Fractionation of Lignocellulosic Fibrous Straw Digestate by Combined Hydrothermal and Enzymatic Treatment

Abstract

In industrial-scale biogas production from cereal straw, large quantities of solid fiber-rich digestate are produced as residual material. These residues usually contain high amounts of cellulose, hemicellulose and lignin and thus have potential for further utilization. However, they also contain impurities such as ammonia and minerals, which could negatively affect further utilization. Against this background, the present study investigates how this fibrous straw digestate can be fractionated by a combined hydrothermal and enzymatic treatment and what influence the impurities have in this process. Therefore, it is analyzed how the fractions cellulose, hemicellulose and lignin are modified by this two-stage treatment, using either raw digestate (including all impurities) or washed digestate (containing only purified fibers) as the substrate. For both substrates, around 50% of the hemicellulose is solubilized to xylans after 50 min of hydrothermal treatment using steam at 180 °C. Furthermore, by subsequent enzymatic treatment, around 90% and 92% of the cellulose and hemicellulose still contained in the solids are hydrolyzed to glucose and xylose, respectively. Lignin accumulates in the remaining solid but structurally degrades during the hydrothermal treatment, which is indicated by decreasing ether and ester bond contents with increasing treatment times. Impurities contained within the raw digestate do not hinder this fractionation; they even seem to positively affect hemicellulose and cellulose valorization, but apparently lead to a slightly higher lignin degradation.

1. Introduction

In view of limited biomass resources, a growing world population and thus an increasing demand for resources, the goal must be to utilize biomass as completely as possible once it has been withdrawn from nature in a sustainable manner. Often, however, only part of the harvested biomass is processed, resulting in numerous organic waste streams available throughout our overall economy. Anaerobic digestion is a simple possibility for the valorization of these organic waste streams as it can convert almost any kind of organic waste into biogas useable as a raw material for the chemical industry as well as a “green” energy carrier [1]. However, lignocellulosic biomasses, representing an abundant organic waste stream, especially in agricultural crop production, are only converted to a lesser extent by anaerobic digestion compared with other plant biomasses due to the complexity and recalcitrance of their structure [2,3,4].

Lignocellulose is a matrix of three polymers: cellulose, composed of glucose monomers; hemicellulose, mainly composed of pentoses; lignin, mainly composed of phenylpropanoids. The polymeric carbohydrates cellulose und hemicellulose (together referred to as holocellulose) are degradable under anaerobic conditions. This is true if they are accessible by the fermentation bacteria. However, they are significantly less degradable when they are incorporated into the lignocellulosic matrix [3,4,5]. Lignin itself is hardly degradable under anaerobic conditions; i.e., typically the lignin to holocellulose ratio increases during the anaerobic digestion of lignocellulose [6,7,8,9]. The consequence is that lignin and not easily degradable carbohydrates accumulate in the fibrous digestate during the anaerobic digestion of a lignocellulosic biomass and rather low biogas yields are achieved. In order to increase the carbohydrate availability, and thus the biogas yields out of a lignocellulosic biomass, there are numerous pretreatment options for the disintegration of the lignocellulosic matrix, which have already been extensively reviewed [2,10,11,12].

Among these pretreatment options are hydrothermal methods that apply just water at certain temperatures (typically 150 to 230 °C) and respective pressures. Typically, no other chemicals are used. Thus, hydrothermal treatments have the advantage that no additional chemical waste is produced [13,14]; however, these processes are energy-intensive. Hydrothermal processes can be divided into two different groups, depending on the state of aggregation in which water is used: either as steam (i.e., steam treatment) or as liquid water (i.e., liquid hot water treatment).

During such hydrothermal methods, a selective solubilization and further degradation of hemicellulose takes place, being catalyzed by an auto-hydrolysis reaction. This is based on two effects. Firstly, higher temperatures lead to stronger water auto-ionization and thus a higher concentration of hydronium ions [13]. Secondly, under typical reaction conditions, the cleavage of acetyl groups from hemicellulose at high temperatures releases acetic acid. This acid decreases the pH value and thus causes further hydrolysis of the hemicellulose [15].

Cellulose is hardly solubilized at temperatures below 210 °C [13,16]. However, the biodegradability of cellulose is significantly enhanced by such a hydrothermal treatment due to an increased available surface area and changes in its microstructure (e.g., loss of crystallinity) [13]. The consequence is that the carbohydrates from the lignocellulosic biomass become more available to enzymes and therefore hydrothermal treatments are often used as pretreatment methods before anaerobic digestion or enzymatic hydrolysis [10,17].

Little to no lignin is removed from the biomass feedstock by such hydrothermal treatments; nevertheless, liquid-hot water processes apparently remove slightly more lignin than processes applying steam [18,19,20]. However, such a hydrothermal treatment leads to several structural changes in the molecular structure of the lignin, including the cleavage of ether bonds, deacetylation and repolymerization reactions [18,19,21,22].

Hydrothermal treatments have already been applied to various organic waste materials such as grass cuttings [23], foliage [24], sewage digestate [25], manure [26], beet pulp [27], food waste [28] or lignocellulosic wastes such as corn stalk and corn stalk digestate [29]. In all these studies, the investigations were focused on increasing the potential for energy use by either increasing the heating value or the biomethane potential of the substrate. However, some studies indicate that hydrothermal pretreatment to increase energy potential is not economically viable. Andersen et al. [30] showed that a simple mechanical pretreatment of straw before anaerobic digestion is more viable than a hydrothermal or an alkaline pretreatment. Zieliński et al. [31] even observed a negative energy balance if liquid hot water pretreatment of the silage of Sida hermaphrodita mixed with cattle manure was applied before biogas production.

