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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.

Additional file 3. Figure 3. Bioreactor cultivation of R. toruloides CBS 14 in triplicates, (A) CG50 medium, (B) HH10CG50 medium. Glucose, xylose and acetic acid are presented on secondary Y axis.

Additional file 2. Figure 2. R. toruloides CBS 14, HH40CG60 media grown in duplicates, dry weight and change of compounds concentration in media over time, average lipid concentration was 10.57 g/L after 70h. Glucose, xylose and acetic acid are presented on secondary y axis.

Additional file 1: Figure S1. Photographs of the investigated strains on agar plates.

Additional file 2: Table S1. Screening of Rhodotorula and Lipomyces strains for growth on glucose, xylose and a mixture of both. All strains were grown on either a glucose- (G), xylose (X) or a glucose/xylose mixture (M) containing media. Growth was measured by OD- determination, Lipids were measured using FTIR spectroscopy. Lipid content was not determined (n.d.) for some strains due to exclusion of these strains from further investigation based on their poor growth performance. Experiments were performed in duplicates and average deviation is displayed, (*) only one replicate available.

Additional file 1. Figure 1. Specific lipid production rates of R. toruloides CBS 14 and R. glutinis CBS 3044 in 55 g/L crude glycerol media and 55 g/L crude glycerol media with 10% hemicellulose hydrolysate. Negative values are due to a decrase of lipid concentrations during the measuring interval.

Background Use of bio-based diesel is increasing in Europe. It is currently produced from oilseed crops, but can also be generated from lignocellulosic biomass such as straw. However, removing straw affects soil organic carbon (SOC), with potential consequences for the climate impact of the biofuel. This study assessed the climate impacts and energy balance of biodiesel production from straw using oleaginous yeast, with subsequent biogas production from the residues, with particular emphasis on SOC changes over time. It also explored the impact of four different scenarios for returning the lignin fraction of the biomass to soil to mitigate SOC changes. Climate impact was assessed using two methods, global warming potential (GWP) and a time-dependent temperature model (∆Ts) that describes changes in mean global surface temperature as a function of time or absolute temperature change potential (AGTP). Results Straw-derived biodiesel reduced GWP by 33–80% compared with fossil fuels and primary fossil energy use for biodiesel production was 0.33–0.80 MJprim/MJ, depending on the scenario studied. Simulations using the time-dependent temperature model showed that a scenario where all straw fractions were converted to energy carriers and no lignin was returned to soil resulted in the highest avoided climate impact. The SOC changes due to straw removal had a large impact on the results, both when using GWP and the time-dependent temperature model. Conclusions In a climate perspective, it is preferable to combust straw lignin to produce electricity rather than returning it to the soil if the excess electricity replaces natural gas electricity, according to results from both GWP and time-dependent temperature modelling. Using different methods to assess climate impact did not change the ranking between the scenarios, but the time-dependent temperature model provided information about system behaviour over time that can be important for evaluation of biofuel systems, particularly in relation to climate target deadlines. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0907-9) contains supplementary material, which is available to authorised users.

Background Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. Results In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. Conclusions The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0813-1) contains supplementary material, which is available to authorized users.

Additional file 1: Figure S1. Estimates of Klason lignin from py-MBMS versus values obtained by wet chemistry. Figure S2. Results of initial test runs to assess optimal pretreatment severity and enzyme loading. Figure S3. Results of second test run to better assess optimal enzyme loading.

Figure S1. Partial protein alignment of the closest neighbors of PEL12 and separation of the group A and B for further reverse conservation analysis. C. rosea paralogs BN869_T0006915 (PEL12) and BN869_T00007653 (PEL13) align to the group B, whereas the protein BN869_T0008859 (PEL2), Fusaria and some members of Sordariales are separated by several hallmarks forming the group A. Figure S2. SDS-PAGE gel showing the putative expression of the PEL12 protein (arrows) at 37 kDa in the fermentation broths of two independent T. reesei PEL12 overexpression strains pel12OE-1 and pel12OE-2. The strains were cultivated for 48 h in Mandels-Anderotti medium supplemented with 1% glucose. Figure S3. Carbon source utilization of T. reesei WT and pel12OE mutants on 95 different carbon sources based on BIOLOG Phenotypic assay for filamentous fungi. Mycelial density is shown for 96 h of incubation at 28 °C in darkness. Black, gray and white circles represent mycelial density of T. reesei QM9414 (wt) and the mutants pel12OE-1 and − 2, respectively. Figure S4. Validation of pel12 mutant strains using PCR and RT-PCR. A: PCR verification of Δpel12 using primers located within the hygB cassette (Hyg F /Hyg R) in combination with primers located upstream and downstream regions from the deletion cassette (6915 ko F / 6915 ko R). A PCR product of ~ 2.5 kb using primers 6915 ko F / Hyg R and 6915 ko R / Hyg F were expected from a correct gene replacement. M, gene ruler DNA ladder mix; 1–3, independent Δpel12 mutants; 4, WT strain. B: RT-PCR analysis of pel12 gene expression in WT and deletion strains using pel12 specific primers. A PCR product of 92 bp was expected from the WT. M, gene ruler DNA ladder mix; 1, WT; 2–4, independent deletion strains. Primer combinations used for PCR and RT-PCR are given above the images. Figure S5. Carbon source utilization of C. rosea WT and ∆pel12 mutants (∆pel12 KOA, KOB, KOC) on 95 different carbon sources based on BIOLOG Phenotypic assay for filamentous fungi. Mycelial density is shown for 144 h of incubation at 28 °C in darkness. Figure S6. Measurement of B. cinerea necrotic lesions area on detached leaves of Arabidopsis thaliana. The leaves were inoculated with C. rosea strains (A) or T. reesei (B) strains spores 30 min before application of B. cinerea spores. Necrotic lesion area was measured under the microscope using a DeltaPix camera and software 56 h post inoculations. Only B. cinerea inoculated leaves were used as control. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistically significant differences (P ≤ 0.05) based on the Fisher test. (PDF 633 kb)

