A Parametric Study of the Organosolv Fractionation of Norway Spruce Sawdust

Abstract

Lignocellulosic biomass represents an excellent alternative to fossil fuels in terms of both energy production and raw material usage for a plethora of daily-use products. Organosolv pretreatment is a fractionation technique able to separate lignocellulosic biomass into individual streams of cellulose, hemicellulose, and lignin under controlled conditions. Sawdust, the by-product of sawmill processing of Picea abies wood, was the subject of our investigation in this work. The aim was to evaluate the effects of different parameters of the organosolv process of spruce sawdust on the yield of components and how this affects the enzymatic saccharification of cellulose. Sixteen distinct pretreatments were performed with ethanol concentrations of 50 and 60% v/v at 180 and 200 °C for 15 and 30 min. Half of the pretreatments contained 1% sulfuric acid as a catalyst, while the other half were acid-free. Thereafter, the effects of different variables on the yield of products were assessed and compared to determine the ideal pretreatment condition. The results showed that cellulose-rich pulps, with cellulose content as high as 55% were generated from an initial mass of 37.7% spruce sawdust with the reactor operating at 180 °C for 30 min using 60% ethanol and 1% sulfuric acid. With the pretreatments performed with the catalyst at 200 °C, hemicellulose was almost entirely removed from the pulps obtained. The recovered hemicellulose fraction was composed mainly of monomers achieving up to 10 g/100 g of biomass. Delignification values of up to 65.7% were achieved with this pretreatment technique. Fractionated lignin presented low levels of sugar and ashes contamination, with values as low as 1.29% w/w. Enzymatic saccharification of the pretreated pulps yielded 78% cellulose hydrolysis, with glucose release higher than 0.54 g/g of biomass, indicating the potential of the pulps to be applied in a fermentation process.

1. Introduction

Biomass utilization and valorization aimed at the production of a variety of materials like chemicals, energy, and other bio-based products have received a great deal of attention in recent times. This has been largely sparked by the growing public interest in sustainable development. Biomass can be categorized into lignocellulosic (such as wood) and non-lignocellulosic biomass (like sewage sludge). This classification is essential to understanding the various forms of biomass and the circumstances surrounding its application [1]. The lignocellulosics are the main biomass types and are the dominant raw material in the production of liquid and gaseous biofuels in Sweden [1,2]. This significant source of biogenic raw material also enables the manufacture of chemicals and other bio-based products without threatening food supply. The conversion of lignocellulosic biomass into these high-value products is achieved via the use of various technologies whose configurations and operations are mostly dictated by the characteristics of the lignocellulosic material [3]. One such technology is organosolv pulping, which is an emerging biorefinery technique used to fractionate lignocellulosic biomass into its individual components (cellulose, hemicellulose, and lignin) using water and miscible organic solvents like ethanol in the temperature range 100–250 °C [3]. The separated components can then be further processed into a variety of value-added products such as fuels, chemicals, and other biomaterials through a range of techniques like thermochemical and biochemical techniques [3,4]. A great advantage of the organosolv technique over other methods like steam explosion is the fact that the organic solvent used can be recovered and reused in the process, which represents an important step in terms of process economics; other advantages include the fact that the technique enables the use of a wide variety of organic solvents (such as acetone, methanol, propanol, and butanol) and allows for the recovery of substantial quantities of high-purity lignin, including the production of a cellulose-rich pulp that can be easily digested under enzymatic conditions [5,6]. The organosolv process typically permits efficient separation of lignocellulosic biomass into its three main components, which include a solid fraction rich in cellulose, a mono- and oligosaccharides-based aqueous hemicellulose liquid, and a solid dry organosolv lignin fraction [3,7].

Spruce sawdust is a type of lignocellulosic feedstock that has the potential to be fractionated using the conventional organosolv technique described above. The forest and timber industry in Sweden generates substantial amounts of lignocellulosic by-products in the form of logs, wood residues, and wood chips, as well as sawdust. Of these by-products of wood processing, the annual generation of sawdust alone (from wood such as spruce) is more than a million tons on a dry matter basis [1,8]. However, the type of biomass and pretreatment parameters, like temperature and solvent ratio, as well as the intended application of the fractions obtained from the organosolv process of biomass are among the factors that determine whether acid or base catalysts, like sodium hydroxide or sulfuric acid, are required in the organosolv fractionation process. The organosolv can also be performed without the use of a catalyst. For instance, feedstocks like spruce sawdust would require the addition of an acid catalyst in the solvent–water mixture, with the reactor (organosolv) operating at temperatures below 200 °C, for optimum fractionation in terms of yield [3,9,10]. This is because spruce sawdust is a naturally recalcitrant softwood that has low concentrations of acetyl groups, which can reduce self-catalyzed processes. As a result, an acid catalyst is frequently needed during the organosolv process for these kinds of biomass materials in order to compensate for their low natural acidity [10]. Moreover, using sawdust as feedstock in lieu of other materials like woodchips in the organosolv process is beneficial because sawdust particles are already uniformly sized, thus mitigating the energy needs and expenses that are often associated with woodchips [11]. Furthermore, smaller particle sizes have larger pores and greater surface area per unit of mass, which allows for faster rates of heat transfer and reaction.

