Salicornia dolichostachya organosolv fractionation: towards establishing a halophyte biorefinery

Halophytes are a potential source of lignocellulosic material for biorefinery, as they can be grown in areas unsuitable for the cultivation of crops aimed at food production. To enable the viable use of halophytes in biorefineries, the present study investigated how different organosolv process parameters affected the fractionation of green pressed fibers of Salicornia dolichostachya. We produced pretreated solids characterized by up to 51.3% ± 1.7% cellulose, a significant increase from 25.6% ± 1.3% in untreated fibers. A delignification yield of as high as 60.7%, and hemicellulose removal of as high as 86.1% were also achieved in the current study. The obtained cellulose could be completely converted to glucose via enzymatic hydrolysis within 24 h. The lignin fractions obtained were of high purity, with sugar contamination of only 1.22% w/w and ashes below 1% w/w in most samples. Finally, up to 29.1% ± 0.4% hemicellulose was recovered as a separate product, whose proportion of oligomers to total sugars was 69.9% ± 3.0%. To the best of our knowledge, this is the first report in which Salicornia fibers are shown to be a suitable feedstock for organosolv biomass fractionation. These results expand the portfolio of biomass sources for biorefinery applications.


Introduction
The increasingly limited availability of non-renewable energy sources and raw materials requires a more sustainable use of natural resources and, consequently, better understanding of upcycling processes. 1,2 The use of lignocellulosic biomass in biorenery represents a sustainable alternative to fossil resources for the production of chemicals and energy. Biomass is composed primarily of carbohydrates, including cellulose, hemicellulose, and lignin, as well as varying amounts of extractives. Hence, biomass fractionation is of paramount importance for a holistic biomass use. [3][4][5] In particular, successful fractionation of lignocellulosic substrates enables the valorization of all biomass components for their use in the manufacturing of biofuels, prebiotics, pharmaceuticals, chemicals, and cosmetics. [6][7][8] Organosolv fractionation has attracted increasing interest owning to its ability to separate lignocellulosic biomass into high-quality cellulose, hemicellulose, and lignin streams. 9,10 During organosolv, lignocellulosic biomass is treated at high temperatures with different combinations of solvents (e.g., ethanol) and water. The resulting fractions can be used in downstream applications based on their properties, generating either high-volume and low-value or low-volume and high-value bio-based products. [11][12][13] Salicornia is a genus of halophyte herbs belonging to the Amatanthaceae family, with species endemic to every continent except South America and Australia. 14 The genus Salicornia includes 117 species, with S. herbacea, S. bigelovii, S. europea, S. prostata, S. ramosissima, and S. verginica being the most widespread. The plants are distributed extensively throughout Europe's shorelines, from the Arctic to the Mediterranean and including the Caspian and Black Sea. 15 Salicornia dolichostachya is a species native to European boreo-temperate biomes, where it acts as a pioneer plant in coastal areas, acquiring most of the nutrients from ooding seawater. 16 Salicornia plants are rich in carbohydrates, lignin, fatty acids, proteins, as well as vitamins A, C, and E; 17,18 whereas inorganic compounds include mainly sodium and potassium, plus other minerals. 17,19,20 Some species of Salicornia are used as animal feed, and the culinary use of stems and seeds has also been described. 21,22 Indeed, dried ground Salicornia plants are sold as a substitute for table salt. 23 Some species can tolerate water with more than 1000 mM NaCl, which is higher than the average salt concentration in the oceans. Hence, these plants could be grown on a large scale and irrigated with seawater. 24,25 Because growth in high-salinity soils is not suitable for the cultivation of other plans, Salicornia species could become a valuable crop in coastal areas affected by the intrusion of seawater. In Sweden, salty groundwater in continental areas may come from fossil seawater, water-rock interaction, freezing of seawater, and anthropogenic activities. 26 Climate change may cause the sea level to rise, and hydrological cycles will lead to more areas with increased soil salinity. Because halophytes grow in areas with high salinity, and as such there is no competition with food production, 27 their utilization as a renewable resource commands further investigation. Studies have reported yields ranging from 2.51-6.07 tons per hectare for S. brachiata to 35 tons per hectare for S. bigelovii. [28][29][30] The aim of the current study was to establish an organosolvbased fractionation method for the treatment of S. dolichostachya bers within a biorenery concept. Organosolv process parameters, such as reaction temperature, treatment duration, and solvent type, can signicantly affect fractionation efficiency. Here, we screened several organosolv process parameters to identify optimal conditions for maximal fractionation of S. dolichostachya bers. To the best of our knowledge, no previous study on organosolv pretreatment of Salicornia exists, making this the rst attempt towards establishing a biorenery concept that uses Salicornia biomass as feedstock.

