3.1. Effect of pretreatment conditions on the fractionation of almond tree pruning
The raw material has the following composition on a dry basis: 31.9 ± 0.5% cellulose, 22.1 ± 1.0% hemicelluloses, 26.5 ± 1.4% acid insoluble lignin, 14.2 ± 0.7% water extractives, 3.4 ± 0.4% organic extractives, and 1.28 ± 0.04% ash. From 100 g of dry raw material, hydrolysis of the hemicellulosic fraction could theoretically generate 16.6 g of xylose, 2.5 g of arabinose, 1.4 g of galactose, and 1.3 g of mannose. The theoretical maximum amount of glucose obtainable from 100 g of dry raw material would be 35.4 g. These data agree with a previous study on almond tree pruning (Cuevas et al. 2014) which reported contents of cellulose, hemicelluloses and acid insoluble lignin of 31.3%, 23.0%, and 28.7%, respectively. It is known that diluted sulphuric acid, acting at high temperatures and for short periods of time, can cause a significant modification in the chemical composition of lignocellulosic biomasses. Normally, the acid catalyst causes severe hydrolysis of hemicellulose and partial hydrolysis of cellulose. This leads to a sharp decrease in solid recovery ("total gravimetric recovery," or TGR) after the treatment. When almond pruning was pretreated with 0.025 M sulphuric acid under the operating conditions specified in Table 1, the TGR values ranged from 54.7–60.1%, corresponding to pretreatments P11 (214.1 ºC-5 min) and P2 (190 ºC-2 min), respectively (Table 2). The small variation in the amount of solid recovered can be due to the application of a narrow temperature range (185.9 ºC–214.1 ºC) and short reaction times (0.8 min–9.2 min). Anyway, it is observed how, in general, an increase in the severity of the pretreatment (higher temperatures and reaction times) led to a decrease in the TGR values. Table 2 also shows that all pretreatments produced a strong reduction in hemicellulose content. Thus, the pretreatment performed at 185.9 ºC-5 min (P1) generated WIS with 3.4% hemicellulose. This means that only 9.4% of the original polymer was maintained, while in the solids pretreated in the P5-P11 assays, the polyose contents were less than 1.5%. The strong loss of hemicellulose during pretreatment caused the percentages of cellulose in the WIS (41.0–47.1%) to be clearly higher than those of the raw material (31.9%). However, the acid attack also caused some loss of cellulose, whose conversion was increased under the most severe pretreatment conditions. Likewise, while P1 pretreatment (185.9 ºC-5 min) only eliminated 11.3% cellulose, P10 pretreatment (210.0 ºC-8.0 min) resulted in a 28.8% biopolymer conversion (Table 2). Acid insoluble lignin was, in general, the structural material that underwent the least variation during pretreatment. It ranges from 38.1–41.1% in the WIS, with conversions in the range of 13.2–17.2%.
Table 2
Total gravimetric recovery and composition of water insoluble solids resulting from sulphuric acid pretreatmentsa.
Run | TGR (%) | Hemicellulose (%) | Cellulose (%) | Acid insoluble lignin (%) |
P1 | 60.0 | 3.4 (90.6)b | 47.1 (11.3) | 38.1 (13.8) |
P2 | 60.1 | 2.9 (92.0) | 46.8 (11.8) | 38.3 (13.2) |
P3 | 59.2 | 2.5 (93.2) | 43.5 (19.2) | 38.4 (14.1) |
P4 | 57.8 | 1.5 (96.2) | 45.1 (18.2) | 38.6 (15.7) |
P5 | 57.2 | 1.4 (96.3) | 43.9 (21.2) | 39.0 (15.8) |
P6 | 57.0 | 1.0 (97.4) | 44.8 (19.9) | 38.9 (16.3) |
P7 | 57.3 | 1.2 (96.9) | 43.3 (22.2) | 39.0 (15.8) |
P8 | 56.6 | 0.0 (100.0) | 45.0 (20.2) | 39.2 (16.2) |
P9 | 55.9 | 0.2 (99.6) | 45.4 (20.5) | 39.3 (17.2) |
P10 | 55.4 | 1.3 (96.8) | 41.0 (28.8) | 41.1 (14.2) |
P11 | 54.7 | 0.0 (100.0) | 44.1 (24.3) | 41.1 (15.2) |
a Percentages expressed on a dry basis.
b The percentage of biopolymer (hemicellulose, cellulose, or acid insoluble lignin) transformed during pretreatments is shown in brackets.
