Aiming for the complete utilization of sugar-beet pulp: Examination of the effects of mild acid and hydrothermal pretreatment followed by enzymatic digestion
© Kühnel et al; licensee BioMed Central Ltd. 2011
Received: 11 February 2011
Accepted: 31 May 2011
Published: 31 May 2011
Biomass use for the production of bioethanol or platform chemicals requires efficient breakdown of biomass to fermentable monosaccharides. Lignocellulosic feedstocks often require physicochemical pretreatment before enzymatic hydrolysis can begin. The optimal pretreatment can be different for different feedstocks, and should not lead to biomass destruction or formation of toxic products.
We examined the influence of six mild sulfuric acid or water pretreatments at different temperatures on the enzymatic degradability of sugar-beet pulp (SBP).
We found that optimal pretreatment at 140°C of 15 minutes in water was able to solubilize 60% w/w of the total carbohydrates present, mainly pectins. More severe treatments led to the destruction of the solubilized sugars, and the subsequent production of the sugar-degradation products furfural, hydroxymethylfurfural, acetic acid and formic acid. The pretreated samples were successfully degraded enzymatically with an experimental cellulase preparation.
In this study, we found that pretreatment of SBP greatly facilitated the subsequent enzymatic degradation within economically feasible time ranges and enzyme levels. In addition, pretreatment of SBP can be useful to fractionate functional ingredients such as arabinans and pectins from cellulose. We found that the optimal combined severity factor to enhance the enzymatic degradation of SBP was between log R'0 = -2.0 and log R'0 = -1.5. The optimal pretreatment and enzyme treatment solubilized up to 80% of all sugars present in the SBP, including ≥90% of the cellulose.
Sugar-beet pulp (SBP) consists of up to 75% w/w of carbohydrates (dry matter). Arabinose, glucose and galacturonic acid (GA) are the main sugar moieties present in complex polysaccharide structures . After sugar extraction, the pressed pulp has a dry-matter content of 18-23% w/w. To date, SBP has been used mainly as animal feed or, in regions with no livestock farming, dumped in landfill [2, 3]. The low dry-matter content makes combustion of SBP for heat and power production unfavorable; however, its low lignin and high sugar content make it an interesting candidate for biorefinery and/or bioethanol production. The availability of new, pentose-fermenting yeast strains allows the efficient use of biomass for bioethanol production ; however, no yeast strains that allow the fermentation of uronic acids to ethanol are currently available.
For fermentation, the cell-wall material needs to be degraded into fermentable monosaccharides. To this end, lignocellulosic feedstocks are often structurally modified by a mild pretreatment before enzymatic release of fermentable monosaccharides . The severity of this pretreatment can be measured by a combined severity factor (log R″0) that takes into account pretreatment time, pretreatment temperature, and acid concentration (w/w%) and pH after pretreatment [6, 7, 47]. In general, a pretreatment has to be strong enough to disrupt the cellulose-hemicellulose network and, if present, the cellulose-lignin network; however, as the severity of the pretreatment increases, more biomass is degraded to non-fermentable products and products toxic to yeast, such as furfural or hydroxymethylfurfural (HMF) . Along with release of furfural and HMF, weak acids (such as acetic acid) which can have a negative effect on yeast growth, viability and fermentation, may also be released [9, 10]. Although yeasts can adapt to moderate levels of furfural and detoxification, as shown previously , it is economically and ecologically favorable to avoid the formation of such compounds.
During the past decades, much work has been performed on the degradation of SBP, which has shown good yields of solubilized carbohydrates; however, in these studies, high levels of enzymes or chemicals were required [12–15]. To our knowledge, there are no data available for the degradation of SBP in commercially reasonable time spans and with economically feasible enzyme levels.
In this study, we examined the effect of hydrothermal or mild acid pretreatment on enhancing the enzymatic degradation of SBP. Several different pretreatments were used, with different temperatures and pH values. The pretreated samples were characterized and used for an enzymatic saccharification study to analyze the amounts of fermentable monosaccharides produced.