Additional file 1: Figure S1. SDS-PAGE analyses of T. atroviride culture filtrate and purified Trichoderma spp. Cel7A enzymes. Figure S2. Substrate dependence plots and Hanes-Wolff plots from enzyme kinetics experiments with TatCel7A, ThaCel7A and TreCel7A, using pNP-Lac as substrate and cellobiose as inhibitor. Additional information regarding the mathematical model for quasi-steady state kinetics of processive cellulose hydrolysis by GH7 cellobiohydrolases and the derivation of kinetic parameters by non-linear regression fitting to real-time progress curves of the initial stage of cellulose hydrolysis. Figure S3. A) Real-time progress curves. B) Derivative of the progress curves in A). Figure S4. A) Simplified reaction scheme for a processive cellulase. B) Illustration of the molecular steps involved in the reaction scheme. Figure S5. Non-linear regression fit to real-time progress curves. Figure S6. Bar diagram of kinetic parameters derived from initial hydrolysis of BMCC. Additional information regarding correlation of kinetic parameters derived by non-linear regression fit to initial hydrolysis data. Table S1. Parameter correlation matrix for TreCel7A. Figure S7. Kinetic parameter fit to simulated data with 2.5% random noise added, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Table S2. Comparison of kinetic parameters from the fit to simulated data with 2.5% random noise, and to experimental data recorded for TreCel7A during initial hydrolysis of BMCC. Figure S8. Sequence alignment of the GH7 CBH catalytic domains used for RCA analysis. Figure S9. Phylogenetic tree of GH7 catalytic domain protein sequences from Trichoderma spp. and Fusarium spp. Table S3. S scores from RCA analysis for residues of interest for TatCel7A, ThaCel7A and TreCel7A. Additional MD simulation results Figure S10. RMSD as a function of time for each 100-ns, ligand-bound MD simulation of TatCel7A, ThaCel7A and TreCel7A catalytic domains.

Additional file 1: Figure S1. Cyclic voltammograms of (A) HT-A-WS, (B) SE-S, (C) SE-B, (D) OS-S, (E) OS-B, (F) OS-A-S, (G) OS-A-B, (H) OS-S-LF, (I) OS-B-LF, (J) OS-A-S-LF and (K) OS-A-B-LF immobilized on a glassy carbon electrode with the use of Nafion in 100 mM tartrate buffer pH 5.0, at 50 oC between -200 and 600 mV vs Ag/AgCl for a scan rate of 5 mV/s. Figure S2. FTacV 1st to 6th Harmonic of HT-A-WS lignin immobilized on a glassy carbon electrode with the use of Nafion for a v = 50 mV/s, A = 110 mV, f = 12.3 Hz in de-aerated 100 mM tartrate buffer pH 5.0 at 50ÎżC. Figure S3. The products of MtLPMO9 action on PASC after addition of ascorbic acid 1 mM or lignin isolated from pretreated materials 10 mg/mL. Products at 5-13 min correspond to neutral sugars, 13-19 min to C1 oxidized sugars, 19-25min to C4 oxidized sugars and 25-30 min to mixed C1/C4 oxidized sugars. Peaks at 12.3, 21.8 and 31.6 min are assigned to ascorbic acid. Figure S4. The products of PcLPMO9D action on PASC after addition of ascorbic acid 1 mM or lignin isolated from pretreated materials 10 mg/mL. Products at 5-13 min correspond to neutral sugars and 13-19 min to C1 oxidized sugars. Figure S5. The products of NcLPMO9C action on PASC after addition of ascorbic acid 1 mM or lignin isolated from pretreated materials 10 mg/mL. Products at 5-13 min correspond to neutral sugars and 19-25 min to C4 oxidized sugars. Peaks at 12.3, 21.8 and 31.6 min are assigned to ascorbic acid. Figure S6. GPC chromatograms for HT-A-WS lignin before and after 24h-incubation with buffer phosphate-citrate 100 mM pH 5.0 and buffer/MtLPMO9. Table S1. Estimated aliphatic and aromatic groups content (mmol/g) of HT-A-WS lignin before and after 24h-incubation with buffer phosphate-citrate 100 mM pH 5.0 and buffer/MtLPMO9, as evaluated from 31P NMR. Table S2. Identified extractives from HT-A-WS lignin in different solvents.