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Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass
Industrial Crops & Products 123 (2018) 84–92
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Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass
T
⁎
Brigitte Chabberta, , Christine Terrync, Mickaël Herbauta, Alankar Vaidyab, Anouck Habranta, ⁎ ⁎ Gabriel Paësa, , Lloyd Donaldsonb, a
FARE Laboratory, INRA, University of Reims Champagne-Ardenne, 51100 Reims, France Scion, Private Bag 3020, Rotorua 3046, New Zealand c PICT platform, University of Reims Champagne-Ardenne, 51100 Reims, France b
A R T I C LE I N FO
A B S T R A C T
Keywords: Confocal fluorescence microscopy Pretreatment Poplar Pine Accessibility FLIM PEG probe Cellulases
The conversion of cell wall biomass to sugar for fermentation to ethanol requires chemical or physical pretreatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Multiscale study of biomass deconstruction gives access to key insights into the cell wall and lignin changes induced by pretreatment. Few studies have compared the effect of pretreatment of different biomass species on the cell wall accessibility. Considering representative softwood (pine) and hardwood (poplar) biomass, we studied the impact of two pretreatments on enzymatic saccharification and cell wall structure and accessibility. Lignin fluorescence properties were investigated by measuring fluorescence lifetime in addition to chemical analysis, and the accessibility of biomass was assessed using fluorescent probes consisting of rhodamine labeled polyethylene glycol (PEG) molecules ranging from 10 to 40 kDa. Hot water treatment and chlorite delignification altered chemical structure and fluorescence lifetime, which was positively correlated with glucose conversion and negatively correlated with lignin and β-O-4′ contents. Imaging distribution of the probes indicated that chlorite pretreatment resulted in a more uniform distribution of probe in the cell wall compared to hot water treatment. The interaction between cell wall and fluorescent PEG probes was evaluated using Förster Resonance Energy Transfer (FRET) and fluorescence microscopy. The FRET efficiency showed a high negative correlation with the probe size and was greatly increased by chlorite delignification, reflecting increased accessibility to the probe and interaction. Thus the accessibility and interactions of small probes in pretreated biomass could be a relevant indicator of potential for saccharification, whereas fluorescence lifetime provides a new criteria for assessing relevant cell wall structural modifications related to enzymatic conversion of lignocellulosic biomass.
1. Introduction There is increasing interest in the use of plant biomass for the production of sustainable liquid fuels and chemicals to replace fossil carbon, due to declining supply, and to mitigate increasing atmospheric CO2 (Menon and Rao, 2012). Plant cell wall biomass consists of a framework of cellulose surrounded by a matrix of complex polymers including hemicellulose and lignin. These polymers are interconnected by non-covalent and covalent linkages resulting in a complex structural and chemical network (Burton et al., 2010) with physical and chemical barriers limiting cellulase
enzyme penetration and progression (Paës et al., 2017), leading to decreased enzyme activity (Arantes and Saddler, 2011; Zhao et al., 2012). Cellulase activity is limited by the crystallinity of cellulose and its restricted accessibility inside the cell wall matrix of hemicelluloses and lignin (Ding et al., 2012). In addition to being a structural obstacle, lignin can give rise to non-productive enzyme binding, so it is considered to be the main factor responsible for lignocellulose recalcitrance (Vaidya et al., 2014; Zeng et al., 2014). The conversion of cell wall biomass to monosaccharides for fermentation into ethanol thus requires chemical or physical pretreatments to disrupt the plant cell walls and increase porosity in
Abbreviations: FLIM, fluorescence lifetime imaging microscopy; FRET, Förster (or Fluorescence) resonance energy transfer; MW, molecular weight; RhPEG1, rhodamine labeled polyethylene glycol of 1 kDa; RhPEG3.4, rhodamine labeled polyethylene glycol of 3.4 kDa; RhPEG10, rhodamine labeled polyethylene glycol of 10 kDa; RhPEG40, rhodamine labeled polyethylene glycol of 40 kDa ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Chabbert), [email protected] (G. Paës), [email protected] (L. Donaldson). https://doi.org/10.1016/j.indcrop.2018.06.058 Received 2 April 2018; Received in revised form 12 June 2018; Accepted 14 June 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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treatments induce alteration of the residual lignin structure (Kumar et al., 2013; Ma et al., 2014; Paës et al., 2017), thus allowing comparison of cell wall accessibility in hardwood and softwood biomass. In particular, interactions of probes with pretreated biomass was assayed by FRET measurements, while FLIM was used to assess cell wall modifications. Such complementary microscopic techniques provide new insights for cell wall accessibility which cannot be obtained by porosity measurements.
order to make the cellulose accessible to cellulolytic enzymes while minimizing the formation of by-products (Silveira et al., 2015). A wide variety of pretreatments has been proposed and each has advantages and limitations (Agbor et al., 2011; Singh et al., 2015). Some pretreatments improve the accessibility of cellulose by removal of matrix components such as lignin (delignification associated with pulping) and hemicelluloses (hot water treatment, steam-explosion or acid treatments). Removal of lignin alone is usually insufficient to optimize sugar yield because hemicelluloses are covalently linked to lignin and/or closely interact with the surface of elementary cellulose, thus forming a physical and chemical barrier that limits enzymatic accessibility and cellulose conversion yields (Ding et al., 2012; Meng et al., 2015; Penttilä et al., 2013; Trajano et al., 2013). Improvement of cellulose conversion is related to the severity and the type of pretreatment and depends on the biomass species, due to variation in the content and composition of lignin and hemicellulose (Arantes and Saddler, 2011; Asada et al., 2015; Singh et al., 2015). For instance, hardwoods and softwoods differ in terms of lignin composition (mixed syringyl-guaiacyl vs guaiacyl) and hemicellulose composition (xylan vs mannan) (Ralph et al., 2004; Scheller and Ulvskov, 2010). These differences contribute to the effectiveness of biomass pretreatment, with xylans being readily extractable by hot water whilst mannans require more intensive treatment (Meng et al., 2015; Nitsos et al., 2016). Syringyl-rich hardwood lignins are more easily chemically degraded than guaiacyl-type softwood lignin due to a lower degree of condensation, a less complex structure, smaller polymer size and higher content of β-ethers (Mottiar et al., 2016; Ralph et al., 2004; Studer et al., 2011). Apart from chemical and structural characterization, microscopy has been an effective tool for the study of biomass pretreatment at multiscale levels (Donohoe and Resch, 2015). Light microscopy and microspectrometry combining UV, infrared and Raman analysis have provided critical information on cell wall chemical changes caused by pretreatments and enzyme hydrolysis (Belmokhtar et al., 2013; Chundawat et al., 2011; Ma et al., 2014; Singh et al., 2015). Among microscopy approaches, confocal fluorescence microscopy is widely used to visualize biomass deconstruction, and gives key insights into cell wall and lignin changes induced by pretreatment (Singh et al., 2015). For example, confocal fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) were used to characterize lignin modification after pretreatments in sugar cane bagasse and eucalyptus fiber (Coletta et al., 2013) and in poplar, wheat straw and miscanthus (Auxenfans et al., 2017b; Zeng et al., 2015). Confocal microscopy is also a fast method to provide a spatial and temporal visualization of fluorescently labeled enzyme and to characterize enzyme-substrate interactions within biomass (Ding et al., 2012; Donaldson and Vaidya, 2017; Luterbacher et al., 2013; Moran-Mirabal, 2013; Thygesen et al., 2011). Confocal fluorescence microscopy can also reveal cell wall modification and cellulose porosity by determining the distribution and mobility of fluorescent probes that vary in molecular weight (MW) or affinity towards cell wall polymers (Donaldson et al., 2015; Moran-Mirabal, 2013; Paës, 2014; Paës et al., 2017; Yang et al., 2013). Few studies have compared the effect of pretreatment of different biomass species on the accessibility of the cell wall. Donaldson et al. (2014) have shown that rhodamine labeled polyethylene glycol (PEG) penetrates steam-exploded wood. The fluorescent PEGs can then be localized by direct imaging of the rhodamine dye and their strength of interaction can be evaluated by measuring the Förster (or Fluorescence) Resonance Energy Transfer (FRET) between rhodamine and cell wall lignins at the molecular level. In this work, we use a set of fluorescence microscopy techniques to study the effects of two different pretreatments (chlorite delignification and hot water extraction) on cell wall accessibility in poplar (hardwood) and pine (softwood) using rhodamine labeled PEGs of various sizes as probes. Chlorite delignification reduces lignin content whilst hot water extraction mainly reduces hemicellulose content; both