The acid pretreatment of hemicellulose and cellulose in almond pruning produced a prehydrolysate with varied amounts of simple sugars. Table 3 shows the yields of glucose, xylose, arabinose, and the sum of mannose plus galactose for the eleven prehydrolysates. In addition, the total monomeric sugar (TMS) yield, calculated as the sum of the yields of the five monosaccharides analysed, is incorporated. Xylose, glucose and arabinose were the most abundant monosaccharides in prehydrolysates, reaching yields of 14.50–4.87 g, 5.23–1.33 g, and 2.50–0.89 g per 100 g of dry raw material, respectively. The yield of galactose and mannose was very low for all operating conditions, and these compounds were not detected in the P10 and P11 tests. The TMS yield ranged from 7.38 g (P11) to 22.50 g (P6) per 100 g of dry raw material. For all monosaccharides, maximum yields were obtained with intermediate severity pretreatments which lead to a high generation of simple sugars (by hydrolysis of polysaccharides) while controlling losses of these monosaccharides by thermal degradation (to products such as 5-hydroxy-methyl-furfural). Concerning the monosaccharide degradation products, the P11 pretreatment, performed at the maximum temperature tested (214.1 ºC), resulted in a relatively low yield of 5-hydroxy-methyl-furfural (0.45 g/100 g dry raw material).
Table 3
Products yields (as g/100 g dry raw material) in the prehydrolysates obtained at different pretreatment conditions.
Run | Yglucose | Yxylose | Yarabinose | YGAL+MAN | YTMS |
P1 | 3.91 | 11.59 | 1.83 | 0.74 | 18.06 |
P2 | 4.51 | 13.43 | 1.45 | 1.66 | 21.06 |
P3 | 5.23 | 13.02 | 0.92 | 2.33 | 21.49 |
P4 | 3.78 | 11.13 | 2.02 | 0.40 | 17.34 |
P5 | 4.17 | 14.50 | 2.50 | 0.70 | 21.87 |
P6 | 4.88 | 14.34 | 2.28 | 1.00 | 22.50 |
P7 | 4.72 | 13.82 | 2.48 | 0.63 | 21.65 |
P8 | 2.34 | 8.20 | 0.89 | 0.89 | 12.32 |
P9 | 3.59 | 8.50 | 1.89 | 0.03 | 14.01 |
P10 | 2.29 | 5.42 | 1.43 | nd | 9.14 |
P11 | 1.33 | 4.87 | 1.17 | nd | 7.38 |
GAL + MAN: galactose plus mannose. TMS: Total Monomeric Sugars. nd: not detected.
Data from Tables 1 and 2 was analysed in the Modde 6.0 program to determine the effect of temperature (TR) and reaction time (tR) during the acid pretreatment of the almond pruning. Mathematical models with statistical validity were checked to obtain the following responses: TGR for the pretreated solids; and xylose yield (Yxylose), glucose yield (Yglucose), arabinose yield (Yarabinose) and total monomeric sugar yield (YTMS) for the prehydrolysates. The model coefficients (ai in Eq. 1) were obtained from the ANOVA analysis along with the standard deviations for the different responses (Table 4). For all models, correlation coefficients (R2 and R2adjust) were acceptable. The value of R2 was 0.934 in the most unfavorable case (Yglucose), implying that only 6.6% of the total variations in the response were not explained by the model.
Table 4
Model parameters (ai), standard errors (SE), and significance level (p) for the mathematic models.
