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

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

Effect of Physicochemical Properties of Carboxymethyl Cellulose on Diffusion of Glucose

Soluble dietary fibers (SDF) are known to reduce the post-prandial plasma glucose levels. However, the detailed mechanisms of this reduced glucose release in the human gut still remain unclear. The aim of our study was to systematically investigate the effect of different types of SDF on glucose release in an in vitro model as a prerequisite for the selection of fibers suitable for application in humans. Three types of carboxymethyl cellulose (CMC) were used to investigate the correlations between fiber concentration, molecular weight (MW), and viscosity on diffusion of glucose using a side-by-side system. CMC solutions below the coil overlap (c*) influenced the glucose diffusivity only marginally, whereas at concentrations above c* the diffusion of glucose was significantly decreased. Solutions of lower MW exhibited a lower viscosity with lower glucose diffusion compared to solutions with higher MW CMC, attributed to the higher density of the solutions. All CMC solutions showed a systematic positive deviation from Stokes-Einstein behavior indicating a greater rise in viscosity than reduction in diffusion. Therefore, our results pave the way for a new approach for assessing glucose diffusion in solutions comprising dietary fibers and may contribute to further elucidating the mechanisms of post-prandial plasma glucose level reduction.

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

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

Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass

Background Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. Results In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. Conclusions We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

Background Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24–0.90 mg/h per well. Conclusions The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.