2. Materials and methods 2.1. Sample preparation Small wood blocks (3–4 mm width × 2 cm long) were isolated from the basal region of 3-year-old short rotation poplar coppice (Orléans, France) and from mature pine wood (Rotorua, New Zealand) which were subsequently dried at 40 °C for 2 days. Pretreatments were performed as previously described (Paës et al., 2017). Hot water treatment was performed for 1 h at 170 °C using mineralization reactors equipped with Teflon tubes (PARR, USA). Sodium chlorite-acetic acid delignification treatment (Wise et al., 1946) was performed on 1 g poplar samples using acetic acid and sodium chlorite at 70 °C for 1 h and the reaction was repeated 5 times. Untreated control samples were also obtained after water washing at 4 °C (1 h). After pretreatment, samples were washed several times with deionized water until the pH of the wash was about 6.0. Then samples were dried at 40 °C in an air forced oven for 2 days. One sample fraction was ground to 200 μm size prior to chemical analysis and enzymatic saccharification, and the other fraction was sectioned with a sledge microtome at a thickness of 30 μm for confocal fluorescence microscopy. 2.2. Chemical analysis and enzymatic saccharification The sugar monomer composition was determined using a two-step sulfuric acid hydrolysis (Seaman et al., 1954) followed by high-performance anion-exchange chromatography (HPAEC-PAD) with 2deoxy-D-ribose as internal standard (Paës et al., 2017). Lignin content was quantified using a spectrophotometric method after acetyl bromide dissolution of the lignocelluloses (Iiyama and Wallis, 1990) and determination of the monomer composition of the alkyl aryl ether lignin structures was achieved by thioacidolysis as previously described (Belmokhtar et al., 2013; Lapierre et al., 1986). Enzymatic saccharification was performed on the ground samples (2% w/v) using a commercial cellulase preparation, Cellic ® CTec2 kindly provided by Novozymes A/S (Bagsværd, Denmark), in 10 mL sodium citrate buffer (0.1 M; pH 5.0) for 48 h at 50 °C and 200 rpm using an enzyme loading of 40 FPU/g-glucan. The reaction mixtures were pre-incubated for 1 h at 50 °C and 200 rpm. Enzymatic hydrolysis was initiated by addition of the cellulase solution and stopped after 72 h by heating for 15 min at 100 °C. Control experiments without enzyme were also carried out. The amount of glucose released was determined using high-performance anion-exchange chromatography (HPAECPAD). Conversion yields were expressed as a percentage of initial glucose amount (Auxenfans et al., 2017a). 2.3. Confocal fluorescence microscopy Samples of untreated control and pretreated pine and poplar wood were sectioned in the transverse plane with a sledge microtome at a thickness of 30 μm. Sections were used for fluorescence spectroscopy, FLIM, and FRET measurements. Fluorescence imaging was performed using a Leica SP5 II confocal microscope at 1024 × 1024 pixel resolution with a 63× glycerol immersion lens. FLIM measurements were performed on the untreated control and pretreated pine and poplar wood sections mounted in 50% glycerol in 10 mM phosphate buffer at pH 7 (Donaldson and Radotic, 2013) using a Zeiss LSM710 multiphoton 85
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microscope equipped with a Becker and Hickl lifetime spectrometer. Decay spectra were acquired with a biphoton excitation at 750 nm and emission between 420 and 722 nm. Data were analyzed using a multiexponential decay model with 2 components. Measurements of average fluorescence lifetime were made on 10 fields of view for each species/ pretreatment combination. For FRET analysis, sections from wood samples were imaged without treatment to act as controls for lignin fluorescence. Other sections were treated with 2 mL of 1.6 μM solutions of rhodamine B labeled PEGs (RhPEG, NANOCS, New York, USA) in water at 1, 3.4, 10 and 40 kDa (RhPEG1, RhPEG3.4, RhPEG10, RhPEG40) for 1 h at room temperature (22 °C) in the dark, followed by washing in distilled water (5 min twice). All sections were mounted in 50% glycerol in phosphate buffer at pH 7. The fluorescence spectrum of rhodamine B was measured with the confocal microscope using a 1.6 μM solution in distilled water. Hydrodynamic radius (RH) of the fluorescent probes was determined by quasi-elastic light scattering (QELS) (Paës et al., 2017). FRET measurements were performed using the acceptor photobleaching technique, using sequential excitation at 488 nm for the donor (lignin in wood samples) and 561 nm for the acceptor (rhodamine in RhPEG probes) with emission at 500–570 and 570–700 nm, respectively (Donaldson et al., 2014). Bleaching of rhodamine label for FRET measurements was performed with 100% laser power at 561 nm for 50 scans. FRET measurements were performed at 512 × 512 pixel resolution but some additional FRET imaging was done at 1024 × 1024 for presentation purposes. FRET calculations were performed using Leica LAS AF software. Images of FRET efficiency on a 0–100% scale were calculated using Digital Optics V++ software. FRET measurements were repeated on 10 fields of view for each probe/treatment combination. FRET efficiency was also measured on sections with no probe treatment and in areas outside the region where photobleaching was performed on probe treated sections to act as additional controls.
Fig. 1. Enzymatic conversion yield (% initial glucose) for pine and poplar. Error bars show the 95% confidence interval.
and xylan content in pine and poplar, respectively) and pectins (lower content of arabinose, galactose, and galacturonic acid), whereas chlorite removed lignin. Pretreated samples contained a higher proportion of glucan compared to control biomass. Lignin content increased after hydrothermal pretreatment as a consequence of hemicellulose removal. Nevertheless, the residual lignin displayed structural alterations as a lower proportion of lignin monomers was obtained by thioacidolysis for both pine and poplar, indicating a reduction in labile ether β-O-4′ linkages content. This modification was even more pronounced after extensive removal of lignin by chlorite pretreatments. Enzymatic hydrolysis of untreated poplar and pine controls gave similar glucan conversion yields of 32% (Fig. 1). Chlorite pretreatment greatly enhanced conversion yields (close to 100%), with a three-fold increase in comparison to controls. Hot water pretreatment was less efficient on pine (40% conversion yield, only a slight increase compared to the control sample), while poplar showed a 2-fold increase in conversion (ca. 80%).
2.4. Data treatment Significant differences between treatments were evaluated using Student’s t-test (Excel) at 0.95 confidence level. Pearson correlation coefficients were calculated to measure the degree of the relationship between the chemical and spectral properties of biomass and their saccharification yields.
3.2. Fluorescence lifetime measurement The fluorescence lifetime of poplar was higher than that of pine in controls (Fig. 2). For hot water pretreated samples, lifetime increased significantly for pine and poplar. Chlorite-treated samples showed the highest increase in fluorescence lifetime: 5-fold for pine and 2-fold for poplar in comparison to control samples, respectively. Lifetime variations could be clearly visualized in the corresponding lifetime colorcoded images of cell walls and were not significantly different between cell corner and secondary cell walls except for the hot water treated poplar that showed 15% higher fluorescence lifetime in the secondary wall layer (Fig. 2).
3. Results 3.1. Chemical composition and enzymatic saccharification Chemical analysis showed differences in polysaccharide and lignin compositions between pine and poplar (Table 1). Following pretreatments, weight losses close to 22% were obtained for pine (19% after hot water and 25% after chlorite) and 30% for poplar (27% after hot water and 29%, after chlorite). Similar effects were observed for the two biomass species: hot water removed mainly hemicelluloses (mannan
3.3. Probe distribution Distribution of the RhPEG probes in wood sections was visualized
Table 1 Chemical composition of untreated and pretreated pine and poplar samples. Lignin and sugar data are expressed as percent of dry matter and β-O-4′ lignin is expressed as percent of lignin. Glc (glucose), Xyl (xylose), Man (mannose), other sugars refer to the total content of minor monosaccharides (arabinose, galactose, uronic acids).
Pine
Poplar
Control Hot water Chlorite Control Hot water Chlorite
Total Lignin (% dry matter)
β-O-4’ Lignin
27.28 ± 2.70 34.32 ± 1.73 7.00 ± 0.34 25.60 ± 0.16 32.55 ± 1.10 6.90 ± 0.73
30.62 ± 0.97 20.00 ± 1.09 < 1.00 39.71 ± 0.86 25.46 ± 3.72 3.03 ± 0.43
Total Sugar
Glc
Xyl
(% lignin)
Man
Ara + Gal + GalU
10.33 ± 0.34 5.12 ± 0.03 10.70 ± 0.38 2.04 ± 0.17 1.76 ± 0.05 2.64 ± 0.03
1.09 0.10 0.89 0.29 0.05 0.23
(% dry matter) 58.80 58.36 66.96 53.20 51.15 66.46
± ± ± ± ± ±
2.05 0.16 3.17 4.26 1.08 1.19
86
41.91 48.61 49.37 35.73 39.05 44.81
± ± ± ± ± ±
1.56 0.20 2.57 2.87 0.75 0.89
3.43 ± 0.09 3.58 ± 0.03 4.07 ± 0.16 13.02 ± 1.06 9.65 ± 0.23 16.43 ± 0.43
± ± ± ± ± ±
0.03 0.01 0.04 0.02 0.01 0.02
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was essentially no detectable FRET in cell walls incubated only with water (data not shown). Similarly, cell walls incubated with probes but not subjected to acceptor photobleaching did not show any FRET. Although equimolar concentrations of RhPEG probes were used, it was apparent that the different probes had different rhodamine labelling ratios depending on their size, as shown by their color (Additional file 1: Figure S1). To assay the impact of labelling ratio on FRET efficiency, FRET was determined for a dilution series of the RhPEG1 probe ranging from 0.5 μM to 2.5 μM. FRET measurements for control pine sections treated with each of the 5 dilutions showed a very small variation in FRET efficiency (from 14% for 0.5 μM to 18% for 2.5 μM) (Additional file 2: Figure S2), demonstrating that labelling ratio of RhPEG probes has a very moderate influence on FRET. FRET measurements were performed considering the whole cell wall region to compare the overall effect of probe size regarding pretreatment and biomass. FRET efficiency generally declined with increasing MW of the probe in both wood species (Fig. 5). Untreated poplar showed higher FRET efficiency (10–30%) than pine (2–20%) regardless of the probe size. For the hot water pretreatment, FRET efficiency in comparison to the untreated samples was either unchanged or slightly increased depending on the probe size in pine, whereas it was decreased in poplar for all probes. Finally, for the chlorite pretreatment, FRET efficiency was greatly increased in both species for all probes in comparison to the controls, with the exception of RhPEG40 which was unchanged for poplar. These results can be further interpreted considering FRET values and the hydrodynamic radii (RH) of the probes, which is representative of their size as diffusing molecules. Indeed, the increasing MW (from 1 to 40 kDa) of the RhPEG probes corresponds to an increase in their RH from 1.3 to 4.8 nm as determined by QELS. Thus considering poplar or pine wood samples, the FRET efficiency showed a strong negative correlation with the probe size (Fig. 6). Chlorite pretreatment results in a sharper increase of FRET efficiency with decreasing size of the probe (Fig. 6) as compared to control and hot water pretreatment.