SBP was obtained as frozen pressed pulp (23% dry-matter content, harvest 2006; Suiker Unie, Dinteloord, The Netherlands). The pulp contained 68% w/w carbohydrates (dry weight). The constituent sugars were arabinose (18% w/w), glucose (22% w/w), uronic acids (UAs) (18% w/w), galactose (5% w/w), rhamnose (2% w/w), xylose (2% w/w) and mannose (1% w/w), respectively, and the pulp contained 4% w/w residual saccharose. Other components were protein (8% w/w), lipids and salts. Ferulic acid (0.5% w/w), acetic acid (1.6% w/w) and methanol (0.4% w/w) were present as ester-linked substituents of polysaccharides.
Pretreatment conditions of sugar-beet pulp
R'0 (R'0 = t × (10-pH) × exp × ((T-100)/14.75)),
and expressed as log R'0 according to Kabel et al. ). This factor is an indicator for the severity of a pretreatment, and takes into account the pretreatment temperature, time, acid concentration and final pH.
Freeze-dried samples (20-40 mg) were weighed into aluminum cups (diameter 50 mm) and dried in an oven at 70°C for up to 3 days, until the weight was constant.
Chemical characterization of pretreated SBP
After freeze-drying, the P1 pellets were weighed and ground (4 minutes at 100 Hz) in a swinging mill (Model M2200; Retsch GmbH, Haan, Germany) using 25 ml stainless steel containers with two stainless-steel metal balls (diameter 10 mm) per container.
The freeze-dried S1 supernatants were treated in the same way except for the supernatants S1 0-0, S1 170-0 and S1 170-1, which did not crystallize but instead formed a syrup. For these fractions, the syrup was dissolved in distilled water, and the volume was determined. An aliquot (1 ml) was freeze-dried and subsequently oven-dried to calculate the dry-matter content of the solutions. The different crystallization behavior did not affect the subsequent results.
Glycosidase activities in the experimental enzyme preparation C1-G1 were measured by determining the hydrolysis of p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-xylopyranoside or p-nitrophenyl-β-l-arabinofuranoside at 37°C after 10 minutes of incubation. The reaction mixture consisted of 25 μl substrate solution (4 mg/ml) and 10 μl of the suitable enzyme concentration in 50 mmol/l sodium acetate buffer pH 5.0. The reaction was stopped by adding 40 μl of reaction mixture to 60 μl of 250 mmol/l TRIS-HCl buffer pH 8.8. The color formation was measured at 410 nm in a microtiter plate reader. One unit of activity was defined as 1 μmol of glucose, xylose or arabinose liberated per minute under the conditions specified. The molar extinction coefficient under these assay conditions was 0.00158/μmol/l/mm. Xylanase and cellulase activities were determined by measuring the amount of reducing sugars released from the hydrolysis of medium-viscosity wheat arabinoxylan (Megazyme International Ireland Limited, Bray, Wicklow, Ireland) and phosphoric acid-treated cellulose . The reaction mixture consisted of 50 μl substrate solution (15 mg/ml) and 20 μl of the suitable enzyme concentration in 60 mmol/l sodium acetate buffer pH 5.0. The reaction mixtures were preheated at 37°C before the enzyme was added. After an incubation time of 10 minutes, 25 μl of the reaction mixture were used to determine the amount of reducing sugars using a PAra-Hydroxy Benzoic Acid Hydrazide (PAHBAH) assay .
The activities of polygalacturonase, rhamnogalacturonase, arabinanase and galactanase were determined by measuring the amount of reducing sugars released from the hydrolysis of poly-GA (Fluka, Sigma-Aldrich, Schnelldorf, Germany), linear arabinan (Megazyme) and potato galactan (Megazyme).The reaction mixture consisted of 180 μl substrate (5 mg/ml) in 50 mmol/l sodium acetate buffer pH 5.0, which was preheated to 37°C before 20 μl of the relevant enzyme concentration were added. The reaction mixture. After an incubation time of 20 minutes, 10 μl of the reaction mixture were used to determine the amount of reducing sugars using the PAHBAH assay . Calibration curves of the different monosaccharides (50 to 750 μg/ml) were used for quantification.
