The contribution of G‑layer glucose in Salix clones for biofuels: comparative enzymatic and HPLC analysis of stem cross sections

Background Interest on the use of short rotation willow as a lignocellulose resource for liquid transport fuels has increased greatly over the last 10 years. Investigations have shown the advantages and potential of using Salix spp. for such fuels but have also emphasized the wide variations existing in the compositional structure between different species and genotypes in addition to their effects on overall yield. The present work studied the importance of tension wood (TW) as a readily available source of glucose in 2-year-old stems of four Salix clones (Tora, Björn, Jorr, Loden). Studies involved application of a novel approach whereby TW-glucose and residual sugars and lignin were quantified using stem cross sections with results correlated with HPLC analyses of milled wood. Compositional analyses were made for four points along stems and glucose derived from enzyme saccharification of TW gelatinous (G) layers (G-glucose), structural cell wall glucose (CW-glucose) remaining after saccharification and total glucose (T-glucose) determined both theoretically and from HPLC analyses. Comparisons were also made between presence of other characteristic sugars as well as acid-soluble and -insoluble lignin. Results Preliminary studies showed good agreement between using stem serial sections and milled powder from Salix stems for determining total sugar and lignin. Therefore, sections were used throughout the work. HPLC determination of T-glucose in Salix clones varied between 47.1 and 52.8%, showing a trend for higher T-glucose with increasing height (Björn, Tora and Jorr). Using histochemical/microscopy and image analysis, Tora (24.2%) and Björn (28.2%) showed greater volumes of % TW than Jorr (15.5%) and Loden (14.0%). Total G-glucose with enzyme saccharification of TW G-layers varied between 3.7 and 14.7% increasing as the total TW volume increased. CW-glucose measured after enzyme saccharification showed mean values of 41.9–49.1%. Total lignin between and within clones showed small differences with mean variations of 22.4–22.8% before and 22.4–24.3% after enzyme saccharification. Calculated theoretical and quantified values for CW-glucose at different heights for clones were similar with strong correlation: T-glucose = G-glucose + CW-glucose. Pearson’s correlation displayed a strong and positive correlation between T-glucose and G-glucose, % TW and stem height, and between G-glucose with % TW and stem height. Conclusions The use of stem cross sections to estimate TW together with enzyme saccharification represents a viable approach for determining freely available G-glucose from TW allowing comparisons between Salix clones. Using stem sections provides for discrete morphological/compositional tissue comparisons between clones with results consistent with traditional wet chemical analysis approaches where entire stems are milled and analyzed. The four clones showed variable TW and presence of total % G-glucose in the order Björn > Tora > Jorr > Loden. Calculated in terms of 1 m3, Salix stems Tora and Björn would contain ca. 0.24 and 0.28 m3 of tension wood representing a significant amount of freely available glucose

Enzymatic hydrolysis of the gelatinous layer in tension wood of Salix varieties as ameasure of accessible cellulose for biofuels

Background Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing D-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free D-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). Results Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of D-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free D-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. Conclusions The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available D-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with D-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with D-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced D-glucose release in Salix varieties

Novel hydrophobization of wood by epoxidized linseed oil. Part 1. Process description and anti-swelling efficiency of the treated wood

The known method of wood modification by epoxidized linseed oil (ELO) has a limiting practical application due to the rapid polymerization of ELO in the presence of acetic acid (AA) needed as a catalyst. The present study was designed to develop an alternative method by means of a two-step process to avoid the rapid polymerization. The treatment options were tested on Scots pine sapwood, with the dimensional stability (DS) of the treated samples in focus. The new method provided an anti-swelling efficiency (ASE) in the range of 40-57%, which was even better than the thermally modified (TM) reference samples with 40% ASE. The developed two-step process is a feasible and practical approach for ELO treatment of wood

Novel hydrophobization of wood by epoxidized linseed oil. Part 2. Characterization by FTIR spectroscopy and SEM, and determination of mechanical properties and field test performance