Response variable | ai | SE | p-value (Prob > F) | R2 | R2adjust |
TGR, % | a0: 57.380 | 0.109 | 1.955·10–19 | 0.967 | 0.959 |
| a1: − 1.679 | 0.128 | 1.104·10–6 | |
| a2: − 1.036 | 0.128 | 4.081·10–5 | |
Yglucose(1) | a0: 4.438 | 0.159 | 1.905·10–8 | 0.934 | 0.906 |
| a1: − 0.913 | 0.133 | 2.429·10–4 | |
| a2: − 0.386 | 0.133 | 2.314·10–2 | |
| a3: − 1.007 | 0.152 | 2.946·10–4 | |
Yxylose(1) | a0: 14.22 | 0.210 | 6.936·10–10 | 0.994 | 0.989 |
| a1: − 2.556 | 0.128 | 1.046·10–6 | |
| a2: − 1.434 | 0.128 | 3.089·10–5 | |
| a3: − 2.982 | 0.153 | 1.178·10–6 | |
| a5: − 1.717 | 0.153 | 2.984·10–5 | |
Yarabinose(1) | a0: 2.420 | 0.050 | 4.978·10–9 | 0.987 | 0.978 |
| a1: − 0.260 | 0.030 | 1.384·10–4 | |
| a2: 0.310 | 0.030 | 5.141·10–5 | |
| a3: − 0.461 | 0.036 | 1.426·10–5 | |
| a5: − 0.509 | 0.036 | 8.050·10–6 | |
YTMS(1) | a0: 22.007 | 0.389 | 2.035·10–9 | 0.991 | 0.985 |
| a1: − 4.006 | 0.238 | 2.807·10–6 | |
| a2: − 2.185 | 0.238 | 9.398·10–5 | |
| a3: − 4.712 | 0.283 | 3.014·10–6 | |
| a4: − 2.196 | 0.283 | 2.422·10–4 | |
Total gravimetric recovery (TGR), glucose yield (YGlucose), xylose yield (YXylose), arabinose yield (YArabinose) and total monomeric sugars yield (YTMS) for the sulphuric acid pretreatment of almond tree pruning.
Significance level was defined as p < 0.05.
(1) Products yields are expressed as grams of product per 100 grams of dry raw material.
The parameters given in Table 4 were used to represent the response surface plots shown in Fig. 2. For the recovery of total solids (Fig. 2-A), it was observed that both the temperature and the reaction time exerted a negative and linear effect on the response, with no quadratic terms and no interaction terms between the two factors. The same behaviour was earlier reported for the diluted acid pretreatment of some lignocellulosic biomasses at high temperatures (Saleh et al. 2014). In the case of sugar production, the glucose yield depended more on the temperature than the reaction time, reaching its maximum value at 195.4 ºC for a fixed time (Fig. 2-B). This could be explained by considering that below 195.4 ºC no significant hydrolysis of cellulose occurred, whereas above that temperature the rate of glucose degradation exceeds the rate at which the monosaccharide is obtained. The model predicts a maximum value for Yglucose equal to 5.03 g/100 g dry raw material under the conditions of 195.4 ºC-2.0 min. Maximum yields of xylose (15.07 ± 1.3 g/100 g dry raw material), arabinose (2.50 ± 0.2 g/100 g dry raw material), and TMS (23.4 ± 2.1 g/100 g dry raw material) were reached under the conditions of 195.7 ºC-3.8 min, 197.1 ºC-5.9 min, and 195.8 ºC-3.46 min, respectively (Figs. <link rid="fig2">2</link>-C, <link rid="fig2">2</link>-D and <link rid="fig2">2</link>-E). Therefore, the pretreatment allowed 90.8% extraction of the xylose present in almond wood. This value is in close range to the data reported by different authors for the acid treatment of different biomasses: 89.3% for olive stone (Saleh et al. 2014), 90.95% for pinewood (Cao et al. 2018), and 94% for giant reed (Shatalov and Pereira 2012).
