Bioresource Technology 169 (2014) 206–212
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Pulp properties resulting from different pretreatments of wheat straw and their influence on enzymatic hydrolysis rate Christine Rossberg a,⇑, Doreen Steffien b, Martina Bremer a, Swetlana Koenig c, Florbela Carvalheiro d, Luís C. Duarte d, Patrícia Moniz d, Max Hoernicke c, Martin Bertau b, Steffen Fischer a a
Institute of Plant and Wood Chemistry, Dresden University of Technology, Pienner Str. 19, 01723 Tharandt, Germany Institute of Chemical Technology, Freiberg University of Mining and Technology, Leipziger Str. 29, 09595 Freiberg, Germany Saxon Institute for Applied Biotechnology, Leipzig University, Permoserstr. 15, 04318 Leipzig, Germany d Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal b c
h i g h l i g h t s Process parameters for grinding and pulp storage were optimized. Comparison of pulp digestibility after three very different pretreatments. Pulp hydrolysis using the novel Penicillium verruculosum cellulase complex. Lignin removal is not crucial for enzymatic hydrolysis. Autohydrolysis pretreatment enables complete conversion of cellulose to glucose.
a r t i c l e
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Article history: Received 8 April 2014 Received in revised form 23 June 2014 Accepted 26 June 2014 Available online 3 July 2014 Keywords: Alkaline pulping Natural pulping Autohydrolysis Wheat straw Enzymatic hydrolysis
a b s t r a c t Wheat straw was subjected to three different processes prior to saccharification, namely alkaline pulping, natural pulping and autohydrolysis, in order to study their effect on the rate of enzymatic hydrolysis. Parameters like medium concentration, temperature and time have been varied in order to optimize each method. Milling the raw material to a length of 4 mm beforehand showed the best cost–value-ratio compared to other grinding methods studied. Before saccharification the pulp can be stored in dried form, leading to a high yield of glucose. Furthermore the relation of pulp properties (i.e. intrinsic viscosity, KLASON-lignin and hemicelluloses content, crystallinity, morphology) to cellulose hydrolysis is discussed. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Wheat straw is a fast-growing and abundant agricultural by-product. For instance, 8–13 million tons per year are readily available in Germany without risking humus reduction (Zeller et al., 2011). As it has no application in the food industry, it could serve as an excellent starting material for the production of cellulose, basic chemicals, lignin and bioethanol in the biorefinery framework. Especially the latter attains increasing importance, since the increasing level of greenhouse gases, the depletion of fossil fuels and the unstable oil market lead to a general interest
⇑ Corresponding author. Tel.: +49 (0) 35203 38 31877. E-mail address: [email protected] (C. Rossberg). http://dx.doi.org/10.1016/j.biortech.2014.06.100 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
in alternative transportation fuels (Solomon et al., 2007; Talebnia et al., 2010). For bioethanol production using lignocellulosic material at least four operations including pretreatment, hydrolysis, fermentation and distillation are necessary (Talebnia et al., 2010). Different pretreatment procedures have been developed in order to increase the accessibility of cellulose by removing hemicelluloses, by breaking down the lignin structure and disrupting the crystalline structure of cellulose (Wang et al., 2012). Furthermore, the removal of lignin can be of advantage, as it has been reported that high lignin contents in the substrates may exert inhibitory effect on the enzymes used for hydrolysis (Rahikainen et al., 2011; Sewalt et al., 1997). Thus different methods have been investigated in order to prepare the carbohydrate fraction for enzymatic hydrolysis, which can be classified into physical (e.g. milling), physico-chemical
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(e.g. autohydrolysis, steam explosion), biological (e.g. white rot-fungi) and chemical processes (e.g. organosolv, alkaline pulping) (Kumar et al., 2009; Talebnia et al., 2010). This study focuses on chemical and physico-chemical treatments. One of the advantages of using annual plants is that traditional pulping methods like Kraft-pulping are not necessary. Thus sulfurfree and hence more environment-friendly procedures can be applied. That is why several studies have been engaged with less severe pulping methods like alkaline pulping using sodium hydroxide or calcium hydroxide (Epelde et al., 1998; García et al., 2009; Khristova et al., 1998). The alkaline condition leads to the dissociation of phenolic hydroxyl groups in the lignin and increases its solubility. The latter is furthermore enhanced by lignin fragmentation and release of methanol, which, though released in small quantities, can work as an additional solvent (Epelde et al., 1998; Gierer, 1985). Due to this method a very good delignification of the pulp can be achieved and the accessibility of cellulose is increased due to swelling and removal of hemicelluloses. On the other hand fermentable sugars are lost and, because of the high silica content of annual plants, irrecoverable salts are formed and incorporated into the biomass (Kumar et al., 2009). However, Lei et al. (2010) showed that the silica content can be effectively lowered by a hot-water pretreatment. Thus, with further studies the process and the recovery of chemicals could still be improved. In contrast to alkaline pulping the use of organic solvents for pulping has the advantage of easily recovering the initial chemicals. Furthermore, very pure lignin can be obtained, so that a complete usage of the lignocellulosic material is possible. Among the organic solvents two main groups have been in the focus of study: organic acids (e.g. formic acid, acetic acid) and alcohols (e.g. ethanol). The so called NATURAL PULPING process, which is similar to the MILOX process, uses formic acid and hydrogen peroxide to generate peroxyformic acid in situ (Siegle, 2001). This leads to an oxidation and depolymerization of lignin and thus enhances its solubility (González et al., 