Scots pine samples were impregnated with epoxidized linseed oil (ELO) by means of a two-step process, and the effect of treatments has been studied concerning the Fourier transform infrared (FTIR) spectra, mechanical properties, moisture uptake, and field test performance. FTIR analysis of ELO-treated samples revealed that part of the ELO epoxy reactive group was chemically bound to the hydroxyl groups of wood. ELO-treated samples have improved dimensional stability, while the mechanical properties were slightly reduced and the moisture uptake was significantly lowered. The field performance of lap joints treated with ELO (90 kg m(-3)) after 60 months' exposure showed great improvements in performance, as the average annual moisture content (MC) was maintained at the level of 19.3% compared to 34.6% for lap joints treated with linseed oil (LO). The lap-joint area was not stained, and less discoloration by staining fungi on the external surfaces was observed in ELO-treated samples compared to samples treated with LO

The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome

Carbadox is a quinoxaline-di-N-oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction in vitro; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the in vivo longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams (n = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism (q \u3c 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox (q ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration

Family A DNA Polymerase Phylogeny Uncovers Diversity and Replication Gene Organization in the Virioplankton

Shotgun metagenomics, which allows for broad sampling of viral diversity, has uncovered genes that are widely distributed among virioplankton populations and show linkages to important biological features of unknown viruses. Over 25% of known dsDNA phage carry the DNA polymerase I (polA) gene, making it one of the most widely distributed phage genes. Because of its pivotal role in DNA replication, this enzyme is linked to phage lifecycle characteristics. Previous research has suggested that a single amino acid substitution might be predictive of viral lifestyle. In this study Chesapeake Bay virioplankton were sampled by shotgun metagenomic sequencing (using long and short read technologies). More polA sequences were predicted from this single viral metagenome (virome) than from 86 globally distributed virome libraries (ca. 2,100, and 1,200, respectively). The PolA peptides predicted from the Chesapeake Bay virome clustered with 69% of PolA peptides from global viromes; thus, remarkably the Chesapeake Bay virome captured the majority of known PolA peptide diversity in viruses. This deeply sequenced virome also expanded the diversity of PolA sequences, increasing the number of PolA clusters by 44%. Contigs containing polA sequences were also used to examine relationships between phylogenetic clades of PolA and other genes within unknown viral populations. Phylogenic analysis revealed five distinct groups of phages distinguished by the amino acids at their 762 (Escherichia coli IAI39 numbering) positions and replication genes. DNA polymerase I sequences from Tyr762 and Phe762 groups were most often neighbored by ring-shaped superfamily IV helicases and ribonucleotide reductases (RNRs). The Leu762 groups had non-ring shaped helicases from superfamily II and were further distinguished by an additional helicase gene from superfamily I and the lack of any identifiable RNR genes. Moreover, we found that the inclusion of ribonucleotide reductase associated with PolA helped to further differentiate phage diversity, chiefly within lytic podovirus populations. Altogether, these data show that DNA Polymerase I is a useful marker for observing the diversity and composition of the virioplankton and may be a driving factor in the divergence of phage replication components

Optimized utilization of Salix-Perspectives for the genetic improvement toward sustainable biofuel value chains

Bioenergy will be one of the most important renewable energy sources in the conversion from fossil fuels to bio-based products. Short rotation coppice Salix could be a key player in this conversion since Salix has rapid growth, positive energy balance, easy to manage cultivation system with vegetative propagation of plant material and multiple harvests from the same plantation. The aim of the present paper is to provide an overview of the main challenges and key issues in willow genetic improvement toward sustainable biofuel value chains. Primarily based on results from the research project "Optimized Utilization of Salix" (OPTUS), the influence of Salix wood quality on the potential for biofuel use is discussed, followed by issues related to the conversion of Salix biomass into liquid and gaseous transportation fuels. Thereafter, the studies address genotypic influence on soil carbon sequestration in Salix plantations, as well as on soil carbon dynamics and climate change impacts. Finally, the opportunities for plant breeding are discussed using willow as a resource for sustainable biofuel production. Substantial phenotypic and genotypic variation was reported for different wood quality traits important in biological (i.e., enzymatic and anaerobic) and thermochemical conversion processes, which is a prerequisite for plant breeding. Furthermore, different Salix genotypes can affect soil carbon sequestration variably, and life cycle assessment illustrates that these differences can result in different climate mitigation potential depending on genotype. Thus, the potential of Salix plantations for sustainable biomass production and its conversion into biofuels is shown. Large genetic variation in various wood and biomass traits, important for different conversion processes and carbon sequestration, provides opportunities to enhance the sustainability of the production system via plant breeding. This includes new breeding targets in addition to traditional targets for high yield to improve biomass quality and carbon sequestration potential