An alternative to the pure energetic utilization is the fractionation of the lignocellulosic biomass via a hydrothermal treatment with possible non-energetic valorization strategies for the resulting single fractions. For example, it has been investigated how xylo-oligosaccharides can be obtained from hemicellulose in wheat bran, brewery spent grain and corn cobs by hydrothermal treatment [32]. It was also demonstrated that the cellulose fraction of such residual materials can be converted to glucose via subsequent enzymatic hydrolysis [20,33]. For the example of wheat straw, it was shown that the lignin fraction is included in the remaining solids after hydrothermal and enzymatic treatments of wheat straw. Some studies also assessed further valorization strategies for this lignin fraction by investigating its structural properties [18,34].

Despite the wide range of investigations in the field of hydrothermal and enzymatic fractionations of biomass, there are only a few studies dealing with the application of such combined hydrothermal and enzymatic processes on residual biomass streams resulting from previous biological treatments such as ensiling [31,35] or anaerobic digestion (no related publications found). During such a fermentation treatment, impurities and/or trace components such as proteins, minerals and ammonia can be formed or accumulate, whereby the composition may differ fundamentally from unprocessed agricultural residues. With regard to a subsequent hydrothermal treatment and enzymatic fractionation, this can lead to significant changes in the reactions taking place and it is hypothesized that the fractionation efficiency decreases due to these impurities.

In order to investigate this further, the hydrothermal and enzymatic fractionation of a separable fibrous digestate remaining after an anaerobic digestion of cereal straw is presented below. Following on from this fermentation of cereal straw, which is already implemented at the industrial scale, the focus of this study is on the further material utilization of the resulting digestate, which still contains significant amounts of carbohydrates and lignin and consequently a high potential for further value creation. The aim is to show in detail what happens to the individual fractions of the digestate, namely hemicellulose, cellulose and lignin, during hydrothermal and enzymatic treatments. Furthermore, it is the aim to verify the hypothesis formulated above and thus to assess the influence of impurities on this fractionation process. This is done by comparing the fractionation effects for raw and washed fibrous digestate. A detailed look is also taken at structural changes (i.e., changes in the ether and ester bond content) caused by the hydrothermal treatment in the lignin fraction, which accumulates in the digestate during anaerobic digestion, as already described above.

In this way, the present research article demonstrates that both raw and washed fibrous straw digestate can be fractionated into a hydrolysate containing mainly hemicellulose-derived sugars, a hydrolysate containing mainly cellulose-derived glucose and a solid enriched in partially degraded lignin by combined hydrothermal and enzymatic treatments.

2. Materials and Methods

In the following, all the materials, treatment methods and analytical methods are presented. In order to keep this part compact, more detailed descriptions of already published methods and calculations are moved to Appendix A. As a starting point, a graphical overview of the experimental procedure is given in Figure 1.

2.1. Materials

The fibrous straw digestate was obtained from an industrial cereal straw mono-fermentation biogas plant in Brandenburg (Germany). This original material is named as the raw digestate in the following. A portion of this raw digestate was extensively washed with tap water and repeatedly pressed off using a hydraulic press (40 L, Speidel, Germany) until a fully clear effluent was obtained. The solids obtained in this way are named the “washed digestate”. Tap water was added to the washed digestate until its water content was the same as the raw digestate (80 wt%) to allow for similar starting conditions. The enzyme mix Cellic^® CTec2 was received from Novozymes A/S (Bagsværd, Denmark). All further chemicals were purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany).

2.2. Treatments

2.2.1. Hydrothermal Steam Treatment

The hydrothermal steam treatment was performed in a high-pressure reactor with a void volume of 3 L (with a height of 0.47 m and an inner diameter of 0.09 m). A schematic drawing of the reactor system is given in Figure 2. Around 506.8 g of moist digestate, corresponding to 100 g dry matter, was loaded into a steel cartridge. The loaded cartridge was then introduced into the reactor, which was preheated to 195 °C by a heating oil jacket. The cartridge was left inside the reactor for five minutes for preheating, while the outlet to the condenser was kept open in order to allow hot expanding air to escape. The valve towards the steam generator was slightly opened to pressurize the reactor with saturated steam at 180 °C and to flush the remaining air from the reactor. As soon as the first condensate came out of the condenser, the outlet valve to the condenser was closed, while the valve towards the steam generator was fully opened and the reaction time started. The temperature of the heating oil jacket was reduced to 188 °C in order to maintain a temperature of about 180 °C inside the reactor. Both the pressure at the bottom and the temperatures at the top and at the bottom of the reactor were monitored using the software LabVIEW. After a defined treatment time (between 20 to 120 min), the treatment was stopped by closing the valves to the steam generator and slowly opening the valve towards the condenser to release the pressure and to condense the steam. Once the atmospheric pressure had been reached (which was typically achieved after about 1 min), the cartridge was removed from the reactor and externally cooled with tap water.

The cartridge was weighed in order to calculate the water mass balance (neglecting compounds that have passed into the gas phase). Subsequently, some hydrolysate was pressed off the treated digestate for pH measurement and further sugar analysis. The treated digestate was freeze dried, milled below 1 mm and stored for further compositional analysis (Section 2.3.1), enzymatic hydrolysis (Section 2.2.2), thioacidolysis and saponification (both Section 2.3.3). All experiments were conducted in duplicates.