Feedstock
S. dolichostachya was collected from the Wadden Sea on the Danish coast (55.307733, 8.652292). Aer collection, the samples were rinsed with freshwater. Using a single horizontal auger screw press (Omega, Sana, Czech Republic), two fractions were obtained: liquid juice and solid de-juiced biomass. The latter was dried in an oven at 95 C for 24 h until constant weight was attained. The dried bers were milled to particles smaller than 1 mm using a size reduction cutting mill (Retsch, Haan, Germany) and stored in plastic bags at room temperature until further use. The composition of the untreated biomass was 25.6% AE 1.3% w/w cellulose, 30.7% AE 1.0% w/w hemicellulose, 13.9% AE 0.1% w/w lignin, 5.3% AE 0.4% w/w ashes (partially comprised in the extractives), 9.49% AE 0.29% w/w water extractives, and 2.36% AE 0.61% w/w ethanol extractives.

Organosolv fractionation
The milled de-juiced S. dolichostachya bers were pretreated in an air-heated multidigester system comprising six 2.5-L batch autoclave reactors. For each pretreatment, 90 g dry biomass was added to a solution of ethanol : water at a 10 : 1 v/w ratio. The conditions were designed to analyze the effect of temperature (160, 180 or 200 C), treatment time (15,30,45 or 60 min), and solvent composition (40%, 50%, 60% or 70% v/v) as displayed in Table 1. At the end of pretreatment, the reactors were cooled to below 40 C and the slurry was vacuum-ltered. Next, the slurry was washed with the same solvent as above, producing an insoluble pulp fraction, liquor, and a wash phase. The latter two were processed in a rotary evaporator (Heidolph, Schwabach, Fig. 1 Processing of Salicornia biomass and recovery of cellulose, hemicellulose, and lignin fractions after organosolv pretreatment. Germany) to remove ethanol and precipitate lignin. The aqueous solution obtained from the ltrate and wash was centrifuged at 12 000 Â g for 10 min at 4 C (5804R; Eppendorf, Hamburg, Germany) to precipitate the remaining lignin, which was then added to the previously produced lignin stream, freeze-dried, and stored at room temperature. The liquid fraction containing hemicellulose-derived sugars and obtained aer centrifugation was stored at 4 C. The solid pulp was airdried and stored in plastic bottles at room temperature. The experimental procedure is summarized in Fig. 1.

Analysis
Untreated and pretreated biomass were analyzed in terms of cellulose, hemicellulose, and lignin composition according to the National Renewable Energy Laboratory protocol for the determination of structural carbohydrates and lignin in biomass. 31 The sugar concentration was measured on a high-performance liquid chromatography (HPLC) apparatus (PerkinElmer, Waltham, MA, USA) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and a refractive index detector. The column was operated at 65 C with 5 mM H 2 SO 4 as mobile phase at a ow rate of 0.6 mL min −1 . Monomeric sugars present in the recovered hemicellulose fraction were determined directly by HPLC; whereas oligomeric sugars were rst hydrolyzed to monomers by adding H 2 SO 4 to a nal concentration of 4% w/w and incubating at 121 C for 1 h. Following neutralization with CaCO 3 , the resulting sugars were analyzed by HPLC. The inorganic ash content was determined gravimetrically by ashing the samples at 550 C for 3 h, with a temperature increase of 1 C min −1 . Moisture content was determined gravimetrically aer drying the samples at 95 C overnight until constant weight was attained. To determine the extractives present in the untreated biomass, Soxhlet extraction was carried out rst with water and then with ethanol. The solvents were evaporated in a rotary evaporator (Heidolph), and the fractions were quantied and stored. The analysis was performed in duplicates. Size-exclusion chromatography was performed with a gel permeation column to determine the molecular weight distribution of lignins. First, acetobromination of lignins was performed by mixing 5 mg lignin with 0.9 mL glacial acetic acid and 0.1 mL acetyl bromide. Next, the mixture was stirred at 500 rpm for 2 h at room temperature in closed vials. The mixture was then transferred to round-bottom asks in a rotary evaporator (Heidolph) and dried at 50 C and 50 mBar. The dried material was washed two times with tetrahydrofuran, the solvent was evaporated, and the sample was solubilized in 1 mL tetrahydrofuran. Aer ltering through 0.22 mm hydrophobic lters, the sample was analyzed directly by HPLC using a Styragel® HR 4E column (Waters, Milford, MA, USA) and a UV detector at 280 nm. The column was operated at 40 C, with tetrahydrofuran as mobile phase at a ow rate of 0.6 mL min −1 . The numbers were rounded up at 100 s due to the resolution of the method.

