locations, revealed that the observed variation was due to a combination of genetic, seasonal and environmental factors, but that some wheat varieties showed consistently higher rates of oxidation. Samples of wheat straw treated with Sphingobacterium sp. T2 were analysed by FT-IR spectroscopy after 7 and 14 days' treatment, showing changes in lignin structure versus time, consistent with lignin breakdown.

Waste biomass is generated during the conservation management of semi-natural habitats, and represents an unused resource and potential bioenergy feedstock that does not compete with food production. Thermogravimetric analysis was used to characterise a representative range of biomass generated during conservation management in Wales. Of the biomass types assessed, those dominated by rush (Juncos effuses) and bracken (Pteridium aquilinum) exhibited the highest and lowest volatile compositions respectively and were selected for bench scale conversion via fast pyrolysis. Each biomass type was ensiled and a sub-sample of silage was washed and pressed. Demineralization of conservation biomass through washing and pressing was associated with higher oil yields following fast pyrolysis. The oil yields were within the published range established for the dedicated energy crops miscanthus and willow. In order to examine the potential a multiple output energy system was developed with gross power production estimates following valorisation of the press fluid, char and oil. If used in multi fuel industrial burners the char and oil alone would displace 3.9 x 10(5) tonnes per year of No. 2 light oil using Welsh biomass from conservation management. Bioenergy and product development using these feedstocks could simultaneously support biodiversity management and displace fossil fuels, thereby reducing GHG emissions. Gross power generation predictions show good potential. (C) 2016 Elsevier Ltd. All rights reserved.

ObjectivesDeveloping screening and diagnosis methodologies based on novel biomarkers should allow for the detection of the lung cancer (LC) and possibly at an earlier stage and thereby increase the effectiveness of clinical interventions. Here, our primary objective was to evaluate the potential of spontaneous sputum as a source of non-invasive metabolomic biomarkers for LC status.Materials and methodsSpontaneous sputum was collected and processed from 34 patients with suspected LC, alongside 33 healthy controls. Of the 34 patients, 23 were subsequently diagnosed with LC (LC+, 16 NSCLC, six SCLC, and one radiological diagnosis), at various stages of disease progression. The 67 samples were analysed using flow infusion electrospray ion mass spectrometry (FIE-MS) and gas-chromatography mass spectrometry (GC–MS).ResultsPrincipal component analysis identified negative mode FIE-MS as having the main separating power between samples from healthy and LC. Discriminatory metabolites were identified using ANOVA and Random Forest. Indications of potential diagnostic accuracy involved the use of receiver operating characteristic/area under the curve (ROC/AUC) analyses. This approach identified metabolites changes that were only observed with LC. Metabolites with AUC values of greater than 0.8 which distinguished between LC+/LC− binary classifications where identified and included Ganglioside GM1 which has previously been linked to LC.ConclusionThis study indicates that metabolomics based on sputum can yield metabolites that can be used as a diagnostic and/or discriminator tool. These could aid clinical intervention and targeted diagnosis of LC within an ‘at risk’ LC− population group. The use of sputum as a non-invasive source of metabolite biomarkers may aid in the development of an at-risk population screening programme for lung cancer or enhanced clinical diagnostic pathways.

Understanding the relationship between ingested plant material and the attached microbiome is essential for developing methodologies to improve ruminant nutrient use efficiency. We have previously shown that perennial ryegrass (PRG) rumen bacterial colonization events follow a primary (up to 4 h) and secondary (after 4 h) pattern based on the differences in diversity of the attached bacteria. In this study, we investigated temporal niche specialization of primary and secondary populations of attached rumen microbiota using metagenomic shotgun sequencing as well as monitoring changes in the plant chemistry using mid-infrared spectroscopy (FT-IR). Metagenomic Rapid Annotation using Subsystem Technology (MG-RAST) taxonomical analysis of shotgun metagenomic sequences showed that the genera Butyrivibrio, Clostridium, Eubacterium, Prevotella, and Selenomonas dominated the attached microbiome irrespective of time. MG-RAST also showed that Acidaminococcus, Bacillus, Butyrivibrio, and Prevotella rDNA increased in read abundance during secondary colonization, whilst Blautia decreased in read abundance. MG-RAST Clusters of Orthologous Groups (COG) functional analysis also showed that the primary function of the attached microbiome was categorized broadly within “metabolism;” predominantly amino acid, carbohydrate, and lipid metabolism and transport. Most sequence read abundances (51.6, 43.8, and 50.0% of COG families pertaining to amino acid, carbohydrate and lipid metabolism, respectively) within these categories were higher in abundance during secondary colonization. Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis confirmed that the PRG-attached microbiota present at 1 and 4 h of rumen incubation possess a similar functional capacity, with only a few pathways being uniquely found in only one incubation time point only. FT-IR data for the plant residues also showed that the main changes in plant chemistry between primary and secondary colonization was due to increased carbohydrate, amino acid, and lipid metabolism. This study confirmed primary and secondary colonization events and supported the hypothesis that functional changes occurred as a consequence of taxonomical changes. Sequences within the carbohydrate metabolism COG families contained only 3.2% of cellulose activities, on average across both incubation times (1 and 4 h), suggesting that degradation of the plant cell walls may be a key rate-limiting factor in ensuring the bioavailability of intra-plant nutrients in a timely manner to the microbes and ultimately the animal. This suggests that a future focus for improving ruminant nutrient use efficiency should be altering the recalcitrant plant cell wall components and/or improving the cellulolytic capacity of the rumen microbiota.