2.2.2. Enzymatic Treatment

The enzymatic hydrolysis was carried out in 50 mL centrifuge tubes filled with 0.825 g dry matter of pretreated digestate and 40 mL of 0.05 M citrate buffer (adjusted to pH 5). Furthermore, Novozymes Ctec2 enzymes with a total activity of 28 FPU were added. Subsequently, the tubes were positioned horizontally in a water bath shaker and shaken continuously (100 min⁻¹) at 50 °C for 72 h. Immediately after the end of the experiment, the tubes were cooled on ice and centrifuged (4776× g for 10 min). The supernatant was decanted and preserved for sugar analysis (Section 2.3.2). The remaining solid was kept in the tube, washed with water twice (40 mL each), freeze dried and subsequently weighed. Its composition/lignin content was analyzed following the procedure described in Section 2.3.1. For each duplicate of hydrothermally pretreated digestate, the subsequent enzymatic hydrolysis was again conducted in duplicates.

2.3. Analytical Methods

2.3.1. Compositional Analysis

The ash content of the biomass samples was determined by ashing at 550 °C according to DIN EN ISO 18122 [36]. The protein contents of the biomass samples were determined based on their nitrogen content by applying a nitrogen-to-protein conversion factor of 6.25. The nitrogen content was determined via a NCHS elemental analyzer (Vario Macro Cube, Elementar, Langenselbold, Germany) by the central laboratory of the TU Hamburg [37]. The content of the structural carbohydrates and lignin in the digestate and treated biomass samples was determined by a two-stage acid hydrolysis according to protocol TP-510-42618 published by NREL (National Renewable Energy Laboratory) [38]. The hydrolysate obtained from this two-stage acid hydrolysis was analyzed for dissolved sugars, acetic acid and degradation products by HPLC (Section 2.3.2). Based on the amount of dissolved sugars and acetic acid, the corresponding cellulose and hemicellulose contents were then calculated according to the protocol TP-510-42618. The acid-soluble lignin (ASL) content was determined using UV spectrometry at a wavelength of 320 nm and calculated with an absorptivity of 30 L/(g cm). The weight of the acid insoluble residue after the two-stage acid hydrolysis was corrected for ash content in the case of all the biomasses analyzed and for protein content in the case of the raw and the washed digestate to obtain the value for acid insoluble lignin (AIL). In the case of the washed and the raw digestate, extractives were removed before analysis by two consecutive Soxhlet extractions using first water and subsequently ethanol (following TP-510-42619 [39]).

While all samples presented up to this point were based on dried samples, the ammonia content in the liquid phase was additionally determined titrimetrically for the moist raw digestate by the central laboratory of the TU Hamburg [40].

2.3.2. Sugar Analysis (Monomers/Oligomers)

For the quantification of sugar monomers, an HPLC system (Infinity II HPLC Series, Agilent, Santa Clara, CA, USA), equipped with a Hi-Plex H+ column (Agilent, Santa Clara, CA, USA) and a refraction index detector, was used. A total of 5 mM sulfuric acid in deionized water was used as the mobile phase. A five point calibration with D-cellobiose, D(+)glucose, D(+)xylose, L(+)arabinose, acetic acid, HMF and furfural was performed. Hemicellulose solubilization yields and enzymatic hydrolysis yields were calculated based on the HPLC results, as described in Appendix A.2 and Appendix A.3.

To quantify oligomeric sugars in the liquid hydrolysate obtained after the hydrothermal treatment, an acidic hydrolysis was conducted before HPLC analysis, using a sample volume of 10 mL and a thermal reactor (TR420 from Merck, Darmstadt, Germany) instead of an autoclave to ensure a temperature of 121 °C during acidic hydrolysis with 4% w/w sulfuric acid according to the protocol in [41]. The degree of oligomerization was determined by dividing the amount of oligomeric xylose by the amount of total xylose, as presented in Appendix A.2.

2.3.3. Analysis of Lignin Structure

Ether-bound lignin monomers were released from the hydrothermally treated biomass samples by thioacidolysis and detected by GC-MS following published procedures [42,43] (for further details see Appendix A.4). Ester-bound phenolic acids were solubilized by saponification and subsequently analyzed by HPLC with UV detection to determine their content in the biomass samples. This was done following the method published by Linh et al. [44], but scaling it down maintaining the same substrate-to-solution ratio of 0.02 g/mL (further details in Appendix A.5).

The molar fraction $x_{mon}$ of the total lignin monomers released by either ether bond cleavage (thioacidolysis) or ester-bond cleavage (saponification) was determined from the amount of monomers $n_{mon,released}$ released from an introduced mass of lignin $m_{lignin}$ by the respective method, assuming an average molecular weight of 5125 µmole/g per lignin monomer and using Equation (1), as further justified in Appendix A.6.

$$x_{mon}=\frac{n_{mon,released}}{5125 \frac{\mu \mathrm{mol}}{g}·m_{lignin}}$$

(1)

The content of the ether linkages in lignin was determined as the root of the molar fraction of lignin monomers released by thioacidolysis [45,46,47,48,49], see Appendix A.6.1 for details. Furthermore, the content of the ester bonds between p-coumaric acid (pCA) and lignin was calculated under the assumption that the molar fraction of pCA released corresponds to the molar fraction of ester bonds with pCA in lignin, as justified in Appendix A.6.2.