Calculations
The delignication yield was calculated according to the following formula: where % lignin indicates the content of lignin (w/w) in either untreated or pretreated biomass, initial biomass MASS indicates the weight in grams of dry biomass used during pretreatment, and pretreated solids MASS indicates the dry weight in grams of pretreated solid biomass. Hemicellulose fractionation was calculated according to the following formula: where % hemicellulose indicates the weight percentage of hemicellulose in either untreated material or pretreated (F) and wash (W) liquids, and volume indicates the liquor volume aer ltration and ethanol evaporation for the ltrate (F) and wash (W) liquids. The solubilization yield of the different fractions was calculated according to the following formula: where FP indicates the fractionated product (cellulose, hemicellulose or lignin) or ashes in either pretreated or untreated solid biomass.

Enzymatic saccharication
Enzymatic saccharication of pretreated biomass was evaluated using the commercial cellulase enzyme solution Cellic® CTec2 (Novozymes A/S, Bagsvaerd, Denmark) at an enzyme load of 20 FPU g solids −1 . The reactions were performed in duplicate inside 2 mL microcentrifuge tubes containing 1.0 mL solutions comprising 3% w/w dry solids in 50 mM citrate buffer (pH 5). The mixture was incubated in a thermomixer at 50 C and 800 rpm for 72 h, with samples taken at 8, 24, 48, and 72 h. Aer collection, the samples were placed in a water bath at 100 C for 5 min to denature the enzyme and then centrifuged at 12 000 Â g for 10 min at room temperature. The supernatant was removed from the solids, ltered through a 0.22 mm syringe lter (Sartorius, Göttingen, Germany) and the sugars were quantied by HPLC as previously described (see section "Organosolv fractionation").