Although a great deal of research has been undertaken in the field of the organosolv fractionation of lignocellulosic biomass and process conditions, in which varying results have been obtained, some of these results are inconsistent and are often not supported by adequate data [12,13]. Additionally, the impact of various pretreatment conditions on the enzymatic saccharification of cellulose has not been well studied in research utilizing the organosolv pretreatment of softwood, such as spruce sawdust. It is for this reason that it was deemed appropriate to confirm the effects of different parameters of the organosolv on the yield of products using spruce sawdust as feedstock. Therefore, the aim of this study was to investigate the influence of several organosolv pretreatment conditions (such as temperature, duration, solvent ratio, and catalyst) on the fractionation efficiency of Spruce sawdust. The investigation was deemed essential in order to establish the ideal pretreatment parameter for spruce sawdust fractionation and to obtain a more profound comprehension of the organosolv system and its working principes.

2. Materials and Methods

2.1. Feedstock

Sawdust of Norway spruce (Picea abies) was used in this study. Spruce was grown and milled in Norrbotten County in northern Sweden. The milled spruce sawdust was air-dried and stored at room temperature. Prior to the pretreatment process, the dry composition of the material was analyzed (see Section 2.3), and the results were 37.67% ± 0.83% cellulose (referring to total glucan), 29.62% ± 1.08% hemicellulose, 29.61% ± 1.03% Klason lignin, and 0.21% ± 0.03% ashes. The particle size distribution was measured on a horizontal sieve shaker (Prüfsieb JEL 200; J. Engelsmann, Ludwigshafen, Germany), yielding the following distribution (in w/w): 2.4% of particles > 8 mm; 8.9% ranging from 4 mm and 8 mm; 27.0% ranging from 2 mm and 4 mm; 29.3% ranging from 1 mm and 2 mm; and 32.4% were <1 mm.

2.2. Pretreatment Procedure

Organosolv pretreatment of the spruce sawdust was performed in a 2.5 L metallic batch reactor with a 1:10 (w/v) solids to liquids ratio and 110 g of biomass (Norway spruce sawdust). Given the small particle size of sawdust, the material was used as received without further milling. This is beneficial for the overall process as it eliminated the energy consumption of the milling process. The solvent concentrations used in the pretreatment process were 50% and 60% v/v aqueous solution of ethanol during a 15- and 30-min reaction time for a total of 16 runs. The effect of the acid catalyst was evaluated by adding 1% w/w_{dry biomass} of sulfuric acid to the biomass/aqueous ethanol mixture in half of the 16 runs, while the other half were acid-free. The different conditions under which the experiment was performed are summed up in

Table 1. The operating conditions of the organosolv pretreatment process of Norway spruce sawdust.

Code	Temperature (°C)	Time (min)	Ethanol (%, v/v)	Catalyst (w/wbiomass)
S-0A5x	200	15	50%	-
S-0A5y	200	15	50%	1%
S-0A6x	200	15	60%	-
S-0A6y	200	15	60%	1%
S-0B5x	200	30	50%	-
S-0B5y	200	30	50%	1%
S-0B6x	200	30	60%	-
S-0B6y	200	30	60%	1%
S-1A5x	180	15	50%	-
S-1A5y	180	15	50%	1%
S-1A6x	180	15	60%	-
S-1A6y	180	15	60%	1%
S-1B5x	180	30	50%	-
S-1B5y	180	30	50%	1%
S-1B6x	180	30	60%	-
S-1B6y	180	30	60%	1%

. After the pretreatment, the reactors were cooled to 40 °C. The insoluble cellulose-rich fibers (pulps) were then filtered from the slurry in a vacuum filter and subsequently washed with either 50% or 60% v/v ethanol aqueous solution and air-dried at room temperature. Using a rotary evaporator (Heidolph, Schwabach, Germany), the liquor obtained from the slurry was then processed downstream with the goal of isolating the lignin fraction and recovering the ethanol used in the process. The liquid solution obtained after the evaporation process was then centrifuged at 12,000× g for 10 min at 4 °C (5804R; Eppendorf, Hamburg, Germany) for the complete separation of the lignin fraction. Thereafter, the lignin fraction was freeze-dried (Telstar, Terrassa, Spain) and stored in plastic bottles at room temperature for further analysis. The leftover liquid, which contained hemicellulose-derived sugars, was also stored in plastic bottles and refrigerated at 4 °C. A simplified overview of the experimental design is presented in Figure 1.