Digestions of pretreated pulp
Activity profile of enzyme mix C1-G1
The neutral sugar composition was determined by analyzing the sugars as their alditol acetate derivatives using GC-FLD. A gas chromatograph (Thermo Focus; Thermo Scientific, Waltham, MA, USA) and autosampler (Focus AS 3000; Thermo Scientific) equipped with a column (15 m × 0.53 mm, 1 μm film; DB-225; J&W Scientific, Santa Clara, IL, USA) was used. The samples were hydrolyzed stepwise with 72% w/w sulfuric acid for 1 hour at 30°C, followed by 1 M sulfuric acid for 3 hours at 100°C. Subsequently, the free monosaccharides were reduced and derivatized to their corresponding alditol acetates . The samples were dissolved in 0.4 ml acetone, and 1 μl was injected with a pre-injection dwell time of 1 second and a post-injection dwell time of 2 seconds. The sample was then eluted using the following temperature profile: 0 to 2 minutes at 180°C, 2 to 17 minutes at a linear gradient from 180 to 210°C, and 17 to 22 minutes at 210°C.
UA content was determined colorimetrically  with 3-phenylphenol using an automated analyzer (Skalar Analytical, Breda, The Netherlands). A GA standard curve (12.5 to 100.0 μg/ml) was used for quantification.
The content of organic acids, HMF, furfural and methanol was analyzed by high performance liquid chromatography with a (Ultimate 3000; Dionex Corp., Sunnyvale, CA, USA) equipped with an ion-exclusion column (300 mm × 7.8 mm; Aminex HPX-87H; Bio-Rad Laboratories, Hercules, CA, USA) in combination with a self-packed guard column (50 mm × 7.8 mm; AG 50W-X4 resin; Bio-Rad Laboratories). Samples were eluted with 5 mmol/l sulfuric acid at a flow rate of 0.6 ml/min at 30°C. The S1 supernatants were filtered through a 0.2 μm round filter (Whatman), and 20 μl samples used for injection. The samples were quantified by single-component standards of known concentrations (0.5 mg/ml). Elution was monitored by refractive index detection (Shodex RI 101; Showa Denko K.K., Kawasaki, Japan).
Monomer and oligomer analysis
The monosaccharide and oligosaccharide levels of the digests were analyzed by HPAEC with pulsed amperometric detection (PAD) (ITS-3000 HLPC; Dionex) equipped with an analytical column (2 mm × 250 mm, CarboPac PA1; Dionex) in combination with a guard column (2 mm × 50 mm, CarboPac PA1; Dionex) at a flow rate of 0.3 ml/min. Arabinose, rhamnose, galactose, glucose, xylose, mannose and GA monosaccharides in the range from 2 to 30 μg/ml were used for quantification. Oligomeric standards included arabinose oligomers (degree of polymerization (DP) 2 to 6, 10 μg/ml; Megazyme), GA oligomers (DP 2 and DP 3, 10 μg/ml), a potato galactan digest containing galactose oligomers (DP 2 to 5; 5 mg/ml potato galactan; Megazyme) partially digested with Aspergillus niger endogalactanase (purified in our laboratory) and cellodextrins (DP2 to 7; kind gift of Dr Vladimir Farkas, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia). The samples (10 μl; 50 to 100 μg/ml) were eluted in post column addition mode with the following elution profile: 0.0 to 30.0 minutes in water, 30.0 to 30.1 minutes in 0.0 to 0.1 mol/l NaOH, 30.1 to 32 in 0.1M NaOH, 32.0 to 62.5 minutes in 0.0 to 0.4 mol/l NaOAc in 0.1 mol/l NaOH, 62.5 to 67.0 minutes in 1.0 mol/l NaOAc dissolved in 0.1 mol/l NaOH, 67.0 to 75.0 minutes in 0.1 mol/l NaOH, and 75.1 to 90.0 minutes in water (equilibration). NaOH (0.5 mol/l, 0.1 ml/min) was added via a post column to allow PAD detection from 0.0 to 30.1 minutes and from 68.5 to 90.0 minutes.
