Straw composition
As seen in Table 1, the main effect of the hydrothermal pretreatment on the composition of the biomass is the partial but substantial removal of hemicelluloses. All measurable arabinan is removed and the xylan content is reduced from 24.5% to 5.2%. Consequently, the overall cellulose content increases. After delignification of the pretreated material, no Klason lignin can be detected. The composition of the straw that has undergone SO2-impregnated steam explosion is similar to that of the hydrothermally pretreated straw except for a slightly higher xylan content at 7.8%.
ATR-FTIR spectroscopic analysis
ATR-FTIR spectroscopy was used as an analytical tool to qualitatively determine the chemical changes in the surface of pretreated straw to complement and understand the microscopic investigations. The FTIR spectra of untreated, hydrothermally pretreated, delignified hydrothermally pretreated and steam-exploded straw samples are shown in Fig. 1A. Excerpts of the four spectra are presented in Fig. 1B.
One of the effects of the pretreatment is the removal of wax from the straw: Fig. 1A shows that the CH2- stretching bands at approximately 2850 and 2920 cm-1 (see [26]) are reduced for the pretreated straw sample, signifying a reduction in the amount of the aliphatic fractions of waxes.
Two interesting features are shown in Fig. 1B. First, it can be seen that the carbonyl band at 1735 cm-1, which has been ascribed to hemicelluloses [27–29] is reduced for the pretreated straw. This is expected as the pretreatment is known to remove a large portion of the hemicelluloses as shown in Table 1 and in Thomsen et al. [16]. Second, lignin bands at approximately 1595 and, in particular, 1510 cm-1 (aromatic ring stretch) [30] are strongly enhanced in the hydrothermally pretreated sample compared with both untreated wheat straw and delignified hydrothermally pretreated straw, where these peaks are reduced (Fig. 1B). One explanation for this could be a relative increase in the amount of lignin due to the removal of hemicelluloses. Another reason could be that lignin is released and re-deposited on the surface (ATR-FTIR spectroscopy is a surface technique; according to [26, 31] the penetration depth in straw is approximately 0.5-3 μm with the signal intensity exponentially decreasing with penetration depth). The increase in lignin is believed to be too significant to be only due to the hemicellulose removal.
One of the strategies employed in increasing enzymatic convertibility is to decrease cellulose crystallinity [15]. Differences between samples with regard to the relative amounts of amorphous and crystalline cellulose have earlier been described through infrared peak ratios. At least four different peak pairs have been proposed [32, 33]. Of these, only the peak pair 1429 cm-1 (crystalline) and 893 cm-1 (amorphous) is seen for the samples of the present study. The peak ratio for the untreated straw was 0.56, while it was 0.52 for the pretreated straw. In the study by Wistara et al. [33], values from 0.46 to 0.56 were reported, and from this and other results the authors claimed that there was no difference in crystallinity between their samples. When comparing their results with ours, it appears that the pretreatment does not adversely affect the degree of cellulose crystallinity. More precise measurements of cellulose crystallinity are needed to confirm this result.
SEM and AFM images
Based on the results from ATR-FTIR spectroscopy, SEM and AFM were used to gather information on the effect of the hydrothermal pretreatment on the ultrastructure and possible disruption of the cell wall.
When untreated, the anatomy of the harvested, chopped wheat straw is easily recognisable, with sheath leaves surrounding the straw itself (Fig. 2A). The various cell types of the straw wall can be seen, including epidermis cells, parenchyma cells, vascular bundles (phloem and xylem) as well as thick-walled fibre cells, as seen in the SEM micrograph presented in Fig. 2B. Imaging by AFM of parenchyma cells lining the straw cavity reveals the appearance of interwoven cellulose microfibrils of the primary wall (Fig. 2C). These particular cells are largely unlignified [34] but microfibrils are partially embedded in what is believed to be hemicellulosic polymers (left-hand side of Fig. 2C).
Initially, the most apparent effect of the hydrothermal pretreatment apart from a colour change from yellow into dark brown is the partial defibration, or separation of individual fibres and cell types of the wheat straw. Although the pretreated material is quite heterogeneous and contains larger pieces (up to about 1 cm) that are easily recognised as straw, a significant fraction consists of cells that are either completely or partially separated from each other (Fig. 2D).
All individual fibres (and most other cell types) seem to be intact despite the hydrothermal treatment, rather than being broken or otherwise disrupted (Fig. 2D and 2E). When looking more closely at the pretreated fibres it becomes apparent that the surface is covered with 'debris' and a thin layer of deposits that seems to be covering the whole surface (Fig. 2E). This debris could be fractions of middle lamellae. When further investigating the pretreated fibre surfaces through AFM, it was not possible to identify any primary or secondary wall cellulose microfibrils (such as seen in untreated fibre cell walls; Fig. 2C). Instead, an uneven surface of spherical and globular shapes was seen (Fig. 2F). These globular shapes (diameter approximately 20-100 nm) are characteristic of lignin deposits as reported in the literature [22, 25, 35], and this interpretation is in accordance with the spectroscopic findings of higher surface lignin concentrations.