2.3. Analytical Methods

The untreated sawdust, as well as fractions of the cellulose-rich pulps, lignins, and the hemicelluloses, were analyzed for their carbohydrates and Klason lignin composition using the National Renewable Energy Laboratory protocols [14,15]. The carbohydrates were quantified using high-performance anion exchange chromatography (HPAEC) (Thermo Scientific, Waltham, MA, USA) using a CarboPac PA-20 column (3 × 150 mm; Dionex™, Thermo Scientific) with a pulsed amperometric detector equipped with a gold electrode. Oligomeric sugars were initially hydrolyzed to monomers using 4% H₂SO₄, whereas monomeric sugars were quantified directly from the liquid. The samples were eluted with a gradient of three eluents for 60 min at 30 °C: Eluent A: deionized water; Eluent B: 200 mM NaOH; and Eluent C: 100 mM NaOAc in 100 mM NaOH in a gradient: 0–18 min, isocratic step (98.8% A and 1.2% B); 18–20 min, 1.2–50% B; 20–30 min, 50% A and 50% B; 30.1–46 min, 100% C; 46.1–50 min, 100% B; and 50.1–60 min, 98.8% A and 1.2% B at 0.4 mL/min.

Inorganic ash content was measured gravimetrically after ashing at 550 °C for 3 h with a heat increase of 1 °C/min. Moisture content was determined gravimetrically on a moisture analyzer (Sartorius, Göttingen, Germany).

Size exclusion chromatography of the lignins was performed to determine the molecular weight of the samples. Incipiently, the acetobromination of the lignin was performed by adding 0.9 mL glacial acetic acid and 0.1 mL acetyl bromide to 5 mg of lignin, followed by stirring at room temperature for 2 h in closed amber vials [16]. The solution was subsequently transferred into a round bottom flask and evaporated using the same rotary evaporator whose model was previously given. This was followed by two steps of tetrahydrofuran (THF) washing and resuspension of the samples in 1 mL THF before filtration in 0.22 µm hydrophobic filters. The samples were then analyzed on a HPLC (PerkinElmer, Waltham, MA, USA) equipped with a UV detector and a Styragel^® HR 4E column (Waters, Milford, MA, USA) set at 280 nm, operating at 40 °C, with THF as mobile phase and 0.6 mL/min flow. The results were rounded to the nearest hundreds, and calibration was performed with polystyrene (Sigma-Aldrich, St. Louis, MO, USA).

Sugar degradation compounds were evaluated using HPLC (PerkinElmer, Waltham, MA, USA) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and a UV detector (set to λ = 205 for formic acid, 227 for acetic acid, or 280 nm for levulinic acid, hydroxymethylfurfural (HMF), and furfural) operated at 65 °C using 5 mM H₂SO₄ as the mobile phase with a flow of 0.6 mL/min.

2.4. Calculations

The recovery of cellulose was calculated according to the following formula:

$$\% Recovery=100\times \left(\frac{{\%LC}_{Pretreated}\times {solids recovery}_{MASS}}{{\%LC}_{Untreated}\times {initial biomass}_{MASS}}\right),$$

where “LC” indicates the composition of each lignocellulosic component (cellulose, hemicellulose, or Klason lignin) in both pretreated and untreated biomass. “Solids recovery” represents the pulp recovered after the pretreatment, whereas “initial biomass” represents the pretreated mass.

2.5. Enzymatic Saccharification

The cellulose pulps were saccharified enzymatically in 2 mL Eppendorf tubes with 3% w/w biomass. The following were used in the process: 20 FPU/g of biomass of Cellic^® CTec2 enzyme solution (Novozyme A/S, Bagsvaerd, Denmark) and citrate buffer at pH 5 with 0.02% w/v sodium azide. The mixtures were incubated on a thermomixer (Eppendorf, Hamburg, Germany) for 48 h at 50 °C, and samples were collected every 24 h. The glucose content was determined on a HPAEC, as described previously (see Section 2.3).