Ferulic acid analysis
The amount of ester-linked ferulic acid was determined after alkaline hydrolysis and ethyl ether extraction as described previously  using reversed phase ultra-high-performance liquid chromatography (UHPLC) on a UHPLC system (Accela; Thermo Scientific) equipped with a column (2.1 mm × 150 mm, 1.9 μm particle size; Hypersyl GOLD; Thermo Scientific). Elution was monitored with the built-in polydiode array multi-wavelength detector set at 335 nm.
Molecular mass distribution
HPSEC was performed on a system (Ultimate 3000; Dionex) equipped with four columns (TSK-Gel superAW; Tosoh Bioscience, Tokyo, Japan) in series: a guard column (6 mm × 40 mm) and three separation columns (4000, 3000 and 2500; 6 mm × 150 mm). The samples (10 μl, 5 mg/ml) were eluted with 0.2 mol/l sodium nitrate at 40°C at a flow rate of 0.6 ml/min. Molecular masses were estimated with the help of molecular mass standards (Pullulan; Polymer Laboratories, Varian Inc., Palo Alto, CA, USA). Elution was monitored by refractive index detection (Shodex RI 101) and UV detection (internal detector) at four wavelengths (λ = 235 nm, 310 nm, 325 nm and 345 nm). The relative peak area distribution was calculated by the integration of the peak area in three time increments. The total peak area was calculated as the total peak area from 7.0 to 18.0 minutes without the negative peak (15.5 minutes) plus the peak area difference between the negative peak of a water blank and the negative peak of the supernatant sample.
The protein content of the pretreated samples was determined by combustion (the Dumas method) with a nitrogen analyzer (FlashEA 1112 series; Thermo Scientific) in accordance with the manufacturer's instructions, using methionine as a standard and a nitrogen factor of 6.25.
Results and Discussion
Mass of pretreated SBP
Generally, more material was solubilized with increasing pretreatment severity, but a loss of mass was seen for all samples. These losses were mainly attributable to the mechanical loss of material during blending and sample handling. When this mechanical loss was averaged over the first five pretreatments, it was estimated to account for roughly 8% w/w. Pretreatments with log R'0 ≥-0.86 (sample 140-1) led to major losses in mass, probably due to chemical decomposition, and no further increase in the level of soluble compounds in the supernatant (Figure 2). The pellet and supernatant curves were both sigmoidally shaped, which could imply that the range between solubilization and chemical degradation of the pulp is narrow, and hence requires good process control. If more severe treatments are required for effective disruption of the cellulose network, solubilized temperature-labile compounds should first be removed from the reactor to minimize their destruction. Such removal of soluble compounds could be carried out by using a flow-through reactor setting or an extruder.
Sugar composition of released and residual carbohydrate material
High solubilization of arabinan and galacturonan from SBP after exposure to temperatures of 150 to 175°C has been reported previously [14, 27]. Temperatures above 160°C led to decreasing recovery of GA, whereas arabinose recovery was stable up to 167°C , and most of the cellulose remained insoluble under the conditions used. Our data are in line with these observations.
We found that the sugar yields in the pellets decreased in line with the increasing severity of pretreatment (Figure 3B). Approximately 30% of the total sugars present in the starting material were left in the pellet fraction of sample 170-1. The absolute amounts of glucose remained almost constant, so that the relative glucose content of the pellet fractions increased up to 90% (moles) (see Additional file 2, Table s1). Even though cellulose was not solubilized during pretreatment, the solubilization of arabinan might also have had a positive effect on the enzymatic degradation of cellulose. It has been shown previously that arabinan can absorb to cellulose in vitro, thus removal of arabinan would be expected to lead to greater exposure of the cellulose surface to enzymes.
When the yields of the individual sugars present in the pellet and supernatant fractions were summed (Figure 3C), a decrease was seen for the pretreatments with log R'0 ≥-0.86 (samples 170-0 and 170-1), which was similar to the yield of the total material after pretreatment (Figure 2).