Investment in new energy technologies is inadequate relative to the timescale on which greenhouse emissions need to be reduced. This raises questions concerning the policy instruments intended to facilitate the spread of lower carbon energy supply technologies. This paper theorises the role of patent practices and relationships between firms of different size and power, drawing on what little evidence is available in relation to biofuels. We bring firm-level theory of value creation together with a critical perspective of selected innovation theory, to discuss the ways in which patents may be used such that the consequences are contrary to the public good. Considering the implications for clean energy technology transfer, particularly the case of biofuel technology, we conclude that while there is relatively little information on the ways in which patents may hinder clean energy technology transfer, there are certainly sufficient grounds for concern. Keywords: Biofuels, intellectual property, patent strategy, innovation, value creation.

Plant cell wall biomass is an abundant and renewable organic resource. Of the polymers it encloses, cellulose and hemicellulose are regarded as a raw material for the production of fuels and other products (Klemm et al., 2005; Slavov et al., 2013). Nonetheless, current usage of lignocellulosic biomass is still below its full potential due to a series of limiting factors mainly related to the cell wall recalcitrance to saccharification, a severe constraint to maximum biomass usability in downstream processing (Pauly and Keegstra, 2008).As a strategy to optimise bio-energy and bio-refining applications, an increasing amount of effort is being put into the advancement of our knowledge concerning the cell wall compositional roots of recalcitrance. Fourier transform mid-infrared spectroscopy (FTIR) represents a very useful tool on this enterprise, as it allows for a high-throughput, non-destructive and low unit cost procedure for the examination of cell wall biomass (Allison et al., 2009; Carpita and McCann, 2015). Furthermore, the use of Attenuated Total Reflection (ATR) in conjunction with infrared spectroscopy (IR) enables cell wall biomass samples to be examined in solid state without extensive preparation. Nonetheless, the analysis of purified cell wall preparations instead of the intact plant biomass is highly recommended, as it minimises or even eradicates interference from biomass components which are not part of the cell wall. Further information regarding the fundamentals of FTIR may be found elsewhere (Smith, 2011). Datasets generated from FTIR spectroscopy can be extensive and complex. In these situations, data-driven modelling techniques are often used as exploratory approaches to identify the most distinctive features of the collected spectra. Here we suggest the use of Principal Component Analysis (PCA), a frequently employed method to transform a large set of variables into a smaller set of new variables (principal components), effectively reducing dataset dimensionality. When the aim is a complete and detailed biomass characterisation, the FTIR-PCA method here described does not exclude the need for parallel wet gravimetric and analytical procedures. However, it does lead to a rapid identification of the major compositional shifts across large sets of samples; thus contributing to steer research pathways, minimise time-draining analytical procedures and reduce overall research costs.

Background Red clover ( Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing ‘transcript tags’. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.

Slavov, G. T., Nipper, R., Robson, P. R., Farrar, K., Allison, G. G., Bosch, M., ... Jensen, E. F. (2014). Genome-wide association studies and prediction of traits related to phenology, biomass and cell wall composition in Miscanthus sinensis.. W422. Abstract from Plant & Animal Genome XXII Conference, San Diego, United States. RONO: Grant Reference: BBSRC BB/J0042/1 (GS, PR, KF, GA, MB, JCB, ID) BB/K01711X/1 (GS)