Results and discussion
3.1 Organosolv pretreatment 3.1.1 Pretreated solids fraction. Organosolv pretreatment of S. dolichostachya bers was designed for the stepwise optimization of different process parameters, including temperature, time, and ethanol content (Table 1). Table 2 details the composition of pretreated solids, alongside recovery of the main biomass fractions (cellulose, hemicellulose, and lignin), and ashes under different conditions. First, we tested the effect of treatment temperature (from 160 C to 200 C) under constant time (30 min) and ethanol content (60% v/v) on the fractionation of Salicornia bers. With increasing temperature, a higher proportion of the initial biomass was solubilized, reducing the yield of pretreated solids from 64.02% to 36.72%, and boosting cellulose content from 31.14% w/w to 51.27% w/w ( Table 2). The nal cellulose content was 2-fold higher than in untreated S. dolichostachya bers. This was matched by the proportional decrease in hemicellulose (from 32.2% w/w to 10.52% w/w) and lignin (from 16.56% w/w to 14.86% w/w) content in pretreated solids.
Increased temperature had a positive impact on delignication. A higher delignication is a desirable outcome as it creates a biomass with less lignin, which facilitates subsequent processing. Nevertheless, the higher delignication (56.39%) observed at 200 C was not coupled to an increase in hemicellulose recovery (see "Hemicelluloses fraction" section), which could be related to sugar degradation into side-products, such as furans and organic acids (levulinic acid, formic acid, and acetic acid). Because such side-products lead to lost sugar mass and inhibit microbial growth, 32 180 C was selected as the optimal temperature for further studies.
Next, we examined the effect of treatment time under constant temperature (180 C) and ethanol content (60% v/v). A longer time promoted biomass solubilization and improved fractionation. The highest delignication rate (60.7%) was achieved with 45 min pretreatment, which was higher than pretreatment at 200 C for 30 min (Table 2). This result demonstrated the importance of testing different parameters to select the best pretreatment conditions for the desired product. When the treatment was extended to 60 min, delignication dropped to 55.60%, resulting in more lignin being recovered from pretreated solids. This can be attributed to the formation of pseudolignin from hemicellulose decomposition, 33 indicating that the conditions were harsh for this biomass stream.
Hemicellulose removal from pretreated solids increased proportionally with the duration of pretreatment. As discussed previously, the depolymerization of hemicellulose sugars is expected to increase with a more severe pretreatment (i.e., with prolonged treatment time). Hence, 45 min was chosen as the optimal treatment time for further studies.
Lastly, the effect of ethanol content was studied under stable temperature (180 C) and treatment time (45 min). As the ethanol content was raised from 40% v/v to 60% v/v, delignication showed a marked increase, but decreased sharply when ethanol content was augmented further to 70% v/v ( Table 2). Achieving an optimal ethanol : water ratio during organosolv fractionation is very important as water facilitates the hydrolysis of bonds between sugars by increasing the hydrogen ion concentration and thus lowering the pH in the solution. 34 While ethanol promotes lignin dissolution into the liquor because of lignin's superior solubility in ethanol, the acidic conditions created by water are also necessary to cleave bonds and liberate lignin. On the one hand, the recovery of hemicellulose in pretreated solids increased as ethanol content went from 40% to 50% v/v, but dropped rapidly thereaer. On the other hand, as discussed above, lignin recovery in pretreated solids decreased as ethanol content rose to 60% v/v, but improved drastically at 70% ethanol. These ndings indicated that the highest ethanol content was not optimal for the pretreatment of Salicornia bers, as it failed to efficiently fractionate lignin from lignocellulosic biomass. Correlating the cellulose content in untreated bers with pretreated solids revealed that the former improved from 66.8% to 83.9% as ethanol content went from 50% to 70% v/v, respectively.