Sugar-degradation products formed during pretreatment
Furfural formation was linked to pentose and UA degradation [25, 31], and only occurred in samples 170-0 (2.1% w/w, 0.2 μmol) and 170-1 (2.6% w/w, 0.25 μmol). This could explain 14 to 18% of the arabinose degradation (around 1.4 μmol in the pulp) or 20 to 25% of the UA degradation (around 1 μmol) we observed. Because 40% of the arabinose and 90% of the GA were decomposed (Figure 3D), it is likely that destruction of arabinose and GA also occurs via different mechanisms or yields different end-products.
For example, pectins can undergo β-elimination at high temperatures to form unsaturated GA and unsaturated GA oligomers . A combination of β-elimination and decarboxylation, resulting in a pyranose ring structure of unsaturated 4,5-l-arabinopyranose and carbon dioxide, has also been proposed. Carbon dioxide could be responsible for the temperature-independent pressure increase we saw during the 170°C pretreatments (see Additional file 1, Figure S1).
Formation of formic acid may be linked to further degradation of furfural and HMF at higher temperatures . Furfural and HMF may also polymerize to form humins under acidic conditions and higher temperatures ; however, humin formation cannot explain the mass loss we found at log R'0 ≥-0.86. In addition, it generally requires higher acid concentrations . The presence of acetic and lactic acid in all our pretreated samples may also indicate contamination of the SBP starting material with acid-releasing bacteria. The release of acetic and lactic acid strongly increased at log R'0 ≥-0.86. The acetic acid release (up to 2.5% w/w) can partly be explained by pectin deacetylation, because SBP contains around 1.6% w/w ester-linked acetic acid. In addition, acetic and lactic acid were also formed during the Maillard reaction.
The data from these experiments suggest that pretreatments at log R'0 ≥-0.86 led to the enhanced destruction of arabinose and GA and to the production of toxic compounds that could negatively affect fermentation.
Compressibility of pretreated pulp
Water-binding capacity of pretreated samples
Bound water, g/g pelleta
Increased swelling and WBC have been described previously for pellets of alkali-extracted beet fibers [36, 37], even though these fibers had strongly reduced levels of pectic arabinan and galacturonan. Analogously, it has been reported for the cell-wall material of wheat flour that enzymatic removal of arabinoxylan increased the WBC of the residual, cellulose-rich material . Possible explanations for the increased water binding include the total polysaccharide content, charge, crystallinity of the cellulose, specific surface area, and hydrophobic components .
These results indicate that dry-matter contents higher than 5% may not be possible for pretreatment and enzymatic digestion of SBP, because it is not possible to pump or stir the pulp efficiently. Bioethanol or biogas production requires higher substrate concentrations to make the downstream processing (for example, distillation/rectification) economically feasible. To allow higher dry-matter concentrations, the WBC could be reduced either enzymatically by the addition of an endopolygalacturonase or thermophysically by a more severe pretreatment. However, there are drawbacks: enzymatic degradation would produce GA, which may be toxic to the yeast, and severe pretreatment would drastically lower the yield by causing biomass degradation and release of inhibiting compounds. A fed-batch setup could be a promising alternative to allow high dry-matter concentrations.
Molecular mass distribution of solubilized material
The 120°C pretreatments both solubilized a polymeric fraction with an average molecular mass of approximately 100 kDa, based on the Pullulan molecular mass standards. Samples pretreated with and without sulfuric acid had the same molecular-mass distributions as the samples pretreated with water (data not shown). The average molecular mass of the solubilized material decreased with increasing temperatures from 50 kDa for the 140°C pretreatments to oligomeric material of ≤1 kDa at 170°C. Along with the solubilization of carbohydrates, an UV signal at 325 nm was detected for these populations, indicating the presence of phenols (Figure 5B).
Ferulic acid (FA) yield in solubilized material after pretreatment
An optimal pretreatment does not necessarily hydrolyze the insoluble material to monosaccharides and oligosaccharides, which are more prone to chemical destruction than are polymers. The molecular mass distributions of the supernatants indicated that the relative proportions of the high and average molecular mass material increased up to log R'0 = -1.65 (sample 140-1), whereas they strongly decreased for pretreatments with log R'0 ≥-0.86 (Figure 5C). In turn, the amount of each compound eluting in the monomeric and oligomeric range increased with increasing severity. These results show, together with the decomposition (Figure 2; Figure 3D) that pretreatments with log R'0 ≥-0.86 favored chemical destruction of sugars and were too severe when aiming for a maximum yield of fermentable sugars.