Fig. 2. Fluorescence lifetime measurements (A) and corresponding lifetime images (B) for control and pretreated pine and poplar. Error bars show the 95% confidence interval. Scale bars = 30 μm.
by fluorescence imaging of the rhodamine at 561 nm excitation (Fig. 3). Wood sections with no RhPEG probe treatment did not show any detectable lignin autofluorescence at 561 nm excitation with the imaging parameters chosen. For the untreated pine, RhPEG was localized in the secondary cell wall with an uneven distribution especially near the cell corners; the distribution was similar for probes whatever their size. In the hot water pretreated pine sample the secondary wall was strongly infiltrated only by the RhPEG1 probe with higher penetration in the S1 layer compared to the untreated sample. In sections treated with larger size probes, a moderate penetration of the RhPEG was observed in the cell wall albeit higher in the S3 layer. Probe distribution in the chloritetreated sample was lower in the secondary wall and higher in the middle lamella, and was not dependent on probe size. For poplar samples, results for the control without pretreatment were similar to those for pine, showing weaker distribution of the probes in the middle lamella and outer part of the secondary cell wall (Fig. 4). There was no obvious trend with increasing probe size. For the hot water pretreatment, all four probes showed a greater affinity for vessel (arrows) cell walls compared to fibers. Also, the distribution of RhPEG was more uniform in the cell wall, with localization in both secondary cell wall and middle lamella; the larger probe seemed to better penetrate into the middle lamella. For the chlorite pretreatment (Fig. 4), the probe distribution was very uniform irrespective of probe size.
4. Discussion 4.1. Lignin is a barrier to cellulose accessibility Pretreatments which facilitate removal of hemicellulose and lignin from lignocellulosic biomass are known to increase enzymatic conversion of cellulose (Arantes and Saddler, 2011; Asada et al., 2015; Silveira et al., 2015; Singh et al., 2015). Our results show that hot water treatment mainly induced a reduction in xylan in poplar samples as previously reported (Assor et al., 2009; Paës et al., 2017; Trajano et al., 2015) and allowed higher glucose conversion yield. In pine, hot water pretreatment removed about 50% of the mannan but did not induce higher enzymatic conversion, suggesting that residual mannan in pine limits saccharification. Unlike poplar, hot water extraction did not reduce the xylan content of pine (3.4 and 3.6% dry matter of control and hot water treated pine respectively). One may hypothesize that the higher recalcitrance of hot water pretreated pine could arise from increased complexity of lignin-carbohydrate (LCC) bonds involving different hemicelluloses in comparison to hardwood LCCs that mostly involve xylan hemicellulose (Balakshin et al., 2011). Residual xylan is more tightly bound to lignin in hot water treated pine in comparison to poplar, and could contribute to pine recalcitrance. In addition interactions between lignin-hemicellulose could be modified as a consequence of the structural changes of lignin induced by the pretreatment. Indeed, residual lignin contained a lower proportion of β-O-4′ ether linkages as lignin undergoes both disruption of labile ether linkage and recondensation mechanisms during hot water treatment (Paës et al., 2017; Trajano et al., 2013). For chlorite-treated samples, delignification of pine and poplar reached the same extent (ca. 75%) and this could be the main reason for the high conversion yield (ca. 100%). Thus, removal of lignin increases cellulose accessibility more
3.4. FRET efficiency RhPEG probes were then used to measure the efficiency of FRET which was assumed to occur between lignin (donor) and rhodamine (acceptor) under conditions of close molecular proximity (Coletta et al., 2013). Measurements were performed on the whole cell walls. There 87
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Fig. 3. RhPEG distribution in pine control, hot water, and chlorite treated substrates. (S1 and S3: secondary cell wall layers). Scale bars = 40 μm.
interlinkages. Fluorescence lifetime is independent of fluorescence intensity and fluorophore concentration (Berezin and Achilefu, 2010). However, lifetime is sensitive to fluorophore structure, composition, and presence of interacting surrounding molecules, so that any change in the fluorophore environment can lead to changes in lifetime. Regarding lignin composition, poplar contains much more syringyl lignin than pine in which lignin is composed mainly of the guaiacyl type. Moreover, poplar is known to have unusual lignin groups (p-hydroxybenzoate) (Ralph et al., 2007). Changes in lifetime have also been associated with different lignin structures in pretreated poplar wood (Auxenfans et al., 2017b; Zeng et al., 2015). Loosely packed lignin with a long lifetime (4 ns) was associated with secondary walls while dense lignin with a short lifetime (0.5–1.0 ns) was associated with lignin distributed throughout all the cell wall layers. Dilute acid pretreatments were found to shorten lignin lifetime by extracting from the secondary wall the low density lignins with long lifetime. In addition the lifetime of a lignin model compound increased with increasing carboxymethyl
effectively than the removal of hemicellulose.
4.2. Fluorescence lifetime characterization reveals changes in lignin organization Strong variations in the fluorescence lifetime were observed for both poplar and pine : low values (0.3-0.6 ns) for untreated samples, intermediate values (0.6-0.8 ns) for hot water treated samples and high values (1.4–1.8 ns) for chlorite-treated samples, these latter values being similar to those obtained for delignified eucalyptus (Coletta et al., 2013). Although variation in FLIM data is also observed between pine and poplar, the fluorescence lifetime differences seem more dependent on the type of pretreatment (i.e. type of polymer removal, especially lignin) than on biomass species. Fluorescence in plant cell walls mainly originates from lignin, which is composed of monolignols. Thus lignin can be seen as a complex fluorescent molecule made of fluorophores varying in their type and 88
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Fig. 4. RhPEG distribution in poplar control, hot water, and chlorite treated substrates. Scale bars = 40 μm.
specific feature so lifetime must be seen as a general indicator of the lignin state.
cellulose content (Zeng et al., 2015), confirming the importance of surrounding polysaccharides on lignin lifetime. Consequently, it seems reasonable to attribute increased fluorescence lifetime of pretreated samples not only to the removal of hemicelluloses by hot water treatment and of lignin by chlorite treatment, but also to the structural modifications of lignin. As a matter of fact, the lignin β-O-4′ content significantly decreased in pretreated pine and poplar, with chlorite treatment being the most impacting (Table 1). The fact that chlorite-treated samples, despite a very low lignin content, display a long lifetime, highlights the influence of chemical structure on lifetime. The very low proportion of non-condensed lignin likely reflects that inter monomer linkages must be largely altered after chloritetreatment, favoring the creation of a residual low density lignin-like network. Overall, modifications of fluorescence lifetime reflect changes in lignin chemical composition and organization in addition to changes in interactions with other polymers, in particular with hemicelluloses. Right now, it does not seem possible to relate lifetime precisely to a
4.3. Assessing structural and chemical factors impacting saccharification The chemical and spectral properties factors that were determined for the biomass samples were correlated to the saccharification yield in order to highlight the relative importance of each factor. Correlation coefficients (r) were calculated considering all samples (untreated and pretreated pine and poplar samples) (Fig. 7). Lignin content and lignin β-O-4′ percentage were negatively correlated with saccharification (r = -0.76 and -0.88, respectively) while hemicellulose and lignin contents also showed a negative value (-0.82). Interestingly, the highest positive correlation is between lifetime and saccharification yield (+0.92), whereas the strongest negative correlations are between lifetime and βO-4′ (-0.88) and between lifetime and lignin content (-0.87). Thus lifetime seems to be an indicator of both the lignin content and the β-O89
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Fig. 7. Correlation between factors characterizing pine and poplar samples: LIG (lignin content as percent dry matter), B-O-4 (β-O-4′content as percent of lignin), HEM (hemicellulose content as the sum of xylose + mannose as percent dry matter), YIELD (saccharification yield as percent initial glucose), LIFET (lifetime as ps). Both color and circle size indicates a strong correlation (blue: positive; red: negative) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Fig. 5. FRET efficiency measurements for the control and pretreated pine and poplar. Error bars show the 95% confidence interval.
4′ content of lignin, reflecting a combination of the lignin structural features as previously hypothesized. The strong positive relation between lifetime fluorescence and saccharification yields suggests that FLIM could be used as a fast method to predict enzymatic conversion of lignocelluloses.
4.4. Fluorescent probe distribution reveals cell wall organization Small PEG probes labelled with rhodamine have previously been demonstrated to bind cell walls in steam-exploded pine (Donaldson et al., 2014). PEG probes were used to reveal the cell wall organization as they can interact to the cell walls thus allowing FRET measurements in contrast to dextran probes which have almost no interaction with cell wall polymers. Using poplar and pine biomass, our results show increasing RhPEG distribution is more dependent on the pretreatment than on the probe’s size. Indeed, considering the structural variations between poplar and pine as well as between wall layers (Donaldson, 2001; Scheller and Ulvskov, 2010), probe distribution suggests differences in chemical features and in reactivity to pretreatments at the nanoscale. The highest penetration in the chlorite samples indicates that the cell wall is more accessible to the probe when lignin is largely removed. This result is in good agreement with a recent study reporting increased porosity in chlorite pretreated poplar and grass species (Herbaut et al., 2018). Regarding hot water pretreatment, there is some evidence for less uniform distribution of probes in pine than in poplar, which may result from variations in cell wall structures after the pretreatment. They can likely be explained by some modifications of the hemicellulose content, which is reduced by 50% (mannan) in pine and only by 25% (xylan) in poplar. Overall, probes showed greater penetration in the secondary walls, except for chlorite pine samples, suggesting that the secondary cell wall is more accessible to probes as compared to the more lignified middle lamella (Donaldson, 2001). Variation in probes penetration may thus reflect possible alteration of the lignin structure and polymer arrangement within the cell wall network, which can vary between softwood and hardwood.