3.2. Effect of pretreatment conditions on the enzymatic hydrolysis
The application of the cellulolytic complex "Celluclast 1.5 L" on pretreated almond pruning using two loads of biocatalyst (10 FPU/g WIS or 15 FPU/g WIS) produced both glucose and total reducing sugars (TRS). Stable values for their concentrations were reached at 96 h (Fig. 3). It is important to note that sugar concentrations depended heavily on pretreatment conditions but not so much on the biocatalyst load used. Thus, for the raw material the final concentrations of total reducing sugars (TRS) were 7.25 ± 1.05 g/L and 8.52 ± 0.37 g/L for the loads of 10 FPU/g WIS and 15 FPU/g WIS, respectively, while with the solid obtain from P11 pretreatment the concentrations were 17.79 ± 0.67 g/L (10 FPU/g WIS) and 18.62 ± 0.11 g/L (15 FPU/g WIS). The maximum glucose concentration (10.95 ± 0.07 g/L) was obtained using an enzyme load of 15 FPU/g WIS on the solids of the P10 pretreatment. Enzymatic digestibility, glucose yield (YGlu/RM), and total reducing sugars yield (YTRS/RM) for raw material and pretreated solids are presented in Table 4. These parameters were calculated using Eqs. 2, 3, and 4, respectively. For the enzymatic digestibility of solids (ED), the minimum values (8.5% and 9.5%) were observed for the hydrolysis of raw material with loads of 10 FPU/g WIS and 15 FPU/g WIS, respectively, whereas for pretreated solids, ED values of 43.5% (P11 with 10 FPU/g WIS) and 46.3% (P10 with 15 FPU/g WIS) were obtained. These data imply an increase of about 400% in the enzymatic digestibility of pretreated cellulose compared to the original polymer. However, acid pretreatment did not allow the complete hydrolysis of the biopolymer. This was possibly due to the high lignin content of WIS and the inhibition of the catalyst by reaction products (cellobiose, glucose, etc.). Earlier studies on the enzymatic hydrolysis of different biomasses pretreated with diluted sulphuric acid have also reported similar aforementioned ED values: 47.5% for almond pruning pretreated at 220 ºC for 5 minutes (Cuevas et al. 2014), and 43.4% for coffee cut-stems pretreated at 120 ºC for 180 min (Solarte-Toro et al. 2020).
The YGlu/RM values (Table 5) were in the range of 3.03–11.68 g glucose/100 g raw material. The lowest value was observed for the raw almond pruning hydrolysed with an enzyme load of 10 FPU/g WIS, whereas the highest value was obtained when the enzymes acted on biomass derived from pretreatment P10 using a load of 15 FPU/g WIS (Table 5). In the latter case, the value achieved is equivalent to a glucose production of 29.09 g per 100 g of WIS. These results indicate that the conditions of acid pretreatment impacted strongly on the enzymatic action. In addition, a slight increase in YGlu/RM was observed as the enzyme load increased from 10 FPU/g WIS to 15 FPU/g WIS. On the other hand, Table 4 also showed an important difference between the yields of glucose and total reducing sugars, even in experiments where the WIS is virtually devoid of hemicelluloses. For example, after pretreatment P11, enzymatic hydrolysis performed with 15 FPU/g WIS led to values of YGlu/RM and YTRS/RM of 11.30 g and 19.65 g/100 g raw material, respectively. The difference between the two yields could be explained by assuming that cellulose hydrolysis not only produces glucose but also generates other carbohydrates (e.g., oligosaccharides), which are quantified together with glucose when applying the DNS method.
Table 5
Glucose and total reducing sugars yields obtained from enzymatic hydrolysis.