3.1.2 Lignin fraction. Lignin samples isolated under different organosolv process parameters were analyzed for impurities, such as sugars and ashes, as well as Klasson lignin content (Table 3). Overall, the purity of the obtained lignins was very high, with only two samples (180 C for 15 min with 60% ethanol and 180 C for 45 min with 50% ethanol) exceeding 6% sugar contamination, which indicated suitable fractionation during pretreatment. Sugar contamination of only 1.22% w/w was obtained during pretreatment at 200 C. The ashes present in lignin aer organosolv pretreatment did not exceed 1.8% w/w, with most samples exhibiting less than 1% w/w ashes content. Altogether, the few impurities found in lignin pointed to highly efficient organosolv fractionation of biomass. Size-exclusion chromatography was performed for lignin samples of S. dolichostachya to determine their molecular weight distribution. Overall, the different pretreatments yielded a number average molecular weight ranging from 600 Da to 700 Da, while the weight average was between 1100 Da and 1900 Da (Table 3). Dispersity was highest (3.00) at 160 C for 30 min with 60% ethanol, which coincided with lower deligni-cation and indicated non-extensive depolymerization of lignin. In our previous study with birch (Betula pendula L.) sawdust, the M w ranged from 1800 Da (180 C for 15 min with 50% v/v ethanol) to 15 900 Da (180 C for 30 min with 60% v/v ethanol). 35 When using birch chips, the M w ranged from 2700 Da (200 C for 15 min with 60% ethanol and 1% w/w biomass H 2 SO 4 ) to 8000 Da (200 C for 30 min with 60% ethanol without catalyst). 36 Pine wood pretreated at 190 C for 60 min with 60% v/v ethanol and 1% w/w biomass H 2 SO 4 generated lignins with M w of 7700 Da; whereas cotton stalks pretreated at 200 C for 45 min with 50% v/v ethanol and 1% w/w biomass H 2 SO 4 , as well as sweet sorghum bagasse pretreated at 180 C for 30 min with 60% v/v ethanol produced lignins with M w of 16 800 Da and 6600 Da, respectively. 37 Comparatively, organosolv-pretreated lignin isolated from Salicornia had a much smaller molecular weight, which can be explained by the catalytic action of transition metals (e.g., iron, cobalt, manganese, platinum, ruthenium, and rhodium) during oxidative cleavage of b-O-4 linkages. [38][39][40] The conversion of lignin into downstream compounds requires homogeneity of the starting material, which can be a challenge in the case of complex fragmented lignin precursors. 41 In this context, low molecular weight lignin is more advantageous as it is more prone to depolymerization, which facilitates its valorization. A plethora of value-added products can be synthesized from low molecular weight lignin, namely vanillin, bioplastic, pigments, resins, dyes, biodiesel, and polymers. 40,42 3.1.3 Hemicelluloses fraction. Fractionated hemicellulose under different organosolv process parameters was analyzed with respect to sugar composition (e.g., monomers and oligomers) and sugar origin (e.g., cellulose or hemicellulose) ( Table  4).
The temperature of 180 C was optimal for the fractionation of hemicellulose as it allowed for the highest percentage of oligomers (69.0%) and hemicellulosic sugars (8.93 g/100 g biomass ) to be recovered. On the one hand, a higher temperature during pretreatment led to fewer oligomers (37.8%), because the harsh conditions (200 C) favored the hydrolysis of hemicellulosic sugars. On the other hand, a lower temperature (160 C) was not optimal for the solubilization of hemicelluloses (only 5.22 g/100 g biomass ) due to lower fractionation of biomass during pretreatment ( Table 2). These ndings conrmed how the increased severity of pretreatment promoted hemicellulose depolymerization into monomers. 43 During pretreatment, hemicellulosic sugars are released within the fractionated biomass and then hydrolyzed into monomeric sugars. Hence, the duration of pretreatment is a decisive factor as it needs to balance delignication with hydrolysis, while minimizing the conversion of sugars into secondary products. 44 Hemicellulose fractionation was optimal at 30 min pretreatment, with yields dropping aer 45 min of pretreatment and the percentage of oligomers aer 60 min ( Table 4).
As explained previously (see "Pretreated solids fraction"), excessive ethanol content lowers biomass hydrolysis, leading to lower delignication and fractionation of hemicellulose. This was observed in our study, where a similar fractionation of hemicellulose ($29%) was observed for 40% and 50% v/v ethanol, followed by consistent reduction (to 25.2% and 23.9%) with increased ethanol concentration. While the oligomer to monomer ratio tended to increase with increasing ethanol content, it dropped dramatically at 70% v/v ethanol, suggesting a more pronounced hydrolysis of oligomers to monomers at this point. A higher yield of oligomers is desired for the use of hemicellulose in prebiotics, feed, food packaging, and food ingredients. Because enzymatic hydrolysis of oligomers into monomers can be easily achieved downstream, lower Table 3 Lignin fractions composition and molecular weight a