The kinetics of total apparent decrease in ether and ester content during the hydrothermal treatment were determined based on the experimental data. First-order reaction kinetics, as derived in Appendix B (Equations (A10) and (A11)), combined with Equation (1) result in Equation (2) used to model the ether and ester content with the assumed first order reaction kinetics as basis.

$$x_{linkage}\left(t\right)=\frac{n_{e,0}·e^{-k_{degr}·t}}{5125 \frac{\mu \mathrm{mol}}{g}·m_{lignin}}$$

(2)

$x_{linkage}$ can be either the ether bond content $x_{ether}$ or the ester bond content $x_{ester}$. $n_{e,0}$ is the initial amount of the respective linkage at $t=0$ and $k_{degr}$ is the reaction rate constant of the apparent decrease of the respective linkage. Based on $k_{degr}$, the half life $t_{1/2}$ of the ether or ester bond content can be calculated according to Equation (3). Further considerations and justifications for kinetic modeling can be found in Appendix B.

$$t_{1/2}=\frac{\ln \left(2\right)}{k_{degr}}$$

(3)

2.3.4. Statistical Analysis

The experimental data were analyzed using the statistical software SPSS Statistics 26 (IBM, New York, NY, USA). The Tukey post hoc test was applied in order to determine statistically significant differences between multiple mean values at a significance level of α = 0.05 [50].

3. Results

The fibrous digestate used had a dry matter content of around 20 wt%. The compositions of the dry mass of the raw digestate and the washed digestate are given in

Table 1. Composition of raw and washed fibrous digestate, including the mean values and standard deviations calculated from duplicates.

	Raw Solid Digestate	Washed Solid Digestate
	In wt% (Based on Dry Mass)
Cellulose	30.6 ± 1.2	37.5 ± 1.7
Hemicellulose	20.2 ± 1.0	25.7 ± 2.1
Lignin	17.9 ± 0.3	23.3 ± 0.7
Total amount extractives *	19.3 ± 1.2	4.5 ± 1.4

* Sum of water and ethanol extractives.

. Washing the digestate fibers removes extractives and thereby relatively enriches the content of cellulose, hemicellulose and lignin within the solid digestate. In the liquid phase of the raw fibrous digestate, an ammonia content of around 2.5 g/L was detected. A total of 506.8 g of moist raw digestate, that was inserted into the reactor for hydrothermal treatment, contained about 406.8 g of liquid phase and thus about 1 g of ammonia. In contrast to this, the liquid phase of the washed digestate did not contain measurable ammonia concentrations. The following subsections show how the constituents of the digestate are distributed into the three fractions: the hydrolysate of the hydrothermal treatment (Section 3.1), the hydrolysate of the enzymatic treatment (Section 3.2) and the remaining solid (Section 3.3).

3.1. Solubilization of Hemicellulose by Hydrothermal Steam Treatment

An important parameter during the hydrothermal treatment is the pH value. At a constant temperature of 180 °C, the final pH values of the hydrolysate and the condensate depend on the hydrothermal treatment time and whether the digestate was applied raw or washed. As can be seen in Figure 3, the pH values for the raw digestate are significantly higher than for the washed digestate. The condensates from the hydrothermal treatment of the raw digestate show a pH value around 9, regardless of the treatment time. The pH values of the hydrolysates obtained from treating the raw digestate start at pH 8 and slowly decrease with longer treatment times. For the washed digestate, the pH values of the condensate and the hydrolysate show a very similar course and are much lower compared with the values achieved for the raw digestate, being already around 5 after 20 min of treatment and further decreasing with longer treatment times approaching a pH = 3.8 after 120 min.

During hydrothermal treatment, hemicellulose solubilizes with a comparable solubilization yield for the raw and the washed digestate, according to Figure 4a. For treatment times within 50 min, solubilization yields increase with treatment time and are slightly higher for the washed digestate than for the raw digestate. However, with higher treatment times, the solubilization yield decreases for the washed digestate, whereas it slightly increases for the raw digestate. Another significant difference is in the proportion of oligomers in the solubilized sugars. While in the case of the raw digestate, irrespective of the reaction time, almost all dissolved sugars are bound in oligomers, increasing hydrolysis to monomers with increasing treatment time, as evident in the case of the washed digestate (Figure 4b).

3.2. Hydrolysis of Cellulose by Enzymatic Treatment

After hydrothermal treatment, the cellulose can be hydrolyzed by enzymes. Using always the same conditions for enzymatic hydrolysis, Figure 5a shows how the glucose yield depends on the hydrothermal treatment time. It is apparent that a treatment duration of 50 min leads to significantly higher yields compared with a treatment duration of 20 min, but longer treatments do not lead to any further significant improvement. For a shorter treatment time of 20 min, hydrolysis yields for the raw digestate are higher than for the washed digestate.

Non-solubilized or non-separated solubilized hemicellulose can also be hydrolyzed enzymatically (see Figure 5b), showing a similar trend as the hydrolysis of cellulose, and even slightly higher yields can be achieved (yields around 90% for cellulose hydrolysis, whereas yields are approaching 100% for hemicellulose).

3.3. Structure of Lignin after Hydrothermal Steam Treatment

After hydrothermal treatment and enzymatic hydrolysis, a solid enriched in lignin remains. With longer hydrothermal treatment times, the lignin content in these solids increases from the starting values of around 18 wt% for the raw digestate and 23 wt% for the washed digestate to around 75 wt% for both digestates (see Figure A3 in the Appendix C). However, there seems to be a limit at around 75 wt% lignin content, since other components, especially ash and proteins, also remain in the solid residue (each with contents up to about 10 wt%, see

Table A3. Acid-insoluble lignin (AIL) content, protein content and ash content in solid residues remaining after hydrothermal and enzymatic treatment (determined as described in Section 2.3.1).