Fig. 6. Effect of probe size on FRET efficiency measurements for the control and pretreated pine and poplar. 90
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4.5. FRET of fluorescence probes can explain cell wall organization and predict saccharification
the cell wall, as determined by FRET measurements, partially explains the susceptibility of cell walls towards enzyme saccharification. Further evaluation of the capacity of probes to diffuse in the cell walls (Paës et al., 2017) and of the ultrastructural effects of the pretreatment could provide a more complete view of the impact of cell wall architecture on enzyme degradation.
Probes interaction with the cell wall was quantified by measuring the FRET that is assumed to occur between lignin (donor) and rhodamine (acceptor) (Donaldson et al., 2014). In addition to classical requirements (dipole orientation, fluorophore vicinity), FRET depends on i) the capacity of the fluorescent probe to penetrate the pores in the samples, ii) the binding interaction between PEG and cell wall: we hypothesize that the longer the PEG chain of the probe is, the stronger it will bind. FRET efficiency was strongly negatively correlated with probe size (Fig. 6), indicating that the overriding factor in FRET efficiency is accessibility rather than binding. Particularly, FRET efficiency increased after chlorite pretreatment irrespective of biomass species and the probe size. This indicates that this pretreatment increases the probes’ penetration and their interaction within the cell wall network. However for the largest probe (40 kDa), chlorite poplar showed almost similar FRET efficiency to the untreated sample albeit imaging RhPEG40 indicated uneven distribution in the cell walls. In contrast imaging RhPEG40 in chlorite treated pine did not show stronger penetration of this probe, suggesting that the higher FRET efficiency as compared to untreated pine could be related to the length of the PEG chain; thus favouring interaction with delignified pine cell walls. For hot water treated samples, the decreased FRET efficiency in poplar for all probes in comparison to the untreated sample, revealed differences in the cell wall accessibility and reactivity between poplar and pine. This cannot be ascribed to the increase in lignin content, which is similar to that of pine after hot water treatment. Rather, it is probably due to complex structural changes at the polymer scale, such as some hemicellulose disruption combined with lignin structural modifications and interactions between cell wall polymers, causing reduced accessibility. The decrease in hemicellulose content (as xylan + mannan) after hot water treatment is lower in poplar (15.1% and 11.4% in control and hot water poplar, respectively) compared to pine (13.7% and 8.7% in control and hot water pine). Thus hemicellulose removal may have different impacts on the cell wall nanoporosity of pine and poplar. Recent study has reported that pretreatments induce different changes in the cell wall nanoporosity depending on the biomass species (Herbaut et al., 2018). Overall, this indicates that the impact of pretreatment on FRET was dependent on wood species. Finally, the correlation between FRET efficiency and saccharification yield was tested, considering each probe size independently. Interestingly, the saccharification yield showed decreasing positive correlation values of +0.80 for RhPEG1 down to +0.45 for RhPEG40. This means that the small probe, whose size is less than the average size of cellulases degrading cellulose (Luterbacher et al., 2013), has increasing interactions with cell walls in samples whose hydrolysis is favored. The measurement of FRET with of such a small 1 kDa probe can thus be considered as a relevant indicator of sample digestibility.
Funding This work was supported by the French Ministry of Foreign Affairs and Ministry of Higher Education and Research, and the New Zealand Ministry of Business, Innovation and Employment within the frame of the Dumont d’Urville programme for facilitating collaboration between France and New Zealand. Authors’ contributions BC, GP and LD designed the study, analysed results and wrote the manuscript. AH and MH performed the pretreatments, enzymatic saccharification, analysed probe size and prepared samples. CT performed FLIM measurements and analysis. AV provided critical reading of the manuscript. All authors read and approved the final manuscript. Acknowledgments The authors thank David Crônier for his technical assistance in chemical analysis and Novozymes A/S for providing the cellulase cocktail. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.06.058. References Agbor, V.B., Cicek, N., Sparling, R., Berlin, A., Levin, D.B., 2011. Biomass pretreatment: fundamentals toward application. Biotechnol. Adv. 29, 675–685. Arantes, V., Saddler, J.N., 2011. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol. Biofuels 4. Asada, C., Sasaki, C., Hirano, T., Nakamura, Y., 2015. Chemical characteristics and enzymatic saccharification of lignocellulosic biomass treated using high-temperature saturated steam: comparison of softwood and hardwood. Bioresour. Technol. 182, 245–250. Assor, C., Placet, V., Chabbert, B., Habrant, A., Lapierre, C., Pollet, B., Perré, P., 2009. Concomitant changes in viscoelastic properties and amorphous polymers during the hydrothermal treatment of hardwood and softwood. J. Agric. Food Chem. 57, 6830–6837. Auxenfans, T., Crônier, D., Chabbert, B., Paës, G., 2017a. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. Biotechnol. Biofuels 10, 36. Auxenfans, T., Terryn, C., Paës, G., 2017b. Seeing biomass recalcitrance through fluorescence. Sci. Rep. 7. Balakshin, M., Capanema, E., Gracz, H., Chang, H.M., Jameel, H., 2011. Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy. Planta 233, 1097–1110. Belmokhtar, N., Habrant, A., Ferreira, N.L., Chabbert, B., 2013. Changes in phenolics distribution after chemical pretreatment and enzymatic conversion of Miscanthus x giganteus internode. Bioenergy Res. 6, 506–518. Berezin, M.Y., Achilefu, S., 2010. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110, 2641–2684. Burton, R.A., Gidley, M.J., Fincher, G.B., 2010. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat. Chem. Biol. 6, 724–732. Chundawat, S.P.S., Donohoe, B.S., Sousa, L.D., Elder, T., Agarwal, U.P., Lu, F.C., Ralph, J., Himmel, M.E., Balan, V., Dale, B.E., 2011. Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment. Energy Environ. Sci. 4, 973–984. Coletta, V.C., Rezende, C.A., da Conceicao, F.R., Polikarpov, I., Guimaraes, F.E.G., 2013. Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy. Biotechnol. Biofuels 6. Ding, S.Y., Liu, Y.S., Zeng, Y.N., Himmel, M.E., Baker, J.O., Bayer, E.A., 2012. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338, 1055–1060. Donaldson, L.A., 2001. Lignification and lignin topochemistry - an ultrastuctural view.
5. Conclusions Fluorescence imaging and FRET microscopy indicate that pretreatments induce cell-wall structural changes in both softwoods and hardwoods, resulting in enhanced enzymatic saccharification yields. However, there are important differences in the behaviour of the two wood species after pretreatment, especially in relation to hot water extraction, which reflect differences in hemicellulose composition. Fluorescence lifetime of lignin is correlated with glucose conversion and lignin traits, and would provide a new criteria to assess relevant cell wall structural modifications related to the enzymatic conversion of lignocellulosic biomass. In addition, the interaction of chlorite-treated cell walls with fluorescent probes (FRET efficiency) using RhPEG probes of different sizes confirms that lignin is the dominant barrier to probe accessibility, which may also reflect accessibility to cellulolytic enzymes. Taken together, these results demonstrate that accessibility of 91
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Phytochemistry 57, 859–873. Donaldson, L.A., Radotic, K., 2013. Fluorescence lifetime imaging of lignin autofluorescence in normal and compression wood. J. Microsc. 251, 178–187. Donaldson, L.A., Vaidya, A., 2017. Visualising recalcitrance by colocalisation of cellulase, lignin and cellulose in pretreated pine biomass using fluorescence microscopy. Sci. Rep. 7, 44386. Donaldson, L.A., Newman, R.H., Vaidya, A., 2014. Nanoscale interactions of polyethylene glycol with thermo-mechanically pre-treated Pinus radiata biofuel substrate. Biotechnol. Bioeng. 111, 719–725. Donaldson, L.A., Kroese, H.W., Hill, S.J., Franich, R.A., 2015. Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy. J. Microsc. 259, 228–236. Donohoe, B.S., Resch, M.G., 2015. Mechanisms employed by cellulase systems to gain access through the complex architecture of lignocellulosic substrates. Curr. Opin. Chem. Biol. 29, 100–107. Herbaut, M., Zoghlami, A., Habrant, A., Falourd, X., Foucat, L., Chabbert, B., Paës, G., 2018. Multimodal analysis of pretreated biomass species highlights generic markers of lignocellulose recalcitrance. Biotechnol. Biofuels 11, 52. Iiyama, K., Wallis, A.F.A., 1990. Determination of lignin in herbaceous plants by an improved acetyl bromide procedure. J. Sci. Food Agric. 51, 145–161. Kumar, R., Hu, F., Hubbell, C.A., Ragauskas, A.J., Wyman, C.E., 2013. Comparison of laboratory delignification methods, their selectivity, and impacts on physiochemical characteristics of cellulosic biomass. Bioresour. Technol. 130, 372–381. Lapierre, C., Monties, B., Rolando, R., 1986. Thioacidolysis of poplar lignins: identification of monomeric syringyl products and characterization of guaiacyl-syringyl rich fractions. Holzforschung 40, 113–119. Luterbacher, J.S., Parlange, J.Y., Walker, L.P., 2013. A pore-hindered diffusion and reaction model can help explain the importance of pore size distribution in enzymatic hydrolysis of biomass. Biotechnol. Bioeng. 110, 127–136. Ma, J., Zhang, X., Zhou, X., Xu, F., 2014. Revealing the changes in topochemical ccharacteristics of poplar cell wall during hydrothermal pretreatment. Bioenergy Res. 7, 1358–1368. Meng, X.Z., Wells, T., Sun, Q.N., Huang, F., Ragauskas, A., 2015. Insights into the effect of dilute acid, hot water or alkaline pretreatment on the cellulose accessible surface area and the overall porosity of Populus. Green Chem. 17, 4239–4246. Menon, V., Rao, M., 2012. Trends in bioconversion of lignocellulose: biofuels, platform chemicals & biorefinery concept. Prog. Energy Combust. Sci. 38, 522–550. Moran-Mirabal, J.M., 2013. The study of cell wall structure and cellulose-cellulase interactions through fluorescence microscopy. Cellulose 20, 2291–2309. Mottiar, Y., Vanholme, R., Boerjan, W., Ralph, J., Mansfield, S.D., 2016. Designer lignins: harnessing the plasticity of lignification. Curr. Opin. Biotechnol. 37, 190–200. Nitsos, C.K., Choli-Papadopoulou, T., Matis, K.A., Triantafyllidis, K.S., 2016. Optimization of hydrothermal pretreatment of hardwood and softwood lignocellulosic residues for selective hemicellulose recovery and improved cellulose enzymatic hydrolysis. ACS Sustain Chem. Eng. 4, 4529–4544. Paës, G., 2014. Fluorescent probes for exploring plant cell wall deconstruction: a review. Molecules 19, 9380–9402. Paës, G., Habrant, A., Ossemond, J., Chabbert, B., 2017. Exploring accessibility of pretreated poplar cell walls by measuring dynamics of fluorescent probes. Biotechnol.