Run | 10 FPU/g WIS | | 15 FPU/g WIS |
ED (%) | YGlu/RM | YTRS/RM | | ED (%) | YGlu/RM | YTRS/RM |
RM | 8.54±1.86 | 3.03±0.66 | 7.25±1.05 | | 9.50±0.80 | 3.37±0.28 | 8.52±0.37 |
P1 | 19.13±0.02 | 6.01±0.01 | 11.64±1.32 | | 22.94±0.59 | 7.21±0.19 | 12.54±1.85 |
P2 | 20.81±2.31 | 6.51±0.72 | 11.56±0.90 | | 23.40±0.43 | 7.32±0.13 | 11.62±0.04 |
P3 | 29.02±1.75 | 8.31±0.50 | 13.39±1.73 | | 27.63±1.05 | 7.91±0.30 | 12.38±0.11 |
P4 | 27.85±2.19 | 8.07±0.63 | 13.67±2.36 | | 28.06±1.74 | 8.13±0.50 | 13.63±0.31 |
P5 | 30.57±0.19 | 8.53±0.05 | 13.93±0.07 | | 32.93±0.36 | 9.19±0.10 | 17.09±1.64 |
P6 | 30.71±0.86 | 8.57±0.24 | 13.98±0.56 | | 31.25±6.37 | 8.87±1.81 | 15.22±0.75 |
P7 | 33.27±5.04 | 9.35±1.51 | 14.86±1.12 | | 33.87±1.65 | 9.51±0.56 | 16.64±1.40 |
P8 | 36.00±1.26 | 10.18±0.36 | 16.29±1.25 | | 34.95±1.72 | 9.88±0.49 | 16.05±0.43 |
P9 | 38.71±0.44 | 10.57±0.12 | 16.14±1.00 | | 37.78±0.72 | 10.32±0.20 | 15.62±0.23 |
P10 | 42.98±0.57 | 10.84±0.14 | 16.40±0.08 | | 46.31±0.32 | 11.68±0.08 | 16.53±0.61 |
P11 | 43.05±0.07 | 11.54±0.02 | 18.78±0.50 | | 42.16±0.61 | 11.30±0.16 | 19.65±0.11 |
RM: Assay carried out with raw material. P1-P11: Assays carried out with pretreated solids (WIS). ED: Enzymatic digestibility, or g glucose by enzymatic hydrolysis/100 g glucose in substrate. YGlu/RM: g glucose by enzymatic hydrolysis/100 g raw material. YTRS/RM: g total reducing sugars by enzymatic hydrolysis/100 g raw material.
The relationship between pretreatment conditions (temperature and reaction time) and the enzymatic digestibility of WIS is described mathematically by Eq. (1). A value of R2 = 0.988 was obtained for 10 FPU/g WIS with the following ai values: 31.182 ± 0.436 (a1), 8.018 ± 0.367 (a2), 3.466 ± 0.367 (a3) and 1.138 ± 0.418 (a5). Whereas for 15 FPU/g WIS, a R2 = 0.990 was achieved with 32.844 ± 0.266 (a1), 7.123 ± 0.312 (a2), 3.797 ± 0.312 (a3) and 1.675 ± 0.442 (a6). From the above values, response surface plots could be represented (Fig. 4). These figures showed that the enzymatic digestibility of WIS is strongly affected by the pretreatment with diluted acid, in a way that the maximum enzymatic digestibility is obtained using the most severe pretreatments. For example, for enzymatic hydrolysis performed with a load of 15 FPU/g WIS, the maximum ED value (45.4%) was reached after a pretreatment carried out at 210 ºC for 8 minutes (Fig. 4-B).
3.3. Optimisation of the sugar production process and mass balance
In order to study the impact of temperature and pretreatment time on the overall production of fermentable sugars, the total sum of yields of glucose, xylose, arabinose, galactose, and mannose obtained in acid pretreatments (YTMS in Table 3) along with glucose yields achieved in enzymatic hydrolysis (YGlu/RM in Table 5) were considered. These global monosaccharide yields generated in the overall process were designated as Yglobal that ranged 18.68–31.37%. By applying Yglobal parameter in the Modde 6.0, the data in Table 6 was generated. This table contains the most relevant information on the two mathematical modes describing the dependence of the overall performance of monosaccharides with temperature and pretreatment time for enzymatic loads of 10 FPU/g WIS and 15 FPU/g WIS. Table 6 revealed the high values of R2 in both models, as well as the existence of quadratic terms for the factors TR and tR.