3. Results and Discussion

3.1. Pulp Composition

Insoluble solids (pulp) obtained after organosolv pretreatment retain most of the cellulose present in the biomass. The composition of the pulps, cellulose, hemicelluloses, and Klason lignin that were retained following the pretreatment procedure are presented in

Table 2. The composition of pretreated solids (pulp).

Codes	Pulp Recovery (% w/w)	Cellulose (% w/w) (% Retained)	Hemicelluloses (% w/w) (% Retained)	Klason Lignin (% w/w) (% Retained)	Ashes (% w/w)	Total (% w/w)
S-0A5x	63.65	48.06 ± 0.25 (73.82)	24.07 ± 0.27 (47.01)	30.69 ± 0.41 (59.97)	0.15 ± 0.00	102.96
S-0A5y	43.19	49.02 ± 0.67 (51.10)	3.69 ± 0.21 (4.89)	36.92 ± 0.37 (48.96)	0.44 ± 0.06	90.07
S-0A6x	67.11	45.55 ± 0.05 (73.77)	28.90 ± 0.07 (59.53)	28.34 ± 0.77 (58.39)	0.17 ± 0.06	102.96
S-0A6y	36.20	50.46 ± 0.77 (44.09)	3.67 ± 0.48 (4.08)	34.06 ± 0.07 (37.86)	0.47 ± 0.06	88.66
S-0B5x	63.47	48.31 ± 0.48 (73.99)	23.85 ± 0.78 (46.46)	28.60 ± 0.31 (55.74)	0.34 ± 0.03	101.10
S-0B5y	40.34	54.47 ± 0.15 (53.02)	5.04 ± 0.35 (6.25)	29.49 ± 0.45 (36.52)	0.83 ± 0.12	89.84
S-0B6x	65.56	48.18 ± 0.63 (76.23)	29.72 ± 0.32 (59.80)	23.79 ± 0.64 (47.88)	0.90 ± 0.08	102.59
S-0B6y	35.02	51.75 ± 1.06 (43.74)	5.64 ± 0.09 (6.06)	31.90 ± 1.31 (34.31)	0.56 ± 0.08	89.85
S-1A5x	74.38	45.50 ± 0.31 (81.67)	30.93 ± 0.24 (70.62)	30.12 ± 1.33 (68.77)	0.18 ± 0.00	106.73
S-1A5y	64.89	53.46 ± 0.88 (83.72)	10.26 ± 0.08 (20.43)	29.73 ± 1.88 (59.22)	0.26 ± 0.02	93.70
S-1A6x	78.82	44.14 ± 0.30 (83.95)	33.11 ± 0.08 (80.11)	26.21 ± 0.05 (63.42)	0.23 ± 0.04	103.69
S-1A6y	53.08	54.80 ± 0.84 (70.20)	9.57 ± 0.13 (15.59)	24.19 ± 0.16 (39.43)	0.55 ± 0.02	89.11
S-1B5x	74.55	43.55 ± 0.36 (78.36)	29.86 ± 0.16 (68.32)	30.76 ± 0.00 (70.40)	0.17 ± 0.00	104.34
S-1B5y	53.51	54.61 ± 0.33 (70.52)	8.17 ± 0.18 (13.42)	28.07 ± 0.01 (46.13)	0.19 ± 0.08	91.05
S-1B6x	74.33	43.52 ± 0.17 (78.07)	30.52 ± 0.11 (69.63)	29.37 ± 0.31 (67.02)	0.93 ± 0.12	104.35
S-1B6y	48.96	55.05 ± 0.43 (65.04)	9.42 ± 0.03 (14.16)	26.35 ± 0.45 (39.61)	0.02 ± 0.00	90.84

Representation of the codes: 0—pretreatment at 200 °C; 1—pretreatment at 180 °C; A—pretreatment time of 15 min; B—pretreatment time of 30 min; 5—50% v/v ethanol content; 6—60% v/v ethanol content; x—catalyst absent; y—1% w/w_BIOMASS H₂SO₄.