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Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Fluorescence techniques can reveal cell wall organization and predict saccharification in pretreated wood biomass
T
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Brigitte Chabberta, , Christine Terrync, Mickaël Herbauta, Alankar Vaidyab, Anouck Habranta, ⁎ ⁎ Gabriel Paësa, , Lloyd Donaldsonb, a
FARE Laboratory, INRA, University of Reims Champagne-Ardenne, 51100 Reims, France Scion, Private Bag 3020, Rotorua 3046, New Zealand c PICT platform, University of Reims Champagne-Ardenne, 51100 Reims, France b
A R T I C LE I N FO
A B S T R A C T
Keywords: Confocal fluorescence microscopy Pretreatment Poplar Pine Accessibility FLIM PEG probe Cellulases
The conversion of cell wall biomass to sugar for fermentation to ethanol requires chemical or physical pretreatments to disrupt the recalcitrant plant cell walls and to make the cellulose accessible to cellulolytic enzymes. Multiscale study of biomass deconstruction gives access to key insights into the cell wall and lignin changes induced by pretreatment. Few studies have compared the effect of pretreatment of different biomass species on the cell wall accessibility. Considering representative softwood (pine) and hardwood (poplar) biomass, we studied the impact of two pretreatments on enzymatic saccharification and cell wall structure and accessibility. Lignin fluorescence properties were investigated by measuring fluorescence lifetime in addition to chemical analysis, and the accessibility of biomass was assessed using fluorescent probes consisting of rhodamine labeled polyethylene glycol (PEG) molecules ranging from 10 to 40 kDa. Hot water treatment and chlorite delignification altered chemical structure and fluorescence lifetime, which was positively correlated with glucose conversion and negatively correlated with lignin and β-O-4′ contents. Imaging distribution of the probes indicated that chlorite pretreatment resulted in a more uniform distribution of probe in the cell wall compared to hot water treatment. The interaction between cell wall and fluorescent PEG probes was evaluated using Förster Resonance Energy Transfer (FRET) and fluorescence microscopy. The FRET efficiency showed a high negative correlation with the probe size and was greatly increased by chlorite delignification, reflecting increased accessibility to the probe and interaction. Thus the accessibility and interactions of small probes in pretreated biomass could be a relevant indicator of potential for saccharification, whereas fluorescence lifetime provides a new criteria for assessing relevant cell wall structural modifications related to enzymatic conversion of lignocellulosic biomass.
1. Introduction There is increasing interest in the use of plant biomass for the production of sustainable liquid fuels and chemicals to replace fossil carbon, due to declining supply, and to mitigate increasing atmospheric CO2 (Menon and Rao, 2012). Plant cell wall biomass consists of a framework of cellulose surrounded by a matrix of complex polymers including hemicellulose and lignin. These polymers are interconnected by non-covalent and covalent linkages resulting in a complex structural and chemical network (Burton et al., 2010) with physical and chemical barriers limiting cellulase
enzyme penetration and progression (Paës et al., 2017), leading to decreased enzyme activity (Arantes and Saddler, 2011; Zhao et al., 2012). Cellulase activity is limited by the crystallinity of cellulose and its restricted accessibility inside the cell wall matrix of hemicelluloses and lignin (Ding et al., 2012). In addition to being a structural obstacle, lignin can give rise to non-productive enzyme binding, so it is considered to be the main factor responsible for lignocellulose recalcitrance (Vaidya et al., 2014; Zeng et al., 2014). The conversion of cell wall biomass to monosaccharides for fermentation into ethanol thus requires chemical or physical pretreatments to disrupt the plant cell walls and increase porosity in
Abbreviations: FLIM, fluorescence lifetime imaging microscopy; FRET, Förster (or Fluorescence) resonance energy transfer; MW, molecular weight; RhPEG1, rhodamine labeled polyethylene glycol of 1 kDa; RhPEG3.4, rhodamine labeled polyethylene glycol of 3.4 kDa; RhPEG10, rhodamine labeled polyethylene glycol of 10 kDa; RhPEG40, rhodamine labeled polyethylene glycol of 40 kDa ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Chabbert), [email protected] (G. Paës), [email protected] (L. Donaldson). https://doi.org/10.1016/j.indcrop.2018.06.058 Received 2 April 2018; Received in revised form 12 June 2018; Accepted 14 June 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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treatments induce alteration of the residual lignin structure (Kumar et al., 2013; Ma et al., 2014; Paës et al., 2017), thus allowing comparison of cell wall accessibility in hardwood and softwood biomass. In particular, interactions of probes with pretreated biomass was assayed by FRET measurements, while FLIM was used to assess cell wall modifications. Such complementary microscopic techniques provide new insights for cell wall accessibility which cannot be obtained by porosity measurements.
order to make the cellulose accessible to cellulolytic enzymes while minimizing the formation of by-products (Silveira et al., 2015). A wide variety of pretreatments has been proposed and each has advantages and limitations (Agbor et al., 2011; Singh et al., 2015). Some pretreatments improve the accessibility of cellulose by removal of matrix components such as lignin (delignification associated with pulping) and hemicelluloses (hot water treatment, steam-explosion or acid treatments). Removal of lignin alone is usually insufficient to optimize sugar yield because hemicelluloses are covalently linked to lignin and/or closely interact with the surface of elementary cellulose, thus forming a physical and chemical barrier that limits enzymatic accessibility and cellulose conversion yields (Ding et al., 2012; Meng et al., 2015; Penttilä et al., 2013; Trajano et al., 2013). Improvement of cellulose conversion is related to the severity and the type of pretreatment and depends on the biomass species, due to variation in the content and composition of lignin and hemicellulose (Arantes and Saddler, 2011; Asada et al., 2015; Singh et al., 2015). For instance, hardwoods and softwoods differ in terms of lignin composition (mixed syringyl-guaiacyl vs guaiacyl) and hemicellulose composition (xylan vs mannan) (Ralph et al., 2004; Scheller and Ulvskov, 2010). These differences contribute to the effectiveness of biomass pretreatment, with xylans being readily extractable by hot water whilst mannans require more intensive treatment (Meng et al., 2015; Nitsos et al., 2016). Syringyl-rich hardwood lignins are more easily chemically degraded than guaiacyl-type softwood lignin due to a lower degree of condensation, a less complex structure, smaller polymer size and higher content of β-ethers (Mottiar et al., 2016; Ralph et al., 2004; Studer et al., 2011). Apart from chemical and structural characterization, microscopy has been an effective tool for the study of biomass pretreatment at multiscale levels (Donohoe and Resch, 2015). Light microscopy and microspectrometry combining UV, infrared and Raman analysis have provided critical information on cell wall chemical changes caused by pretreatments and enzyme hydrolysis (Belmokhtar et al., 2013; Chundawat et al., 2011; Ma et al., 2014; Singh et al., 2015). Among microscopy approaches, confocal fluorescence microscopy is widely used to visualize biomass deconstruction, and gives key insights into cell wall and lignin changes induced by pretreatment (Singh et al., 2015). For example, confocal fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) were used to characterize lignin modification after pretreatments in sugar cane bagasse and eucalyptus fiber (Coletta et al., 2013) and in poplar, wheat straw and miscanthus (Auxenfans et al., 2017b; Zeng et al., 2015). Confocal microscopy is also a fast method to provide a spatial and temporal visualization of fluorescently labeled enzyme and to characterize enzyme-substrate interactions within biomass (Ding et al., 2012; Donaldson and Vaidya, 2017; Luterbacher et al., 2013; Moran-Mirabal, 2013; Thygesen et al., 2011). Confocal fluorescence microscopy can also reveal cell wall modification and cellulose porosity by determining the distribution and mobility of fluorescent probes that vary in molecular weight (MW) or affinity towards cell wall polymers (Donaldson et al., 2015; Moran-Mirabal, 2013; Paës, 2014; Paës et al., 2017; Yang et al., 2013). Few studies have compared the effect of pretreatment of different biomass species on the accessibility of the cell wall. Donaldson et al. (2014) have shown that rhodamine labeled polyethylene glycol (PEG) penetrates steam-exploded wood. The fluorescent PEGs can then be localized by direct imaging of the rhodamine dye and their strength of interaction can be evaluated by measuring the Förster (or Fluorescence) Resonance Energy Transfer (FRET) between rhodamine and cell wall lignins at the molecular level. In this work, we use a set of fluorescence microscopy techniques to study the effects of two different pretreatments (chlorite delignification and hot water extraction) on cell wall accessibility in poplar (hardwood) and pine (softwood) using rhodamine labeled PEGs of various sizes as probes. Chlorite delignification reduces lignin content whilst hot water extraction mainly reduces hemicellulose content; both