Table 6
Model parameters (ai), standard errors (SE), and significance level (p) for the models.
Response variable | ai | SE | p-value (Prob > F) | R2 | R2adjust |
Yglobal (A) | a0: 30.823 | 0.407 | 3.543·10–10 | 0.984 | 0.974 |
| a1: − 2.224 | 0.249 | 1.099·10–4 | |
| a2: − 1.508 | 0.249 | 9.166·10–4 | |
| a3: − 4.784 | 0.296 | 3.594·10–6 | |
| a4: − 1.936 | 0.296 | 6.154·10–4 | |
Yglobal (B) | a0: 31.197 | 0.307 | 6.082·10–11 | 0.992 | 0.986 |
| a1: − 2.647 | 0.188 | 7.961·10–6 | |
| a2: − 1.434 | 0.188 | 2.630·10–4 | |
| a3: − 4.802 | 0.224 | 6.647·10–7 | |
| a4: − 2.168 | 0.224 | 6.892·10–5 | |
Y global for enzymatic hydrolysis carried out with biocatalyst loads of 10 FPU/g WIS (A) and 15 FPU/g WIS (B).
Response surface plots representing the effect of TR and tR on Yglobal are shown in Fig. 5. This helped in identifying the maximum values of Yglobal obtained under the studied pretreatment conditions. For enzymatic hydrolysis with Celluclast 1.5 L (loads equal to 10 FPU/g WIS) the maximum Yglobal value (31.37%) was obtained with pretreatment conditions of 197.6 ºC and 3.8 min. While for enzyme loads of 15 FPU/g WIS, the maximum value of Yglobal (31.80%) was achieved with a pretreatment at 197.2 ºC and 4.0 min. The pretreatment conditions obtained for the two enzyme load series were very close. So, from a practical point of view, the temperature of 197 ºC and the time of 4.0 minutes can be adopted as appropriate values to maximise the production of sugars from almond tree pruning. Cara (2008) achieved a maximum Yglobal value of 36.3% by pretreating olive prunings with diluted sulphuric acid (1%) and then subjecting the WIS to enzymatic hydrolysis with a mixture of Celluclast 1.5L (15 FPU/g substrate) and Novozym 188 (15 International Unit/g substrate) (Cara et al. 2008). The above value is slightly higher than that obtained in the present study but implies the use of a higher enzymatic load.
To study the effect of the incorporation of the enzyme complex “Novozym 188” in the enzymatic production of glucose, the pruning of almonds was pretreated under previously optimised conditions (197 ºC-4 min), and the obtained WIS was hydrolysed with Celluclast 1.5L (15 FPU/g WIS) supplemented with Novozym 188 (30 IU/g WIS). By this method, an ED value of 52.9% was reached which was equivalent to the production of 14.77 g of glucose by enzymatic hydrolysis per 100 g of raw material. The addition of Novozym188 increased the β-glucosidase activity which led to a Yglobal value of 36.8%. This data is equivalent to a recovery of 64.3% of the sugars present in the raw material. The above Yglobal values are in line with earlier research findings related to high-temperature pretreatments followed by enzymatic hydrolysis of various biomasses: 37.8% using rapeseed straw (Romero et al. 2018) and 37% using olive tree biomass (López-Linares et al. 2013). Figure 6 shows the material balance of the sugar production process from almond tree pruning, including the pretreatment with diluted sulphuric acid under optimal conditions (197 ºC-4 min) and the subsequent enzymatic hydrolysis of WIS using Celluclast 1.5L (15 FPU/g WIS) and Novozym 188 (30 IU/g WIS). The results obtained under optimal conditions also confirmed the validity of the mathematical models used in the present work (interval confidence of 95%).