. Solubilization of the biomass was higher when the pretreatment was performed using the acidic catalyst, and the cellulose retaining yield in the pulp ranged from 43.7% w/w (S-0B6y) to 84.0% w/w (S-1A6x). Pretreatments at a lower temperature of 180 °C resulted in a greater recovery rate among the samples treated with acidic catalyst. The acidic catalyst increased the cellulose content in the pulps across all tested temperatures and times. The recovery of cellulose showed a strong correlation with the presence of catalyst, as evidenced by the differences in pulp recovery in the presence or absence of the catalyst. The use of catalyst resulted in lower cellulose retained across all pretreatments (recoveries with the catalyst ranged from 43.7–83.7%; whereas in absence the absence of the catalyst, the range was 73.8–84.0%) except the treatment at 180 °C for 15 min with 50% ethanol. Finally, in terms of solvent concentration, pretreatment with 50% ethanol at 200 °C presents, in most cases, higher cellulose content, whereas at 180 °C, solvent concentration does not seem to affect the cellulose content. Agnihotri and collaborators studied organosolv pretreatment using 63% v/v ethanol content using formic acid at 0.05 mol/L as a catalyst and obtained the highest degree of delignification (about 65%), which was similar to our results, although this was achieved at a much higher temperature of about 235 °C [17].

Hemicelluloses recovery showed a similar trend, as pretreatment with the catalyst at 200 °C resulted in lower recovery (4.1–6.3%), indicating a solubilization of over 93.7% in all pretreatments performed at this temperature with the acid catalyst; whereas the highest hemicelluloses recovery was attained at a shorter pretreatment time at 180 °C without catalyst (S-1A6x). Delignification was also dependent on the presence of acid, as all pretreatments performed with the acid catalyst resulted in lower recovery of lignin in the pulps, which was further augmented when treatment took place with 60% ethanol. On the other hand, owing to the higher removal of hemicellulose and cellulose, use of the acidic catalyst results in higher lignin content in the pulps after treatment at 200 °C; whereas the opposite results were observed at 180 °C. Finally, lignin exhibits greater solubilization in solutions with higher ethanol contents and, in longer pretreatments, provides the highest biomass fractionation [18]. Hrůzová and collaborators studied the organosolv pretreatment of spruce bark and obtained the highest delignification of 58.6% on the pretreatment performed at 190 °C for 60 min with 60% v/v ethanol content and 1% w/w biomass sulfuric acid [19].

3.2. Hemicellulose Fraction

During organosolv fractionation, the liquid fraction that is left over after the pulp and lignin fractions are removed is primarily hemicellulose-derived sugars. The sugar content of this liquid portion is presented in Figure 2. The pretreatments using the catalyst yielded the highest recovery of hemicellulose sugars, with the bulk of the sugars recovered as monomers. This indicates that the presence of the acid catalyst increased the hydrolysis of the biomass. The highest recovery was from the pretreatment performed at 200 °C for 30 min with 50% v/v ethanol content and the acid catalyst, achieving a 10.01 g/100 g_BIOMASS recovery of monomeric sugars, of which 7.13 g/100 g_BIOMASS was derived from hemicelluloses, which accounts for 20.9% of the initial hemicelluloses. The recovery of hemicelluloses was significantly impacted by the amount of ethanol used in the pretreatment process with seven out of eight pretreatments performed with 50% v/v ethanol yielding higher sugars relative to pretreatments performed with 60% v/v. Dias et al. performed organosolv pretreatment of olive tree pruning biomass and obtained higher derivatives from sugar degradation (i.e., HMF, furfural, and levulinic acid) when the pretreatments were performed with higher water content [20], which is comparable to our previous study using halophytes [21]. Longer pretreatment time favored the recovery of hemicelluloses as an isolated fraction, which correlates to delignification.

The quantification of the sugar degradation products remaining on the liquid fraction after the described methodology (i.e., ethanol evaporation) is shown in

Table 3. The degradation compounds present in the liquid fraction.

Codes	Acetic Acid (g/100 g)	Formic Acid (g/100 g)	Levulinic Acid (g/100 g)	HMF (g/100 g)	Furfural (g/100 g)
S-0A5x	0.00 ± 0.00	0.25 ± 0.02	0.00 ± 0.00	1.66 ± 0.05	0.98 ± 0.02
S-0A5y	6.77 ± 0.26	0.00 ± 0.00	0.00 ± 0.00	4.89 ± 0.04	2.09 ± 0.21
S-0A6x	0.00 ± 0.00	0.28 ± 0.02	0.00 ± 0.00	0.83 ± 0.01	0.25 ± 0.01
S-0A6y	4.58 ± 0.20	0.00 ± 0.00	0.00 ± 0.00	4.25 ± 0.44	4.27 ± 0.68
S-0B5x	0.00 ± 0.00	0.37 ± 0.03	0.00 ± 0.00	1.86 ± 0.01	0.25 ± 0.00
S-0B5y	0.24 ± 0.12	0.00 ± 0.00	0.00 ± 0.00	2.90 ± 0.03	0.85 ± 0.05
S-0B6x	0.00 ± 0.00	0.24 ± 0.01	0.00 ± 0.00	1.07 ± 0.00	0.25 ± 0.01
S-0B6y	7.26 ± 0.62	0.00 ± 0.00	0.00 ± 0.00	3.95 ± 0.15	5.99 ± 0.72
S-1A5x	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.22 ± 0.00	0.35 ± 0.00
S-1A5y	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	4.54 ± 0.30	5.23 ± 0.73
S-1A6x	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.20 ± 0.00	0.09 ± 0.00
S-1A6y	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	3.25 ± 0.01	7.18 ± 0.13
S-1B5x	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.40 ± 0.05	0.30 ± 0.05
S-1B5y	8.33 ± 0.12	0.00 ± 0.00	0.00 ± 0.00	3.64 ± 0.00	6.80 ± 0.01
S-1B6x	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.25 ± 0.01	0.28 ± 0.01
S-1B6y	5.22 ± 0.22	0.00 ± 0.00	0.00 ± 0.00	2.68 ± 0.32	2.32 ± 0.05