2. Materials and methods 2.1. Sample preparation Small wood blocks (3–4 mm width × 2 cm long) were isolated from the basal region of 3-year-old short rotation poplar coppice (Orléans, France) and from mature pine wood (Rotorua, New Zealand) which were subsequently dried at 40 °C for 2 days. Pretreatments were performed as previously described (Paës et al., 2017). Hot water treatment was performed for 1 h at 170 °C using mineralization reactors equipped with Teflon tubes (PARR, USA). Sodium chlorite-acetic acid delignification treatment (Wise et al., 1946) was performed on 1 g poplar samples using acetic acid and sodium chlorite at 70 °C for 1 h and the reaction was repeated 5 times. Untreated control samples were also obtained after water washing at 4 °C (1 h). After pretreatment, samples were washed several times with deionized water until the pH of the wash was about 6.0. Then samples were dried at 40 °C in an air forced oven for 2 days. One sample fraction was ground to 200 μm size prior to chemical analysis and enzymatic saccharification, and the other fraction was sectioned with a sledge microtome at a thickness of 30 μm for confocal fluorescence microscopy. 2.2. Chemical analysis and enzymatic saccharification The sugar monomer composition was determined using a two-step sulfuric acid hydrolysis (Seaman et al., 1954) followed by high-performance anion-exchange chromatography (HPAEC-PAD) with 2deoxy-D-ribose as internal standard (Paës et al., 2017). Lignin content was quantified using a spectrophotometric method after acetyl bromide dissolution of the lignocelluloses (Iiyama and Wallis, 1990) and determination of the monomer composition of the alkyl aryl ether lignin structures was achieved by thioacidolysis as previously described (Belmokhtar et al., 2013; Lapierre et al., 1986). Enzymatic saccharification was performed on the ground samples (2% w/v) using a commercial cellulase preparation, Cellic ® CTec2 kindly provided by Novozymes A/S (Bagsværd, Denmark), in 10 mL sodium citrate buffer (0.1 M; pH 5.0) for 48 h at 50 °C and 200 rpm using an enzyme loading of 40 FPU/g-glucan. The reaction mixtures were pre-incubated for 1 h at 50 °C and 200 rpm. Enzymatic hydrolysis was initiated by addition of the cellulase solution and stopped after 72 h by heating for 15 min at 100 °C. Control experiments without enzyme were also carried out. The amount of glucose released was determined using high-performance anion-exchange chromatography (HPAECPAD). Conversion yields were expressed as a percentage of initial glucose amount (Auxenfans et al., 2017a). 2.3. Confocal fluorescence microscopy Samples of untreated control and pretreated pine and poplar wood were sectioned in the transverse plane with a sledge microtome at a thickness of 30 μm. Sections were used for fluorescence spectroscopy, FLIM, and FRET measurements. Fluorescence imaging was performed using a Leica SP5 II confocal microscope at 1024 × 1024 pixel resolution with a 63× glycerol immersion lens. FLIM measurements were performed on the untreated control and pretreated pine and poplar wood sections mounted in 50% glycerol in 10 mM phosphate buffer at pH 7 (Donaldson and Radotic, 2013) using a Zeiss LSM710 multiphoton 85
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microscope equipped with a Becker and Hickl lifetime spectrometer. Decay spectra were acquired with a biphoton excitation at 750 nm and emission between 420 and 722 nm. Data were analyzed using a multiexponential decay model with 2 components. Measurements of average fluorescence lifetime were made on 10 fields of view for each species/ pretreatment combination. For FRET analysis, sections from wood samples were imaged without treatment to act as controls for lignin fluorescence. Other sections were treated with 2 mL of 1.6 μM solutions of rhodamine B labeled PEGs (RhPEG, NANOCS, New York, USA) in water at 1, 3.4, 10 and 40 kDa (RhPEG1, RhPEG3.4, RhPEG10, RhPEG40) for 1 h at room temperature (22 °C) in the dark, followed by washing in distilled water (5 min twice). All sections were mounted in 50% glycerol in phosphate buffer at pH 7. The fluorescence spectrum of rhodamine B was measured with the confocal microscope using a 1.6 μM solution in distilled water. Hydrodynamic radius (RH) of the fluorescent probes was determined by quasi-elastic light scattering (QELS) (Paës et al., 2017). FRET measurements were performed using the acceptor photobleaching technique, using sequential excitation at 488 nm for the donor (lignin in wood samples) and 561 nm for the acceptor (rhodamine in RhPEG probes) with emission at 500–570 and 570–700 nm, respectively (Donaldson et al., 2014). Bleaching of rhodamine label for FRET measurements was performed with 100% laser power at 561 nm for 50 scans. FRET measurements were performed at 512 × 512 pixel resolution but some additional FRET imaging was done at 1024 × 1024 for presentation purposes. FRET calculations were performed using Leica LAS AF software. Images of FRET efficiency on a 0–100% scale were calculated using Digital Optics V++ software. FRET measurements were repeated on 10 fields of view for each probe/treatment combination. FRET efficiency was also measured on sections with no probe treatment and in areas outside the region where photobleaching was performed on probe treated sections to act as additional controls.
Fig. 1. Enzymatic conversion yield (% initial glucose) for pine and poplar. Error bars show the 95% confidence interval.
and xylan content in pine and poplar, respectively) and pectins (lower content of arabinose, galactose, and galacturonic acid), whereas chlorite removed lignin. Pretreated samples contained a higher proportion of glucan compared to control biomass. Lignin content increased after hydrothermal pretreatment as a consequence of hemicellulose removal. Nevertheless, the residual lignin displayed structural alterations as a lower proportion of lignin monomers was obtained by thioacidolysis for both pine and poplar, indicating a reduction in labile ether β-O-4′ linkages content. This modification was even more pronounced after extensive removal of lignin by chlorite pretreatments. Enzymatic hydrolysis of untreated poplar and pine controls gave similar glucan conversion yields of 32% (Fig. 1). Chlorite pretreatment greatly enhanced conversion yields (close to 100%), with a three-fold increase in comparison to controls. Hot water pretreatment was less efficient on pine (40% conversion yield, only a slight increase compared to the control sample), while poplar showed a 2-fold increase in conversion (ca. 80%).
2.4. Data treatment Significant differences between treatments were evaluated using Student’s t-test (Excel) at 0.95 confidence level. Pearson correlation coefficients were calculated to measure the degree of the relationship between the chemical and spectral properties of biomass and their saccharification yields.
3.2. Fluorescence lifetime measurement The fluorescence lifetime of poplar was higher than that of pine in controls (Fig. 2). For hot water pretreated samples, lifetime increased significantly for pine and poplar. Chlorite-treated samples showed the highest increase in fluorescence lifetime: 5-fold for pine and 2-fold for poplar in comparison to control samples, respectively. Lifetime variations could be clearly visualized in the corresponding lifetime colorcoded images of cell walls and were not significantly different between cell corner and secondary cell walls except for the hot water treated poplar that showed 15% higher fluorescence lifetime in the secondary wall layer (Fig. 2).
3. Results 3.1. Chemical composition and enzymatic saccharification Chemical analysis showed differences in polysaccharide and lignin compositions between pine and poplar (Table 1). Following pretreatments, weight losses close to 22% were obtained for pine (19% after hot water and 25% after chlorite) and 30% for poplar (27% after hot water and 29%, after chlorite). Similar effects were observed for the two biomass species: hot water removed mainly hemicelluloses (mannan
3.3. Probe distribution Distribution of the RhPEG probes in wood sections was visualized
Table 1 Chemical composition of untreated and pretreated pine and poplar samples. Lignin and sugar data are expressed as percent of dry matter and β-O-4′ lignin is expressed as percent of lignin. Glc (glucose), Xyl (xylose), Man (mannose), other sugars refer to the total content of minor monosaccharides (arabinose, galactose, uronic acids).