3.4. Characteristics of biomass and solid fractions for thermochemical applications
The diluted acid pretreatment of almond pruning biomass, followed by enzymatic hydrolysis of the pretreated solids, produces both liquid and solid fractions. The liquid fractions are generally used for the recovery of monosaccharides. But it is also essential to valorise the final solid from sugar production process (a rich-lignin solid residue) to achieve an integral use of the raw material. Thermochemical utilisation of biomass is generally favoured by increasing higher heating value (HHV) and reducing both the percentage of ash and the Equilibrium Moisture Content (EMC). The EMC is indicative of the capacity of adsorption of moisture by a solid under certain environmental conditions and, in this work, it has been expressed as mg of water adsorbed in each gram of dry solid (mg/g). Figure 7 shows the values of the aforementioned three parameters for the raw material, the solids resulting from acid pretreatments (WIS) and the solids resulting from the enzymatic hydrolysis of WIS with an enzyme load of 15 FPU/g WIS. With respect to the higher heating values (Fig. 7-A), the raw material had an HHV of 18.11 ± 0.1 MJ/kg, a value clearly lower than that of the solids generated in acid pretreatments (20.48–22.09 MJ/kg) and enzymatic hydrolysis (21.28–23.01 MJ/kg,). In general, the application of pretreatments with higher temperatures and reaction times led to an increase in the HHV of the WIS. This way, solids derived from pretreatments P1, P6, and P11 had HHV values of 20.77 MJ/kg, 21.60 MJ/kg and 21.99 MJ/kg, respectively (Fig. 7-A). This could be due to the increase in the percentage of AIL in pretreated solids (Table 2), as lignin is the structural component of biomass with the highest heating value (Maksimuk et al. 2021). This fact would also explain how, in general, the solids resulting from the enzymatic hydrolysis showed slightly higher HHV than the WIS, as the enzymes reduce the amount of cellulose available in the solids without altering the lignin fraction. The maximum HHV obtained in the experimental study (5505 kcal/kg) was achieved with the solid that remain after the pretreatment (214.1 ºC-5 min) of the raw material following by the enzymatic hydrolysis of the corresponding WIS. This HHV value represents an increase of 27% over the heating value in the almond pruning. This value implies a higher energy densification of almond wood than that obtained by Aguado et al. [35] when subjecting the same type of biomass to wet torrefaction at 250°C-10 min (23% increase) and at 225°C-60 min (26% increase), i.e. applying much more energetically intensive treatments. On the other hand, Fig. 7-B shows that the ash content of the WIS (0.14–0.75%) was clearly lower than the original biomass (1.28 ± 0.04%). This could be beneficial for the thermochemical use of the pretreated solids. The effect of acid treatments on the ash content of lignocellulosic biomasses has been studied by different authors (Lee et al. 2021) and could be explained by considering that the H+ ion reacts with the alkali components in the biomass via neutralization reactions (Chin et al. 2015). Enzymatic hydrolysis generated solids with ash percentages slightly higher than those of hydrolysed substrates (0.35–0.86%). The reason could be that lignin contained more inorganic material than cellulose. Regarding the moisture adsorption capacity (Fig. 7-C) of the different biomasses under a constant relative humidity atmosphere of 75.5%, the raw material showed an EMC value of 123.9 mg/g, much higher than that of the WIS (81.6–70.1 mg/g) and the solids from enzymatic hydrolysis (79.8–69.9 mg/g). These results could be due to the more hydrophobic nature of lignin compared to cellulose or hemicellulose (Piao et al. 2010). In the case of the process scheme developed under optimised conditions for the production of sugars (Fig. 6), it was found that the residual solid generated after enzymatic hydrolysis had values of HHV, ash percentage, and EMC of 5259 ± 102 kcal/kg (21.4% higher than raw material), 0.87 ± 0.04% (32.0% lower than raw material), and 72.1 ± 4.3 (41.8% lower than raw material), respectively.