Enzymatic degradation of pretreated SBP pellets
For bioethanol production, the aim is to ferment a wide range of monosaccharides. Besides hexose-fermenting yeast strains (mainly glucose and fructose but also galactose), several yeast strains that are able to ferment pentoses have been described in the literature . The efficient enzymatic degradation of sugar-beet arabinan has already been reported . To verify the influence of the pretreatment on enzymatic digestibility of cellulose, we degraded pretreated SBP pellets with an experimental enzyme mixture (C1-G1) that is rich in cellulases and xylanases (Table 2). This mixture has been designed for the degradation of wheat bran, but also has endogalactanase and endoarabinanase activities (Table 2). It is almost devoid of polygalacturonase/pectate lyase activity (Table 2).
Polygalacturonase deficiency could be beneficial for a subsequent yeast fermentation because high concentrations of weak acids have a negative effect on yeast viability, and could therefore lower ethanol production . We were able to recover sugar-beet polygalacturonides after fermentation. These polygalacturonides can be used to produce pectin-derived oligosaccharides enzymatically. Pectin-derived oligosaccharides have been shown to have a number of beneficial biological effects, including prebiotic activity [43–45]. Alternatively, enzymatically released GA may be fermented to ethanol by bacteria .
Yields of individual monosaccharides ending up in the supernatant after enzyme incubation for 48 hours of pretreated pellets with C1-G1 enzyme mixture (1% w/w)a
Total solubilization after pretreatment and enzymatic digestion
The total carbohydrate solubilization was calculated as the sum of all material solubilized after pretreatment plus the material solubilized from the pellet after enzymatic digestion (Figure 7B). The solubilization was inversely correlated with increasing severity, and ranged from 34% for the 0-0 digest to 80% for the 140-0 digest. The pectin-associated sugars arabinose, galactose and GA had 85, 70 and 75% solubilization, respectively, in sample 140-1. Mild pretreatments up to log R'0 = -1.65 generally enhanced the enzymatic degradation of cellulose from SBP (Figure 7B). C1-G1 also released material of high molecular mass from the pellets of samples 0-0, 120-0 and 120-1 (determined by HPSEC analysis, data not shown). It is likely that the solubilized material was pectin, because after enzyme incubation for 48 hours, the pellets had a strong reduction in their UA content (data not shown). Pectin can be solubilized either by the degradation of the cellulose network or by the removal of arabinan sidechains linking it to cellulose .
We found that the combination of a mild hydrothermal pretreatment at 140°C with subsequent enzymatic digestion allowed fast and efficient hydrolysis of SBP. More than 90% of all cellulose could be hydrolyzed within 24 hours, using lower enzyme levels than those reported in earlier studies. In contrast to the chemical pretreatments described in literature, mild pretreatments up to 140°C did not result in a loss of biomass or in production of any compounds toxic to yeast. If the pretreatment process were to be implemented in a sugar factory, the pulp would already be at 70°C after saccharose extraction, and the exhaust heat of the lime ovens could be used in part for the heating of the SBP up to 140°C. The mild pretreatments did not lower the WBC of beet pulp and are therefore unsuitable for increasing the dry-matter content of SBP. Hence, to reduce transportation, pretreated SBP should be fermented in the vicinity of the sugar factory. A fed-batch reactor setting could allow a reasonably high ethanol concentration.
We thank David van Eylen (Royal Nedalco/C5 Yeast Company, Bergen op Zoom, The Netherlands) for help with the pretreatments. Mirjam Kabel (Royal Nedalco/C5 Yeast Company) is gratefully acknowledged for valuable discussion, and Sandra Hinz (Dyadic Netherlands) for the provision and characterization of the C1-G1 enzyme mixture. The work was financed in part by the Dutch Ministry of Economic Affairs via an EOS-LT grant (http://www.agentschapnl.nl).
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