Pine
Poplar
Control Hot water Chlorite Control Hot water Chlorite
Total Lignin (% dry matter)
β-O-4’ Lignin
27.28 ± 2.70 34.32 ± 1.73 7.00 ± 0.34 25.60 ± 0.16 32.55 ± 1.10 6.90 ± 0.73
30.62 ± 0.97 20.00 ± 1.09 < 1.00 39.71 ± 0.86 25.46 ± 3.72 3.03 ± 0.43
Total Sugar
Glc
Xyl
(% lignin)
Man
Ara + Gal + GalU
10.33 ± 0.34 5.12 ± 0.03 10.70 ± 0.38 2.04 ± 0.17 1.76 ± 0.05 2.64 ± 0.03
1.09 0.10 0.89 0.29 0.05 0.23
(% dry matter) 58.80 58.36 66.96 53.20 51.15 66.46
± ± ± ± ± ±
2.05 0.16 3.17 4.26 1.08 1.19
86
41.91 48.61 49.37 35.73 39.05 44.81
± ± ± ± ± ±
1.56 0.20 2.57 2.87 0.75 0.89
3.43 ± 0.09 3.58 ± 0.03 4.07 ± 0.16 13.02 ± 1.06 9.65 ± 0.23 16.43 ± 0.43
± ± ± ± ± ±
0.03 0.01 0.04 0.02 0.01 0.02
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was essentially no detectable FRET in cell walls incubated only with water (data not shown). Similarly, cell walls incubated with probes but not subjected to acceptor photobleaching did not show any FRET. Although equimolar concentrations of RhPEG probes were used, it was apparent that the different probes had different rhodamine labelling ratios depending on their size, as shown by their color (Additional file 1: Figure S1). To assay the impact of labelling ratio on FRET efficiency, FRET was determined for a dilution series of the RhPEG1 probe ranging from 0.5 μM to 2.5 μM. FRET measurements for control pine sections treated with each of the 5 dilutions showed a very small variation in FRET efficiency (from 14% for 0.5 μM to 18% for 2.5 μM) (Additional file 2: Figure S2), demonstrating that labelling ratio of RhPEG probes has a very moderate influence on FRET. FRET measurements were performed considering the whole cell wall region to compare the overall effect of probe size regarding pretreatment and biomass. FRET efficiency generally declined with increasing MW of the probe in both wood species (Fig. 5). Untreated poplar showed higher FRET efficiency (10–30%) than pine (2–20%) regardless of the probe size. For the hot water pretreatment, FRET efficiency in comparison to the untreated samples was either unchanged or slightly increased depending on the probe size in pine, whereas it was decreased in poplar for all probes. Finally, for the chlorite pretreatment, FRET efficiency was greatly increased in both species for all probes in comparison to the controls, with the exception of RhPEG40 which was unchanged for poplar. These results can be further interpreted considering FRET values and the hydrodynamic radii (RH) of the probes, which is representative of their size as diffusing molecules. Indeed, the increasing MW (from 1 to 40 kDa) of the RhPEG probes corresponds to an increase in their RH from 1.3 to 4.8 nm as determined by QELS. Thus considering poplar or pine wood samples, the FRET efficiency showed a strong negative correlation with the probe size (Fig. 6). Chlorite pretreatment results in a sharper increase of FRET efficiency with decreasing size of the probe (Fig. 6) as compared to control and hot water pretreatment.
Fig. 2. Fluorescence lifetime measurements (A) and corresponding lifetime images (B) for control and pretreated pine and poplar. Error bars show the 95% confidence interval. Scale bars = 30 μm.
by fluorescence imaging of the rhodamine at 561 nm excitation (Fig. 3). Wood sections with no RhPEG probe treatment did not show any detectable lignin autofluorescence at 561 nm excitation with the imaging parameters chosen. For the untreated pine, RhPEG was localized in the secondary cell wall with an uneven distribution especially near the cell corners; the distribution was similar for probes whatever their size. In the hot water pretreated pine sample the secondary wall was strongly infiltrated only by the RhPEG1 probe with higher penetration in the S1 layer compared to the untreated sample. In sections treated with larger size probes, a moderate penetration of the RhPEG was observed in the cell wall albeit higher in the S3 layer. Probe distribution in the chloritetreated sample was lower in the secondary wall and higher in the middle lamella, and was not dependent on probe size. For poplar samples, results for the control without pretreatment were similar to those for pine, showing weaker distribution of the probes in the middle lamella and outer part of the secondary cell wall (Fig. 4). There was no obvious trend with increasing probe size. For the hot water pretreatment, all four probes showed a greater affinity for vessel (arrows) cell walls compared to fibers. Also, the distribution of RhPEG was more uniform in the cell wall, with localization in both secondary cell wall and middle lamella; the larger probe seemed to better penetrate into the middle lamella. For the chlorite pretreatment (Fig. 4), the probe distribution was very uniform irrespective of probe size.
4. Discussion 4.1. Lignin is a barrier to cellulose accessibility Pretreatments which facilitate removal of hemicellulose and lignin from lignocellulosic biomass are known to increase enzymatic conversion of cellulose (Arantes and Saddler, 2011; Asada et al., 2015; Silveira et al., 2015; Singh et al., 2015). Our results show that hot water treatment mainly induced a reduction in xylan in poplar samples as previously reported (Assor et al., 2009; Paës et al., 2017; Trajano et al., 2015) and allowed higher glucose conversion yield. In pine, hot water pretreatment removed about 50% of the mannan but did not induce higher enzymatic conversion, suggesting that residual mannan in pine limits saccharification. Unlike poplar, hot water extraction did not reduce the xylan content of pine (3.4 and 3.6% dry matter of control and hot water treated pine respectively). One may hypothesize that the higher recalcitrance of hot water pretreated pine could arise from increased complexity of lignin-carbohydrate (LCC) bonds involving different hemicelluloses in comparison to hardwood LCCs that mostly involve xylan hemicellulose (Balakshin et al., 2011). Residual xylan is more tightly bound to lignin in hot water treated pine in comparison to poplar, and could contribute to pine recalcitrance. In addition interactions between lignin-hemicellulose could be modified as a consequence of the structural changes of lignin induced by the pretreatment. Indeed, residual lignin contained a lower proportion of β-O-4′ ether linkages as lignin undergoes both disruption of labile ether linkage and recondensation mechanisms during hot water treatment (Paës et al., 2017; Trajano et al., 2013). For chlorite-treated samples, delignification of pine and poplar reached the same extent (ca. 75%) and this could be the main reason for the high conversion yield (ca. 100%). Thus, removal of lignin increases cellulose accessibility more
3.4. FRET efficiency RhPEG probes were then used to measure the efficiency of FRET which was assumed to occur between lignin (donor) and rhodamine (acceptor) under conditions of close molecular proximity (Coletta et al., 2013). Measurements were performed on the whole cell walls. There 87
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Fig. 3. RhPEG distribution in pine control, hot water, and chlorite treated substrates. (S1 and S3: secondary cell wall layers). Scale bars = 40 μm.
interlinkages. Fluorescence lifetime is independent of fluorescence intensity and fluorophore concentration (Berezin and Achilefu, 2010). However, lifetime is sensitive to fluorophore structure, composition, and presence of interacting surrounding molecules, so that any change in the fluorophore environment can lead to changes in lifetime. Regarding lignin composition, poplar contains much more syringyl lignin than pine in which lignin is composed mainly of the guaiacyl type. Moreover, poplar is known to have unusual lignin groups (p-hydroxybenzoate) (Ralph et al., 2007). Changes in lifetime have also been associated with different lignin structures in pretreated poplar wood (Auxenfans et al., 2017b; Zeng et al., 2015). Loosely packed lignin with a long lifetime (4 ns) was associated with secondary walls while dense lignin with a short lifetime (0.5–1.0 ns) was associated with lignin distributed throughout all the cell wall layers. Dilute acid pretreatments were found to shorten lignin lifetime by extracting from the secondary wall the low density lignins with long lifetime. In addition the lifetime of a lignin model compound increased with increasing carboxymethyl
effectively than the removal of hemicellulose.
4.2. Fluorescence lifetime characterization reveals changes in lignin organization Strong variations in the fluorescence lifetime were observed for both poplar and pine : low values (0.3-0.6 ns) for untreated samples, intermediate values (0.6-0.8 ns) for hot water treated samples and high values (1.4–1.8 ns) for chlorite-treated samples, these latter values being similar to those obtained for delignified eucalyptus (Coletta et al., 2013). Although variation in FLIM data is also observed between pine and poplar, the fluorescence lifetime differences seem more dependent on the type of pretreatment (i.e. type of polymer removal, especially lignin) than on biomass species. Fluorescence in plant cell walls mainly originates from lignin, which is composed of monolignols. Thus lignin can be seen as a complex fluorescent molecule made of fluorophores varying in their type and 88
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Fig. 4. RhPEG distribution in poplar control, hot water, and chlorite treated substrates. Scale bars = 40 μm.
specific feature so lifetime must be seen as a general indicator of the lignin state.
cellulose content (Zeng et al., 2015), confirming the importance of surrounding polysaccharides on lignin lifetime. Consequently, it seems reasonable to attribute increased fluorescence lifetime of pretreated samples not only to the removal of hemicelluloses by hot water treatment and of lignin by chlorite treatment, but also to the structural modifications of lignin. As a matter of fact, the lignin β-O-4′ content significantly decreased in pretreated pine and poplar, with chlorite treatment being the most impacting (Table 1). The fact that chlorite-treated samples, despite a very low lignin content, display a long lifetime, highlights the influence of chemical structure on lifetime. The very low proportion of non-condensed lignin likely reflects that inter monomer linkages must be largely altered after chloritetreatment, favoring the creation of a residual low density lignin-like network. Overall, modifications of fluorescence lifetime reflect changes in lignin chemical composition and organization in addition to changes in interactions with other polymers, in particular with hemicelluloses. Right now, it does not seem possible to relate lifetime precisely to a
4.3. Assessing structural and chemical factors impacting saccharification The chemical and spectral properties factors that were determined for the biomass samples were correlated to the saccharification yield in order to highlight the relative importance of each factor. Correlation coefficients (r) were calculated considering all samples (untreated and pretreated pine and poplar samples) (Fig. 7). Lignin content and lignin β-O-4′ percentage were negatively correlated with saccharification (r = -0.76 and -0.88, respectively) while hemicellulose and lignin contents also showed a negative value (-0.82). Interestingly, the highest positive correlation is between lifetime and saccharification yield (+0.92), whereas the strongest negative correlations are between lifetime and βO-4′ (-0.88) and between lifetime and lignin content (-0.87). Thus lifetime seems to be an indicator of both the lignin content and the β-O89
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Fig. 7. Correlation between factors characterizing pine and poplar samples: LIG (lignin content as percent dry matter), B-O-4 (β-O-4′content as percent of lignin), HEM (hemicellulose content as the sum of xylose + mannose as percent dry matter), YIELD (saccharification yield as percent initial glucose), LIFET (lifetime as ps). Both color and circle size indicates a strong correlation (blue: positive; red: negative) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Fig. 5. FRET efficiency measurements for the control and pretreated pine and poplar. Error bars show the 95% confidence interval.