Theoretical Determination of Pit Membrane Natural Frequency for Destruction by Resonance Effect

The low permeability of many wood species causes significant problems during processing. Industrial methods used for increasing wood permeability reduce strength properties, are energy consuming, and are not viable economically. Destruction of pit membranes in wood cell walls can provide an increase in wood permeability without affecting wood strength properties. It can be accomplished using resonance applied to the pit membranes. Theoretical analysis and calculations have been performed to determine pit membrane (torus and margo) natural frequency. Membrane natural frequencies of bordered pits of Norway spruce are in the range of 3 to 11 MHz. Water in the pit chamber did not have a significant effect on the resonant frequency of the membrane. The main limitation of the amplitude of membrane fluctuations inside the pit chamber was the width of the chamber. Two methods to initiate resonance frequency for pit membrane destruction have been suggested, namely, alternating electric field application and microwave energy pulsation

Effect of microwave treatment on the wood structure of Norway spruce and Radiata pine

Low permeability of many wood species causes problems during timber manufacturing, including long drying times, material losses after drying, and expensive drying processes. Impregnating low permeability timber with preservatives and resins is extremely difficult. In the pulp and paper industry, use of low permeability wood results in shallow chemical penetration, and it requires the use of small-sized chips, high chemical usage, and high-energy consumption. Microwave (MW) wood modification technology can provide solutions to many of these problems. The wood structural changes in Norway spruce and radiata pine after MW modification with 0.922 and 2.45 GHz of were investigated. High intensity MW application (specific MW power 22 to 25 W/cm3, applied energy 79 to 102 kWh/m3) to moist wood caused the following wood structural changes: pit opening and pit membrane rupture; middle lamella weakening and rupture; and ray cell wall destruction and check (voids) formation mainly in the radial-longitudinal plane caused by the destruction of rays and weak middle lamella regions. Microwave destruction of different wood structure elements provided a significant increase in wood permeability for liquids and gases. Knowledge of the effects of MW treatment to the wood structure elements allows assessment of opportunities for the use of microwave irradiation in wood technology

Evaluation of Wood Quality Traits in Salix viminalis Useful for Biofuels: Characterization and Method Development

Salix (willow) is a well-known coppice plant that has been used as a source for bioenergy for decades. With recent developments in changing from a fossil-based to a circular bioeconomy, greater interest has been orientated towards willow as a potential source of biomass for transport biofuels. This has created increasing interest for breeding strategies to produce interesting genotypic and phenotypic traits in different willow varieties. In the present study, 326 genetically distinct clones and several commercial varieties of S. viminalis were analyzed using complementary approaches including density, chemical, image, histochemical, and morphometric analyses. A systematic approach was adopted whereby the basal regions of harvested stems were separated and used in all studies to aid comparisons. Density analyses were performed on all clone individuals, and from the results, 20 individual plants representing 19 clones were selected for the more in-depth analyses (chemical, image analysis, histochemical, and morphometric). The absolute dry density of the clones selected varied between ca. 300 and 660 kg/m3 with less variation seen in the commercial S. viminalis varieties (ca. 450–520 kg/m3). Selected clones for chemical analysis showed the largest variation in glucose (47.3–60.1%; i.e., cellulose) and total sugar content, which ranged between ca. 61 and 77% and only ca. 16 and 22% for lignin. Image analyses of entire basal stem sections showed presence of tension wood in variable amounts (ca. 7–39%) with characteristic G-fibers containing cellulose-rich and non-lignified gelatinous layers. Several of the clones showing prominent tension wood also showed high glucose and total sugar content as well as low lignin levels. A morphometric approach using an optical fiber analyzer (OFA) for analyzing 1000 s (minimum 100,000 particles) of macerated fibers was evaluated as a convenient tool for determining the presence of tension wood in stem samples. Statistical analyses showed that for S. viminalis stems of the same density and thickness, the OFA approach could separate tension wood fibers from normal wood fibers by length but not fiber width. Results emphasized considerable variability between the clones in the physical and chemical approaches adopted, but that a common aspect for all clones was the occurrence of tension wood. Since tension wood with G-fibers and cellulose-rich G-layers represents an increased source of readily available non-recalcitrant cellulose for biofuels, S. viminalis breeding programs should be orientated towards determining factors for its enhancement