Initial Substrate	Hydrothermal Treatment Time	AIL Content	Protein Content	Ash Content
Washed digestate	20	44.4	7.5	4.1
Washed digestate	70	69.7	10.0	10.0
Washed digestate	120	73.6	10.3	8.5
Raw digestate	120	73.9	10.9	7.7

in the Appendix C).

In order to design possible valorization strategies for these lignin enriched solids, the changes in lignin structure were further analyzed (i.e., changes in the content of the ether and ester bonds before and after hydrothermal treatment). By thioacidolysis, it was shown that the amount of purely ether-bound lignin monomers per lignin mass decreases with an increasing reaction time (see

Table A4. Amount of monomers released (a) from washed digestate and (b) from raw digestate during thioacidolysis and saponification as well as the respective ester and ether bond contents calculated from these results. In (a) experiments were conducted as duplicates, in (b) they were conducted as single determinations.

(a)	Washed Digestate
	Analysis of Ether Bonds		Analysis of Ester Bonds
Treatment Time	Released Monomers by Thioacidolysis	Resulting Ether Bond Content	Released Monomers by Saponification	Resulting Ester Bond Content
[min]	[µmole/gLignin]	[mole%]	[mg/gLignin]	[mole%]
0	620 ± 52	34.8 ± 1.5	31.5 ± 1.7	3.7 ± 0.2
20	409 ± 82	28.2 ± 2.8	25.2 ± 1.4	3.0 ± 0.2
50	296 ± 6	24.0 ± 0.3	18.5 ± 1.8	2.2 ± 0.2
70	207 ± 5	20.0 ± 0.3	14.9 ± 0.9	1.8 ± 0.1
90	190 ± 19	19.2 ± 1.0	14.4 ± 0.7	1.7 ± 0.1
120	167 ± 2	18.0 ± 0.1	12.1 ± 1.0	1.4 ± 0.1
(b)	Raw Digestate
	Analysis of Ether Bonds		Analysis of Ester Bonds
Treatment Time	Released Monomers by Thioacidolysis	Resulting Ether Bond Content	Released Monomers by Saponification	Resulting Ester Bond Content
[min]	[µmole/gLignin]	[mole%]	[mg/gLignin]	[mole%]
0	639	35.5	36.2	4.3
20	307	24.5	20.3	2.4
50	183	18.9	11.0	1.3
70	168	18.1	8.8	1.1
90	175	18.4	8.9	1.1
120	159	17.6	7.0	0.8

in the Appendix C). Similarly, the amount of ester-bound p-coumaric acid (pCA) per lignin decreases with an increasing reaction time (see Table A4 in the Appendix C).

From the results from thioacidolysis and saponification, conclusions about the content of the ether and ester bonds in lignin can only be drawn indirectly. For this purpose, a model conception of lignin is used, which is presented in Section 2.3.3 (for more details see Appendix A.6). The resulting ether and ester contents were fitted to the first-order reaction kinetic model presented in Section 2.3.3 and Appendix B, as shown in Figure 6.

By fitting the degradation of the ether and ester bonds to first-order reaction kinetics via the method of least squares (Appendix B), the degradation rate constant and the half-life of the total apparent decrease in bond types could be determined. However, it is apparent from Figure 6 that the first-order reaction kinetics model does not well describe the decrease in the ether bond content. After 120 min, the content of the ether linkages decreases by about half for both the washed and the raw digestate and appears to approach an asymptote in this range at an ether content around 18 mole%, whereas the kinetic model approaches an asymptote at 0 mole%. While the ether bond contents after 120 min are similar for the raw and the washed digestate, the ether bond content in the raw digestate appears to decrease more rapidly at shorter treatment durations.

The ester bond content can be fitted to the first-order reaction kinetics model with smaller deviations, but, here too, it appears that the values decrease less with longer treatment times than described by the kinetic model. The degradation of the ester bonds seems to be higher for the raw digestate than for the washed digestate, which can be seen in a higher degradation rate constant (3.4 × 10⁻⁴ s⁻¹ for raw digestate and 1.6 × 10⁻⁴ s⁻¹ for washed digestate) and consequently a shorter half-life (34 min for raw digestate and 74 min for washed digestate).

4. Discussion

Having a considerable high content in lignocellulose of nearly 70 wt% in a raw state and nearly 90 wt% when washed, the straw digestate represents an interesting waste stream for further valorization. However, compared with untreated straw, the lignin-to-holocellulose ratio for straw digestate is higher, as holocellulose degrades more than lignin during fermentation (see

Table 2. Comparison of the lignin to holocellulose ratios in different straw varieties (calculated by their average composition given in [30]) and the straw digestate used in this study.

Type of Biomass	Wheat Straw	Barley Straw	Maize Straw	Rice Straw	Straw Digestate
Lignin to holocellulose ratio	0.29	0.26	0.29	0.21	0.42

). Fermentation thus represents a biological method for lignin enrichment, with the special feature that only mild conditions are employed and, accordingly, no lignin degradation is expected.