Representation of the codes: HMF—5-(Hydroxymethyl)furfural; 0—pretreatment at 200 °C; 1—pretreatment at 180 °C; A—pretreatment time of 15 min; B—pretreatment time of 30 min; 5—50% v/v ethanol content; 6—60% v/v ethanol content; x—catalyst absent; y—1% w/w_BIOMASS H₂SO₄.

.

The analysis of the composition of the hemicelluloses fraction is important for the potential applications of the hemicellulose product. When comparing pretreatments carried out under the same circumstances without the catalyst to those carried out with the acidic catalyst, all the acid-pretreated samples showed higher levels of furans (furfural and HMF). Levulinic acid was absent in all samples, while formic acid was only present in samples pretreated without catalyst at 200 °C. Acetic acid was present in samples pretreated under the most severe conditions, i.e., in all pretreatments performed with the acidic catalyst at 200 °C and longer pretreatments (30 min) performed with the acid catalyst at 180 °C.

3.3. Lignin Fraction

The lignin fractions obtained from the organosolv pretreatment process of Norway spruce sawdust under different conditions were analyzed for their contents of sugars, Klason lignin, and ashes. The weight percentages of these components are presented in

Table 4. The composition of the lignin obtained after organosolv pretreatment of Norway spruce sawdust.

Codes	Cellulose (% w/w)	Hemicelluloses (% w/w)	Klason Lignin (% w/w)	Ashes (% w/w)	Total (% w/w)	Mn (g/mol)	Mw (g/mol)	DI
S-0A5x	0.40 ± 0.03	1.74 ± 0.10	97.02 ± 7.38	0.11 ± 0.02	99.27	2000	12,500	6.25
S-0A5y	0.00 ± 0.00	2.48 ± 0.00	93.64 ± 0.00	0.06 ± 0.01	96.18	900	1500	1.67
S-0A6x	0.96 ± 0.02	0.82 ± 0.15	88.63 ± 7.11	0.30 ± 0.18	90.71	1900	13,600	7.16
S-0A6y	0.02 ± 0.00	1.28 ± 0.00	93.49 ± 0.00	0.01 ± 0.01	94.81	1000	1700	1.70
S-0B5x	0.51 ± 0.00	0.00 ± 0.00	85.23 ± 1.32	3.31 ± 0.74	89.04	2000	13,100	6.55
S-0B5y	0.00 ± 0.00	0.00 ± 0.00	85.23 ± 0.07	1.85 ± 0.42	87.08	1000	2700	5.76
S-0B6x	1.00 ± 0.00	2.60 ± 0.01	87.85 ± 0.40	0.07 ± 0.01	91.51	2000	14,900	7.45
S-0B6y	1.34 ± 0.28	0.44 ± 0.08	95.25 ± 4.38	0.16 ± 0.00	97.19	900	1500	1.67
S-1A5x	0.84 ± 0.00	6.09 ± 0.06	81.59 ± 0.99	0.27 ± 0.05	88.80	2000	15,000	7.50
S-1A5y	0.27 ± 0.00	1.68 ± 0.00	91.99 ± 0.00	0.03 ± 0.01	93.97	800	1200	1.50
S-1A6x	1.10 ± 0.03	1.27 ± 0.09	86.68 ± 9.27	0.10 ± 0.03	89.14	1800	11,200	6.22
S-1A6y	0.28 ± 0.00	1.58 ± 0.00	92.68 ± 0.00	0.03 ± 0.01	94.58	1000	1700	1.70
S-1B5x	2.23 ± 0.04	2.03 ± 0.18	85.16 ± 0.67	0.22 ± 0.00	89.64	1800	10,600	5.89
S-1B5y	0.48 ± 0.01	0.65 ± 0.01	93.62 ± 0.13	0.16 ± 0.00	94.91	800	1100	1.38
S-1B6x	0.38 ± 0.09	1.67 ± 0.09	88.21 ± 0.44	0.22 ± 0.00	90.48	1800	11,000	6.11
S-1B6y	2.38 ± 0.11	1.19 ± 0.03	90.10 ± 0.75	0.08 ± 0.00	93.76	900	1300	1.44