Code
Cellulose (% w/w) Hemicelluloses (% w/w) Klasson lignin (% w/w) Ashes (% w/w) M N (g mol −1 ) M w (g mol −1 ) DI  hydrolysis of hemicelluloses during fractionation is preferred. 45 Taking into account the above, treatment with 50% v/v ethanol seems to be the most favorable as it results to the highest hemicellulosic sugar production (8.98 g/100 g biomass ), alongside the highest fractionated hemicellulose (29.1% w/w) and the second highest oligomer ration (55.8% w/w). Cybulska et al. (2013) studied the effects of hydrothermal pretreatment on S. bigelovii at three different temperatures (190,200, and 210 C). They observed a progressive reduction in the recovery of xylose, in both pretreated solids and liquid, accompanied by a concomitant increase in furfural. This nding highlighted the degradation of pentose sugars with increased time and consequent severity of pretreatment. 32,46

Enzymatic saccharication
To assess the suitability of pretreated solids as feedstock for microbial cultivations, the pretreated pulp fractions were subjected to enzymatic saccharication. The hydrolysis of cellulose to glucose was near complete (100%) within 72 h in the majority of tested samples, and within 24 h in six of these samples (Fig. 2). Saccharication yields were high (>70%) also at the initial stage of saccharication (8 h). Pretreatment parameters affected the saccharication results. Both pretreatments at higher temperatures (180 C and 200 C) allowed signicantly higher saccharication within 8 h and complete saccharication within 24 h whereas the one at 160 C resulted in a much lower saccharication rate, likely due to lower delignication and fractionation of hemicellulose (Table 3). These factors hindered the enzymes' action and led to longer incubation times to achieve better cellulose hydrolysis yields, as observed by an increase in the release of glucose towards 72 h ( Fig. 2 and  3). A treatment of 15 min resulted in low cellulose hydrolysis aer 8 h of saccharication. The result improved with a pretreatment of 30 min but did not change any further thereaer. Complete saccharication was achieved with all treatment times except 15 min (Fig. 2), which can be attributed to lower delignication and removal of hemicellulose (Table 2). Finally, ethanol content of up to 50% did not have any impact on the results obtained at 8 h, and only 70% v/v ethanol caused a marginal improvement. In all cases, complete saccharication was achieved when the reactions were extended to 24 h. Cybulska et al. (2013) reported yields of 87-92% following the saccharication of hydrothermally pretreated S. bigelovii biomass. 46 Smichi and collaborators (2018) evaluated organosolv pretreatment of the halophyte Juncus maritimus with H 3 PO 4 at 50 C for 24 h. Saccharication of the resulting cellulosic pulp reached a 90% hydrolysis yield aer 48 h using the same enzyme as in the present study with an initial load of 61.25 cm 3 U mL −1 . 47 Taken together, these ndings indicate that organosolv pretreatment is an outstanding method for processing halophytes, enabling elevated cellulose saccharication due to increased accessibility of the fractionated feedstock to the hydrolyzing enzymes.
Cellulose hydrolysis yield is a very important factor when assessing the suitability of pretreated solids for microbial conversion processes. However, owing to differences in cellulose content, it is sometimes more informative to consider the amount of glucose released per gram of solids. Because most samples achieved total saccharication (Fig. 2), the total release of glucose relative to the initial biomass was calculated (Fig. 3). Pretreatment at 200 C achieved complete cellulose hydrolysis as the majority of the pretreatments tested but, due to the higher amount of cellulose present in this sample (Table 2), it released the highest amount of glucose (0.61 g g biomass −1 ). The sample pretreated at 160 C exhibited the lowest release of glucose during saccharication and the lowest cellulose content, which can be attributed to its lower delignication and fractionation of hemicellulose (Table  3). These ndings highlight the tight link between saccharication and proper biomass fractionation. Larran and collaborators (2015) studied the saccharication of the halophyte Spartina argentinensis following pretreatment with laccase. Using 0.4 U of commercial enzymes, they achieved the release of 0.035 g g biomass −1 of glucose within 24 h. 48 Accordingly, it can be concluded that the majority of pretreated solids are amenable to anaerobic digestion, but the choice of a suitable organosolv pretreatment will strongly affect the process.

Conclusions
The present study demonstrated that Salicornia bers served as an excellent substrate for organosolv fractionation, achieving puried fractions of cellulose, hemicellulose, and lignin. Different process parameters, including pretreatment temperature, duration, and solvent content, were tested. The highest tested temperature achieved excellent delignication, but at the expense of hemicellulose recovery, particularly in the form of  oligomers. Taking into account total biomass recovery and hemicellulose yield, the optimal treatment temperature appeared to be 180 C. When assessing the duration of pretreatment, delignication, cellulose content, and fractionation of hemicellulose were optimal at 45 min, with the proportion of hemicellulose oligomers decreasing following longer pretreatments. Delignication and the oligomers ratio were optimal with 60% ethanol, whereby cellulose content in biomass increased by 79.6% compared to untreated bers.
Finally, enzymatic saccharication trials demonstrated that cellulose from pretreated solids was easily hydrolysable to glucose and in most cases complete conversion of cellulose was attained. In summary, we demonstrate that S. dolichostachya bers can be used as a novel sustainable feedstock for biomass bioreneries, thereby widening the portfolio of renewable biomass sources.

Conflicts of interest
There are no conicts to declare.