This study investigated how the combination of hydrothermal and enzymatic treatments can promote the valorization of the digestate. Below, the respective effects on hemicellulose, cellulose and lignin are discussed individually. The digestate fibers could be used as they are (raw) or they could be previously washed. There are optimized concepts for the washing of biomass fibers; however, washing always implies a further processing effort and the generation of additional wastewater streams [51]. The most influential difference between raw and washed fibrous straw digestate is considered to be the high ammonia content in raw digestate, as ammonia acts chemically as a base and is also used in different pretreatment strategies for lignocellulose fractionation [52,53]. In the following, its presumed influence will be further discussed.

4.1. Solubilization of Hemicellulose by Hydrothermal Steam Treatment

The hydrothermal treatment of lignocellulose leads to the solubilization of hemicellulose. In this context, the pH value does not seem to have a major influence on the extent of the solubilization. Although in the case of the raw digestate, much higher pH values are observed in the condensate and in the hydrolysate, a comparable solubilization of the hemicellulose takes place as in the case of the washed digestate. A maximal solubilization yield of 53 wt% was achieved from the washed digestate after 50 min of hydrothermal treatment corresponding to a severity factor of 4.05 (the severity factor is commonly used to compare results from hydrothermal treatments obtained with different reaction times and temperatures and is calculated as described in Appendix A.1 [54]). No comparative values could be found in the literature for cereal straw digestate. However, for the hydrothermal treatment of cereal straw, similar yields were obtained in other studies, e.g., by treating wheat straw (around 50% for a severity of 3.94 [33]) or corn straw (53% at a severity of 3.75 [55]) under comparable conditions.

However, the pH value has a decisive influence on further hydrolysis. At low pH values in the case of the washed digestate, solubilized oligomers are further broken down into monomers, as can be seen in Figure 4b. Subsequently, further degradation of xylose monomers seems to occur, so that the total sugar yield decreases further with increasing treatment time of the washed digestate, a trend which is also observed in other studies, e.g., for the treatment of wheat straw [56]. It is known that acidic conditions lead to hydrolysis and further xylose degradation to furfural or even humins [57]. For this reason, researchers have developed the strategy of adding a certain amount of sodium hydroxide before the hydrothermal treatment, so that the treatment takes place at neutral conditions and the degradation of the oligomers is prevented [58,59]. The results obtained here show that ammonia is also suitable for buffering the pH value and even keeps it in the slightly alkaline range in the condensate, which hardly affects the solubilization yield but prevents the degradation of the sugar oligomers. The raw digestate used in this study naturally already contained this buffer system with an ammonia content of 2.5 g/L in the liquid phase, as organic nitrogen is converted to ammonia during anaerobic digestion.

However, with regard to further valorization of the xylo-oligosaccharides, it must be noted that the hydrolysate in the case of the raw digestate is a very complex, murky and dark mixture, containing the impurities found in the liquid phase of the raw digestate (see Figure 7). If the isolation of valuable products from this hydrolysate is targeted, this would be a disadvantage, as it makes further product purification challenging. Impurities in the raw digestate could also lead to rapid fouling at reactor, pipe and heat exchanger walls, for example by Maillard reactions occurring at high temperatures in which amino acids and reducing sugars, both present in the hydrolysate, react with each other in an undirected manner. However, if the hydrolysate is to be used for a subsequent fermentation, the increased complexity and higher nutrient content could also be an advantage. Compared with that, the hydrolysate obtained from the washed digestate was brownish but transparent (see Figure 7). This shows that the dark coloration of the hydrolysate is not due to the hydrothermal treatment of the fibers but to the impurities contained in the liquid phase of the raw digestate.

4.2. Hydrolysis of Cellulose by Enzymatic Treatment

The hydrothermal treatment increases the enzymatic degradability of the cellulose contained in the digestate. The results show that a treatment duration of 50 min (severity of 4.05) is sufficient to make about 90% of the cellulose enzymatically hydrolysable and longer treatment durations hardly lead to increased yields. For rape straw, Wang et al. [60] also achieved a glucose yield of around 90% from cellulose by enzymatic hydrolysis after treating rape straw hydrothermally with a severity of 3.99. When making comparisons with the literature data, it should be noted that yields are based on the cellulose content of the original feedstock in most studies, whereas here they are based on the cellulose content of the hydrothermally pretreated feedstock, i.e., possible cellulose losses during hydrothermal treatment must be included. Thomsen et al. [61] indicated a yield of 72% based on the cellulose content of wheat straw during enzymatic hydrolysis after a steam explosion treatment with a severity of 3.72 in a pilot scale. Taking into account that about 4% of the cellulose is solubilized during the treatment and that a recovery of almost 100% of cellulose was realized in these analytics, a yield of about 75% for the enzymatic hydrolysis alone results. This is comparable to the yield for the raw digestate (73 ± 2%), but higher than the yield observed for the washed digestate (48 ± 0.5%) at comparable conditions (20 min treatment time, severity of 3.66). The combined hydrothermal and enzymatic treatment of the fibrous cereal straw digestate is thus well comparable to that of the untreated straw. Additionally, after other biological pretreatments such as ensiling, comparable and even increased yields were observed after hydrothermal and enzymatic treatments compared with the untreated cereal straw [35].

Higher pH values during a hydrothermal treatment of the raw fibrous digestate show a slightly positive effect. Whether the digestate is raw or washed does not seem to affect the maximum achievable yield, but it does affect the yield after a less severe treatment. It was observed that after a shorter treatment time of 20 min, the raw digestate seemed to be better hydrolyzable by enzymes. This finding can also be supported by findings from the literature. Alkaline treatment is generally considered to be efficient in making cellulose available [62]. Specifically for hydrothermal treatment, Imman et al. [63] showed that the addition of sodium hydroxide leads to the increased enzymatic digestibility of cellulose.