4′ content of lignin, reflecting a combination of the lignin structural features as previously hypothesized. The strong positive relation between lifetime fluorescence and saccharification yields suggests that FLIM could be used as a fast method to predict enzymatic conversion of lignocelluloses.
4.4. Fluorescent probe distribution reveals cell wall organization Small PEG probes labelled with rhodamine have previously been demonstrated to bind cell walls in steam-exploded pine (Donaldson et al., 2014). PEG probes were used to reveal the cell wall organization as they can interact to the cell walls thus allowing FRET measurements in contrast to dextran probes which have almost no interaction with cell wall polymers. Using poplar and pine biomass, our results show increasing RhPEG distribution is more dependent on the pretreatment than on the probe’s size. Indeed, considering the structural variations between poplar and pine as well as between wall layers (Donaldson, 2001; Scheller and Ulvskov, 2010), probe distribution suggests differences in chemical features and in reactivity to pretreatments at the nanoscale. The highest penetration in the chlorite samples indicates that the cell wall is more accessible to the probe when lignin is largely removed. This result is in good agreement with a recent study reporting increased porosity in chlorite pretreated poplar and grass species (Herbaut et al., 2018). Regarding hot water pretreatment, there is some evidence for less uniform distribution of probes in pine than in poplar, which may result from variations in cell wall structures after the pretreatment. They can likely be explained by some modifications of the hemicellulose content, which is reduced by 50% (mannan) in pine and only by 25% (xylan) in poplar. Overall, probes showed greater penetration in the secondary walls, except for chlorite pine samples, suggesting that the secondary cell wall is more accessible to probes as compared to the more lignified middle lamella (Donaldson, 2001). Variation in probes penetration may thus reflect possible alteration of the lignin structure and polymer arrangement within the cell wall network, which can vary between softwood and hardwood.
Fig. 6. Effect of probe size on FRET efficiency measurements for the control and pretreated pine and poplar. 90
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4.5. FRET of fluorescence probes can explain cell wall organization and predict saccharification
the cell wall, as determined by FRET measurements, partially explains the susceptibility of cell walls towards enzyme saccharification. Further evaluation of the capacity of probes to diffuse in the cell walls (Paës et al., 2017) and of the ultrastructural effects of the pretreatment could provide a more complete view of the impact of cell wall architecture on enzyme degradation.
Probes interaction with the cell wall was quantified by measuring the FRET that is assumed to occur between lignin (donor) and rhodamine (acceptor) (Donaldson et al., 2014). In addition to classical requirements (dipole orientation, fluorophore vicinity), FRET depends on i) the capacity of the fluorescent probe to penetrate the pores in the samples, ii) the binding interaction between PEG and cell wall: we hypothesize that the longer the PEG chain of the probe is, the stronger it will bind. FRET efficiency was strongly negatively correlated with probe size (Fig. 6), indicating that the overriding factor in FRET efficiency is accessibility rather than binding. Particularly, FRET efficiency increased after chlorite pretreatment irrespective of biomass species and the probe size. This indicates that this pretreatment increases the probes’ penetration and their interaction within the cell wall network. However for the largest probe (40 kDa), chlorite poplar showed almost similar FRET efficiency to the untreated sample albeit imaging RhPEG40 indicated uneven distribution in the cell walls. In contrast imaging RhPEG40 in chlorite treated pine did not show stronger penetration of this probe, suggesting that the higher FRET efficiency as compared to untreated pine could be related to the length of the PEG chain; thus favouring interaction with delignified pine cell walls. For hot water treated samples, the decreased FRET efficiency in poplar for all probes in comparison to the untreated sample, revealed differences in the cell wall accessibility and reactivity between poplar and pine. This cannot be ascribed to the increase in lignin content, which is similar to that of pine after hot water treatment. Rather, it is probably due to complex structural changes at the polymer scale, such as some hemicellulose disruption combined with lignin structural modifications and interactions between cell wall polymers, causing reduced accessibility. The decrease in hemicellulose content (as xylan + mannan) after hot water treatment is lower in poplar (15.1% and 11.4% in control and hot water poplar, respectively) compared to pine (13.7% and 8.7% in control and hot water pine). Thus hemicellulose removal may have different impacts on the cell wall nanoporosity of pine and poplar. Recent study has reported that pretreatments induce different changes in the cell wall nanoporosity depending on the biomass species (Herbaut et al., 2018). Overall, this indicates that the impact of pretreatment on FRET was dependent on wood species. Finally, the correlation between FRET efficiency and saccharification yield was tested, considering each probe size independently. Interestingly, the saccharification yield showed decreasing positive correlation values of +0.80 for RhPEG1 down to +0.45 for RhPEG40. This means that the small probe, whose size is less than the average size of cellulases degrading cellulose (Luterbacher et al., 2013), has increasing interactions with cell walls in samples whose hydrolysis is favored. The measurement of FRET with of such a small 1 kDa probe can thus be considered as a relevant indicator of sample digestibility.
Funding This work was supported by the French Ministry of Foreign Affairs and Ministry of Higher Education and Research, and the New Zealand Ministry of Business, Innovation and Employment within the frame of the Dumont d’Urville programme for facilitating collaboration between France and New Zealand. Authors’ contributions BC, GP and LD designed the study, analysed results and wrote the manuscript. AH and MH performed the pretreatments, enzymatic saccharification, analysed probe size and prepared samples. CT performed FLIM measurements and analysis. AV provided critical reading of the manuscript. All authors read and approved the final manuscript. Acknowledgments The authors thank David Crônier for his technical assistance in chemical analysis and Novozymes A/S for providing the cellulase cocktail. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2018.06.058. References Agbor, V.B., Cicek, N., Sparling, R., Berlin, A., Levin, D.B., 2011. Biomass pretreatment: fundamentals toward application. Biotechnol. Adv. 29, 675–685. Arantes, V., Saddler, J.N., 2011. Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnol. Biofuels 4. Asada, C., Sasaki, C., Hirano, T., Nakamura, Y., 2015. Chemical characteristics and enzymatic saccharification of lignocellulosic biomass treated using high-temperature saturated steam: comparison of softwood and hardwood. Bioresour. Technol. 182, 245–250. Assor, C., Placet, V., Chabbert, B., Habrant, A., Lapierre, C., Pollet, B., Perré, P., 2009. Concomitant changes in viscoelastic properties and amorphous polymers during the hydrothermal treatment of hardwood and softwood. J. Agric. Food Chem. 57, 6830–6837. Auxenfans, T., Crônier, D., Chabbert, B., Paës, G., 2017a. Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment. Biotechnol. Biofuels 10, 36. Auxenfans, T., Terryn, C., Paës, G., 2017b. Seeing biomass recalcitrance through fluorescence. Sci. Rep. 7. Balakshin, M., Capanema, E., Gracz, H., Chang, H.M., Jameel, H., 2011. Quantification of lignin-carbohydrate linkages with high-resolution NMR spectroscopy. Planta 233, 1097–1110. Belmokhtar, N., Habrant, A., Ferreira, N.L., Chabbert, B., 2013. Changes in phenolics distribution after chemical pretreatment and enzymatic conversion of Miscanthus x giganteus internode. Bioenergy Res. 6, 506–518. Berezin, M.Y., Achilefu, S., 2010. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110, 2641–2684. Burton, R.A., Gidley, M.J., Fincher, G.B., 2010. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat. Chem. Biol. 6, 724–732. Chundawat, S.P.S., Donohoe, B.S., Sousa, L.D., Elder, T., Agarwal, U.P., Lu, F.C., Ralph, J., Himmel, M.E., Balan, V., Dale, B.E., 2011. Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment. Energy Environ. Sci. 4, 973–984. Coletta, V.C., Rezende, C.A., da Conceicao, F.R., Polikarpov, I., Guimaraes, F.E.G., 2013. Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy. Biotechnol. Biofuels 6. Ding, S.Y., Liu, Y.S., Zeng, Y.N., Himmel, M.E., Baker, J.O., Bayer, E.A., 2012. How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338, 1055–1060. Donaldson, L.A., 2001. Lignification and lignin topochemistry - an ultrastuctural view.
5. Conclusions Fluorescence imaging and FRET microscopy indicate that pretreatments induce cell-wall structural changes in both softwoods and hardwoods, resulting in enhanced enzymatic saccharification yields. However, there are important differences in the behaviour of the two wood species after pretreatment, especially in relation to hot water extraction, which reflect differences in hemicellulose composition. Fluorescence lifetime of lignin is correlated with glucose conversion and lignin traits, and would provide a new criteria to assess relevant cell wall structural modifications related to the enzymatic conversion of lignocellulosic biomass. In addition, the interaction of chlorite-treated cell walls with fluorescent probes (FRET efficiency) using RhPEG probes of different sizes confirms that lignin is the dominant barrier to probe accessibility, which may also reflect accessibility to cellulolytic enzymes. Taken together, these results demonstrate that accessibility of 91
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