Mn: Number average molecular weight; Mw: weight average molecular weight; DI: Dispersity index (Mw/Mn). Representation of the codes: 0—pretreatment at 200 °C; 1—pretreatment at 180 °C; A—pretreatment time of 15 min; B—pretreatment time of 30 min; 5—50% v/v ethanol content; 6—60% v/v ethanol content; x—catalyst absent; y—1% w/w_BIOMASS H₂SO₄.

. Low levels of sugar contamination were generally observed, with the highest value of 3.6% w/w in samples pretreated at 200 °C, while values as high as 6.93% w/w were found in samples pretreated at 180 °C. Ash contents of the lignin samples were also typically low and depended on the duration of pretreatment, with most cases of longer treatment resulting in higher ash contamination. The amount of ash in the lignin fractions was greater in the samples that were not acid-pretreated.

The content of Klason lignin was higher when the acid catalyst was applied in all applied conditions, except the shortest pretreatment performed at 200 °C for 15 min with 50% v/v ethanol content. As previously discussed, the shorter pretreatments duration (15 min) with lower ethanol concentration (50% v/v) at both temperatures (180 °C and 200 °C) exhibited lower delignification among the pretreatments containing the acid catalyst. The condition at 200 °C (S-0A5x) presented 97.0% Klason lignin, the highest content among the pretreatments without the catalyst, which with the exception of this condition, had less than 89% Klason lignin. The weight average molecular weight (Mw) of lignin ranged from 1100 to 15,000 Da, which decreased after pretreatment with the acidic catalyst. A similar pattern was observed with the number average molecular weight (Mn) value, suggesting that the use of an acidic catalyst increases the depolymerization of lignin and favors its application as a building block in the generation of value-added products [22].

3.4. Enzymatic Saccharification

In order to convey the valorization of organosolv fractionated pulps as feedstock for microbial conversion processes, enzymatic saccharification was performed. Given the different effects of the pretreatment time, solvent concentration (ethanol content), temperature, and the presence or absence of the catalyst on the biomass during the pretreatment, we evaluated the effects of different conditions on the saccharification of the biomass. Pulp samples were incubated with an enzyme load of 20 FPU/g_biomass (Cellic^® CTec2) for 24 h and 48 h (Figure 3). In relation to the hydrolysis, all the samples pretreated with the acidic catalyst achieved higher saccharification yields when compared to pretreatments without the use of the catalyst; in addition, all the pretreatments presented higher saccharification yield when compared to the untreated feedstock. Hydrolysis yielded up to 77.8% (S-1B5y), showing a 7.1-fold increase when compared to the untreated biomass. For the samples pretreated without the acidic catalyst, the highest saccharification yield was 20.6%, and the lowest yield was exactly 12.1%, whereas the untreated biomass reached 11.0%. In most of the cases, longer treatment duration resulted in higher saccharification, which was more pronounced in treatments with acid. Nitsos and collaborators reviewed the organosolv pretreatment and saccharification of softwood biomass and summarized the yield, demonstrating, on average, lower yields on pretreatments without the catalyst when compared to pretreatments with the catalyst [23].

Nevertheless, to normalize the difference between cellulose composition and pulp recovery on the various pretreatments and untreated material, the glucose released in relation to the initial weight of the spruce sawdust used in each pretreatment is represented in Figure 4. After 48 h, untreated biomass released 0.046 g/g of biomass, whereas the value increased up to 0.537 g/g of biomass with the pretreatment conducted at 200 °C for 30 min with 50% v/v ethanol content and 1% w/w H₂SO₄ (S-0B5y). With the exception of the longer pretreatment at 200 °C with 60% ethanol, all the biomasses pretreated with the catalyst yielded more than 0.300 g/g_biomass during saccharification, demonstrating the superior ability of acid-pretreated biomass to be applied in saccharification processes and potentially in microbial cultivations. In their review of the effects of various acid catalysts on the organosolv pretreatment of lignocellulosic biomass, Zhang et al. found a strong positive correlation between the delignification, hemicellulose removal, and cellulose accessibility of acid-catalyzed pretreatments using ethanol as a solvent [24], which is consistent with the findings of the present study.