In conclusion, cellulose in raw or washed fibrous digestate can be enzymatically hydrolyzed to glucose after hydrothermal pretreatment. Here, a steam pretreatment of 50 min is sufficient to achieve yields of 90%. Furthermore, the remaining hemicellulose can be enzymatically hydrolyzed nearly completely at these conditions. Sugar hydrolysates obtained in this way can be used for further fermentations in order to produce propionic acid or bioethanol, as examples.

4.3. Structure of Lignin after Hydrothermal Steam Treatment

Full valorization of the lignocellulose also includes valorization of the lignin. Many studies emphasize that lignocellulosic biorefining will only become economically viable when the lignin is utilized in a high-quality way [22,64]. However, depending on the intended use, different structural and functional properties are desired, and these properties are varying significantly depending on the type of biomass and isolation technique and must therefore be characterized precisely beforehand. One important characteristic, particularly with regard to selective depolymerization to aromatic chemicals, is the ether bond content [45,64,65,66,67].

The ether bond content in the washed digestate is decreased by hydrothermal treatment from around 35% to roughly 18% after 120 min of treatment time, corresponding to a decrease of around 48% for a severity of 4.4. After a treatment time of around 50 min, which might be chosen as the optimum for carbohydrate conversion from the results discussed above, around 296 µmoles of lignin monomers are released per gram of lignin by thioacidolysis. This is comparable with results from Li et al. [22], who could release 185 to 639 µmoles/g lignin monomers by thioacidolysis after steam explosion treatment of different types of wood. Furthermore, it is comparable with technical lignins that were obtained by other processes. From Kraft lignin, for example, around 100 to 400 µmoles/g lignin monomers could be released by thioacidolysis [68,69]. From organosolv lignins, values between 0 to 670 µmoles/g were found [70], depending on the type of acid catalyst added. However, in these studies, higher thioacidolysis yields were obtained from the untreated substrate than in this study, so that the relative decreases in the ether bond contents appear to be higher. All over, there are large deviations in the evaluation in the ether bond content in the literature, depending on the chosen substrate, treatment process, severity of treatment and analysis method.

Another analysis method to evaluate the change in the ether bond content is 2D NMR. Heikkinen et al. [71] detected a decrease in the ether bonds of about 33% after the steam explosion of wheat straw (severity of 3.94), which is comparable to the findings here (31% decrease at a severity of 4.05). Yelle et al. [18] observed a much higher decrease in the ether bonds of 60% after treating wheat straw with steam at a severity of 3.6. The choice of the analytical method and the respective evaluation approach are probably reasons for these discrepancies in ether quantification. After treatment, there might be structurally modified ethers which are not found in the typical spectrum for β-aryl ethers and thus might not be quantified by all 2D NMR investigations [72]. However, there might also be some inaccuracies in the ether bond contents detected by thioacidolysis. Besides the inaccuracies already arising from the rough assumptions about the lignin molecule structure, it also has to be considered that esters are only partially cleaved during thioacidolysis [73,74]. Grass lignin, such as cereal straw lignin, typically has a high content of ester-bound pCA, ferulic acid, uronic acid and acetic acid [74,75], and comparable contents are expected for cereal straw digestate. This can be justified by comparing the ester-bound pCA-contents: In [76], a content of 21.8 mg of ester-bound pCA per gram of lignin was found in wheat straw. Here, 32 to 36 mg of pCA was found per g lignin in cereal straw digestate. An incomplete cleavage of these ester bonds leads to reduced monomer yields during thioacidolysis, as esterified monomers are not detected. This reduction in the thioacidolysis yield due to an incomplete ester cleavage was likely greater with less severely treated substrate, as more ester bonds were detected in the lignin of less severely treated substrate. This might explain the relatively low thioacidolysis yield (and thus calculated ether content) observed for the untreated fibrous straw digestate lignin.

On this basis, it is difficult to make an accurate and comparative statement about the extent of the decline in the ether bonds reported from different treatments and investigated using differing analytical methods. However, a significant decrease in the ether bond content also after hydrothermal treatment is obvious, indicating a lignin depolymerization. Furthermore, due to the large amount of polymeric lignin remaining and the low occurrence of typical lignin degradation products in the hydrolysate, it is assumed that repolymerization and condensation reactions take place to a comparable extent as depolymerization (for a more detailed justification, see Appendix B and Appendix C.1). Many other authors have made similar observations about hydrothermal processes and have reasoned in this way [19,21,71,77], and were able to support this assumption by illustrating how the molecular weight of lignin actually increases with increasing treatment severity [21,71].

Lignin repolymerization (also known as “condensation”) is not only affecting the lignin quality, but also decreasing the carbohydrate degradability as shown by Pielhop et al. [78]. It is assumed that an acid-catalyzed condensation takes place, for example, through the reaction of a highly reactive benzylic carbocation with nucleophilic carbons of the aromatic rings [21,79]. Li et al. [21] concluded that, under the conditions of steam explosion with treatment severities between 3.2 and 4.5, repolymerization seems to dominate over fragmentation. They showed that the repolymerization reaction can be avoided by the addition of 2-Naphtol as a scavenger for carbonium ions. Furthermore, they assumed, that repolymerization could also be prevented by shifting the pH conditions of the steam treatment towards the alkaline side. In contradiction to this, lignin condensation, thus a decreasing ether content and polymeric nature of lignin, was observed also under the alkaline conditions caused by the ammonia contained in the raw digestate in this study. However, the conditions were only slightly alkaline and no precise statement about the actual extent of repolymerization can be made from the experimental data.