Initially, delignification did not have a great effect on the overall structure of the pretreated material apart from a change in colour; the straw was still only partially defibrated (Fig. 2G), presumably due to the hemicellulose content of the middle lamella [34]. However, upon closer observation, the surface of the individual fibres had changed drastically. The uneven surface now appeared smooth and cellulose aggregates (macrofibrils) running in the direction of the fibre could be seen, as in the SEM image in Fig. 2H. When investigating the delignified fibre surfaces with AFM, the globular shapes of deposited lignin were not seen. Instead, intact surfaces believed to be primary and secondary wall lamellae were observed. Due to the mixing of fibres and other cell types during the pretreatment it was not possible to investigate the same straw cavity parenchyma cells as with the untreated straw. However, numerous scans of different cells revealed several surfaces with similar primary walls to the parenchyma cells. The microfibrils of these primary walls displayed the same interwoven structure as previously seen and were partially embedded in non-cellulosic polymers (Fig. 2I). It should be added, that with AFM only relatively smooth surfaces are successfully imaged.
Surprisingly, neither the overall or fibrillar structure of the individual fibres seems to show large structural changes such as the rupture of fibres or a visible increase of porosity, which are believed to be associated with thermal pretreatments. No holes or cracks were seen in the fibres and AFM did not indicate that the accessibility of the internal parts of the cell wall matrix had been improved due to structural dislocations. Rather, the primary and secondary cell walls appeared to be fully intact, except for the pits and simple perforations that already exist in certain cell types [36]. Despite these observations of a substrate where the skeletal structure is intact and the crystallinity of the cellulose does not appear to have been lowered, the hydrothermally pretreated straw has been shown to be easily digestible by enzymes [16, 17]. Consequently, the effectiveness of the pretreatment must be related to hemicellulose removal and lignin re-localisation. This is in spite of the fact that lignin is not removed by the pretreatment and that lignin is known to be responsible for unproductive adsorption of cellulases [37, 38]. It is well known that lignin encases the cellulose in the cell-wall matrix, hindering cellulases from reaching cellulose fibrils. We hypothesise that the migration of lignin to the outer surface exposes internal cellulose surfaces. More investigations are needed in order to confirm this. Selig et al. [39] have also observed the formation and migration of spherical lignin deposits onto the surface of fibres as a result of pretreatment. They also suggest that the deposited lignin can have a negative impact on the enzymatic cellulose hydrolysis. It is possible, however, that the surface lignin layer is easily removed by simple mechanical forces through mixing, due to lignin being less strongly bound to carbohydrate polymers compared with its native linkages. Furthermore, we theorise that the re-located lignin has exposed cellulose inside the cell wall, thus increasing the enzyme accessibility.
Based on these observations, we therefore propose that the re-localisation of lignin as well as partial hemicellulose removal are likely to be important factors in increasing the enzymatic digestibility of wheat straw through hydrothermal pretreatment. It seems that exposing cellulose through manipulation of hemicelluloses and lignin are equally as important as altering the crystallinity and rupture of the skeletal structure of the cell wall.
Comparison with conventional steam explosion
In order to understand whether the factors affecting biomass digestibility through hydrothermal pretreatment are of a more general nature, steam-exploded straw was also investigated microscopically and spectroscopically. Steam explosion is considered one of the most promising pretreatment technologies and is often combined with the addition of chemicals [5, 6]. In our case the straw was impregnated with SO2 prior to steam explosion. In principle, steam explosion is not unlike hydrothermal pretreatment. As such, the effect on compositional changes is also similar (Table 1).
As seen in Fig. 1 the FTIR spectra of the steam-exploded straw are similar to those of hydrothermally treated straw, both in general and in spectral ranges related to wax, hemicellulose and lignin. SEM investigations (Fig. 2J and 2K) showed that steam-exploded straw was more heterogeneous than hydrothermally pretreated straw, containing larger pieces of almost intact straw but also a larger fraction of individual fibres that had been compacted together. Some SEM images also showed droplets on the surface of the fibres (see the arrows in Fig. 2K). These droplets are also believed to be lignin, possibly formed through coalescence of smaller sized lignin deposits during the pretreatment as described in [39]. The difference in amount of larger lignin droplets between the different pretreatments may be due to varying water contents and pH during the treatment. AFM showed globular deposits similar to, but larger than those seen on hydrothermally pretreated straw (Fig. 2L).