For the acid-free pretreatments, with the exception of the longer pretreatment time of 30 min at 180 °C, where the glucose release gave similar values (0.085 g/g and 0.086 g/g of biomass for 50% and 60% v/v ethanol content, respectively), lower ethanol content was related to the highest sugar release during hydrolysis.

3.5. Mass Balances of the Major Biomass Fractions

The aim of this part of the study was to address the recovery of the three primary cell wall constituents (cellulose, lignin, and hemicelluloses) in the three distinct streams that were generated by the organosolv fractionation (Figure 1) to obtain a more holistic understanding of the efficiency of the process. Figure 5 presents the distribution of cellulose recovered in the three different streams from the organosolv fractionation process. The majority of the cellulose was retained in the pulps with lower values on the pretreatments performed at 200 °C with the catalyst and 60% v/v ethanol content (altogether presenting cellulose losses above 40%). When comparing the acid-catalyzed and acid-free pretreatments, a trend was evident, which was that pretreatments with acid catalysts yielded more cellulose in the hemicellulose fractions than the pretreatments without acid. This is supported by the higher biomass solubilization during organosolv fractionation and by the recovery of pulp (Table 2). Harsher pretreatment conditions (presence of the catalyst, higher temperature, and longer duration) resulted in less recovery of cellulose, which can be linked to the breakdown of monomers of glucose into hydroxymethylfurfural and organic acids, as shown in Table 3.

The distribution of lignin in the various streams is presented in Figure 6. In the majority of the samples, most of the lignin was retained in the pulp, with the exception of the pretreatments performed at 200 °C with 60% v/v ethanol content and the catalyst (S-0A6y and S-0B6y). As discussed in a preceding section, the highest ethanol concentration used in this study presents higher efficiency in solubilizing lignin during organosolv, and the presence of acid enhances delignification, which explains the variation in lignin distribution in these samples.

Finally, the distribution of hemicelluloses in the various streams is presented in Figure 7. As the bond between cellulose fibers and lignin polymers on the cellular structure, hemicellulose is the most susceptible to degradation under organosolv fractionation conditions, which impacts its mass balance. The pretreatments carried out under milder conditions (180 °C for 15 min without acid) showed greater recovery of hemicelluloses from the pulp, an indication that milder fractionation conditions of biomass enhance the recovery of hemicelluloses. The hemicellulose liquid fraction from the acid-induced pretreatments had a greater sugar content, which led to the optimum recovery of hemicelluloses from the residual liquid rather than the pulp, as was the case with the pretreatments without the catalyst. This is clearly demonstrated by the data presented in Figure 2.

4. Conclusions

Using the organosolv approach, cellulose-rich fibers were satisfactorily obtained from Picea abies (Norway Spruce) sawdust. Comparison of the effect of temperature, time, and ethanol concentration, as well as the presence and absence of an acid catalyst examined after 16 pretreatment attempts, showed that hemicelluloses removal was higher than 79.6% in all of the conditions evaluated when the pretreatments were conducted with acid; whereas in absence of the catalyst, the highest removal of hemicellulose was 53.5%. Enhanced delignification was obtained from longer pretreatment. Lignin samples had very low ash content and minimal sugar contamination, with only one of the samples having more than 4.3% w/w sugar content.

Enzymatic saccharification of the cellulose-rich pulps showed that the acid-pretreated pulp samples were more susceptible to saccharification and released more glucose during enzymatic saccharification trial. Pretreated spruce sawdust exhibited a 7.1-fold increase in the saccharification yield of cellulose compared to untreated biomass. The glucose release per gram of biomass (pretreated or untreated) increased by up to 11.7-fold among acid-pretreated samples and 3-fold among the pretreatments without the catalyst.

Mass balances showed that the majority of cellulose remains on the pretreated pulps, and higher losses occurred at pretreatments performed with acid at 200 °C. Lignin had higher solubilization during organosolv and was therefore recovered mainly in the lignin fraction in the pretreatments performed at 200 °C using 60% v/v ethanol concentration and an acid catalyst. Acid pretreatment resulted in more hemicellulose losses, which were also reflected in the resultant material’s increased acetic acid and furans content.

Overall, our findings suggest that spruce sawdust exhibits the potential to be used as a feedstock in a biorefinery setting and that various combinations of parameters may be used to obtain the desired final product.