In grass lignin such as lignin from cereal straw digestate, ester bonds lead to further heterogeneity, even if they play only a minor role. Taking the ester bonds to pCA as an example, and in accordance with other studies, it was found that they are partially cleaved during hydrothermal treatment [71,80,81]. After a treatment time of 50 min and thus a severity of 4.05, the content in the pCA ester bonds cleavable by saponification decreased by about 41% for the washed digestate and even by about 64% for the raw digestate. The fact that a higher decrease in esters was generally observed for the raw digestate could be attributed to the catalytic effect of the ammonia on the ester cleavage. No comparable investigations using saponification of hydrothermally treated samples could be found, but pCA esters can also be detected by 2D NMR. Heikkinen et al. [71], for example, found a comparable decrease of 55% after the steam explosion of wheat straw with a severity of 3.94 by 2D NMR. The released pCA could be hardly recovered in the hydrolysate (see Appendix C.1), which raises the research question of what other side reactions occur with pCA. Besides further reactions to other monomers (e.g., via oxidation), repolymerization reactions with lignin or carbohydrates are also possible here. These would again hinder cellulose and lignin valorization.

Figure 6 indicates that the first-order reaction kinetic model used seems to fit the degradation of the pCA ester bonds to some extent. The degradation of the ether bonds, on the other hand, did apparently not fit well to the model and seemed to follow a different kinetics. Based on Figure 6, it appears that the ether bond content initially decreases with an increasing treatment time and then approaches a value of 18% ether bonds for both the washed and unwashed digestate for higher treatment times. One explanation might be that ether bonds with differing stability can be found in the digestate lignin. For example, Heikkinen et al. [71] calculated different bond energies for ether bonds between different monomers, which would consequently result in different depolymerization rates.

In view of lignin valorization to aromatic chemicals and based on the ether and pCA ester bond content, it can be concluded that further depolymerization targeting these weaker linkages in lignin obtained after hydrothermal and enzymatic treatment would result in smaller oligomers and even few amounts of monomers. The longer the hydrothermal treatment, the fewer the amount of monomers and the larger the amount of oligomers are expected to be due to the formation of stable carbon–carbon bonds in the repolymerization reactions. For example, after a steam treatment of 50 min, 296 µmoles (corresponding to about 58 mg) of lignin monomers and 18.5 mg of pCA can be obtained per gram of pure lignin. However, the limited lignin purity, which was also observed in other studies, has to be considered [82]; especially, the contained ashes might act as a catalyst poison in the catalytic depolymerization reactions.

5. Conclusions

Both washed (and thus purified) and raw fibrous straw digestate (containing different impurities such as ammonia, proteins and minerals) can be fractionated by combined hydrothermal and enzymatic treatments, leading to three fractions:

• About half of the hemicellulose contained in the digestate can be solubilized and recovered in the hydrolysate after a hydrothermal steam treatment at 180 °C for at least 50 min. During the hydrothermal treatment of the raw digestate, much higher pH-values are observed than during the treatment of the washed digestate, most likely due to the contained ammonia in the raw digestate. Higher pH values prevent the hydrolysis and further degradation of xylans for long treatment times. However, if xylo-oligosaccharides are a targeted product, it has to be considered that the hydrolysates from the raw digestate are very complex mixtures, making a separation challenging.
• Cellulose mainly stays with the solid fibers and can be enzymatically hydrolyzed to glucose with a yield of around 90% after a hydrothermal steam treatment at 180 °C for 50 min for both the raw and the washed digestate. Furthermore, the remaining hemicellulose can be enzymatically hydrolyzed nearly completely at these conditions. For shorter treatment times, a higher enzymatic degradability of carbohydrates is observed for the raw digestate than for the washed digestate.
• A solid enriched in lignin remains after both treatment steps. The lignin is partially degraded due to depolymerization and repolymerization reactions, which is indicated by a reduced content of ether and ester bonds. The reduced ether content as well as the high ash content should be considered if a catalytic depolymerization is targeted.

The hypothesis that the impurities in raw digestate lead to a decreased fractionation efficiency is not valid, as has been shown. The impurities lead to some changes in the process, but, among these, there are also improvements such as a decrease in the degradation of hemicellulose-derived sugars and a faster increase in cellulose degradability. However, it must be considered that the impurities end up in the product streams.

In order to achieve optimum xylan or xylose and glucose yields and to keep lignin degradation and energy requirements as low as possible, a treatment time somewhere around 50 min seems to be appropriate if a one-step hydrothermal steam treatment at 180 °C is considered. The solubilized and hydrolyzed carbohydrates can be used for the fermentation to bio-based chemicals such as ethanol or propionic acid. Here, the use of the raw digestate might have the advantage that various nutrients for fermentation are already included; however, the actual effects have to be tested in the specific case. In order to achieve a full valorization of the straw digestate, different possibilities for the valorization of the lignin enriched solids should be identified and tested in a next step. Finally, in order to verify the feasibility of the proposed concept, it would be important to perform a techno-economic analysis of an overall concept. As a promising basis, the scalability of steam treatments in general and their transferability to a continuous operation mode has already been demonstrated in practice [83].