2010; Ligero et al., 2010). However, the corrosive character of peroxyformic acid makes it necessary to work with glass or enameled steel. Compared to the pretreatments already described, autohydrolysis is the most environmental friendly procedure, as the biomass is treated only with hot water or steam at temperatures usually in the range of 150–230 °C for only short residence times (Carvalheiro et al., 2008; Garrote et al., 1999; Wang and Sun, 2010). Autohydrolysis leads to specific hemicellulose removal resulting in a cellulose and lignin rich solid fraction. Due to cell penetration and breakdown of the lignocellulosic structure the cellulose accessibility is increased. This process also has the advantage to produce a hemicellulosic liquid stream that can also be used for fermentation purposes or for recovering value-added compounds. However, a careful optimization of the operational conditions is important in order to minimize the formation of degradation compounds like furfural and hydroxymethylfurfural, which might act inhibitory on microorganisms used later on in the process (Gírio et al., 2010). After the pretreatment procedure the lignocellulosic material is prepared for the hydrolysis step. Two main procedures are used to hydrolyze cellulose: acid and enzymatic treatments. Unlike acid hydrolysis, enzymatic hydrolysis is highly specific and occurs under mild reaction conditions. Thus, it is considered to be more promising for the inclusion into a biorefinery concept (Wang and Sun, 2010). For this application a fungal cellulase complex is used. It includes endo-glucanase, exo-glucanase and b-glucosidase activities. They are known to be influenced by the pretreatment of cellulosic material as well as the hydrolyzing conditions. Most cellulase enzyme complexes show an optimum activity at h = 45–55 °C and pH = 4–5 (Talebnia et al., 2010). For this investigation an alternative enzyme complex obtained from Penicillium
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verruculosum strain was used. In comparison to the worldwide employed complex from Trichoderma reesei, P. verruculosum cellulases are characterized by higher content of b-glucosidase and higher resistance towards ethanol (Morozova et al., 2010). The objective of this work was to investigate the influence of pretreatment processes, i.e. alkaline pulping, natural pulping and autohydrolysis on the enzymatic hydrolysis of wheat straw pulp. Pulping parameters have been varied to optimize each method and to examine their impact on hydrolysis rate using an enzymatic complex of P. verruculosum. Furthermore the effect of a mechanical treatment of the wheat straw prior to pulping has also been investigated.
2. Experimental 2.1. Raw material The wheat straw used in this study was obtained from Agrargenossenschaft Rossau eG, Germany. It was prepared in three different ways: chopped to a length of 4 to 10 cm, milled to pass a 4 mm mesh and as thermo mechanical pulp (TMP) using a 12-zoll-refiner and a disk gap of 0.15 mm. The composition of the wheat straw is as follows: cellulose 44.2%, hemicellulose 27.3%, lignin 19.3%, extract 3.9% and ash 3.0% on a dry weight basis. With the parameters used, TMP refining was not found to have a significant effect on the chemical composition of wheat straw.
2.2. Methods 2.2.1. Alkaline Pulping Alkaline pulping was carried out in a 2 L digester. The cooking conditions were: liquid–solid ratio 6.2 mL/g, concentration of sodium-hydroxide (1, 3, 6, 9) wt%, maximum temperature (120, 140, 160) °C, time at maximum temperature (30, 90, 180) min. The pulp was separated from the black liquor over a Büchner funnel, washed with sodium hydroxide solution (0.1 M) and afterwards with deionized water. Enzymatic hydrolysis was conducted directly or after storing the pulp at 18 °C or after drying it at 105 °C to analyze the influence of different storage conditions.
2.2.2. Natural Pulping Natural pulping was carried out according to Siegle (2001) using a 3 L round bottom flask with reflux condenser. The following parameters were used: liquid–solid ratio 27 mL/g, concentration of formic acid (30, 50, 60, 70, 80) wt%, maximum temperature 101 °C, time at maximum temperature (20, 40, 60) min. After cooling down, the pulp was separated, washed and dried.
2.2.3. Autohydrolysis The hydrothermal treatments (autohydrolysis) were performed in a stainless steel reactor with a total volume of 2 L. The reactor was fitted with two four-blade turbine impellers, heated by an external mantle, and cooled by cold water circulating through an internal stainless steel loop. The wheat straw was mixed with water in the reactor in order to obtain a liquid-to-solid ratio of 10 mL/g. A maximum temperature of (180, 190, 200, 210, 220) °C at an average rate of temperature increase (from 100 °C) of approximately 4.7 °C/min was applied. When the desired temperature was reached, the reactor was cooled down and the liquid and solid phases were recovered by pressing in a hydraulic press up to 200 bar. The solid phase was washed with water, pressed again and dried.
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2.2.4. Enzymatic Hydrolysis For pulp hydrolysis 0.5 g biomass on dry weight base were incubated after adding 23.5 mL of citrate buffer solution (0.05 M, pH = 5) and an enzyme complex from Penicillium verruculosumM28-10b (DSM24317) (cellulase: 100 FPU/g dry feedstock and b-glucosidase: 600 IU/g dry feedstock). Incubation was carried out at 50 °C for 24 h in a laboratory shaking device (150 rpm). In terms of relevance for the SSF process, the hydrolysis time of 24 h was examined. 2.2.5. Analysis of hydrolyzate After 24 h of enzymatic hydrolysis 1 mL sample was centrifuged to remove insoluble material. 500 lL supernatant, 200 lL maltitol (20 g/L) and 500 lL acetone were mixed and filtered through 0.2 lm PVDF filter into vials. Soluble sugars were analyzed by HPLC using a NUCLEODURÒ100–5 NH2-RP column (Macherey Nagel). The analysis conditions were: mobile phase ACN:H2O 79:21 v/v, injection volume 5 lL, column temperature 30 °C, flow rate: 1 mL/min. The detector (ELSD) parameters were: evaporation temperature 94 °C, nebulizer temperature 60 °C, carrier flow (N2) 1 slm. The rate of hydrolysis was calculated as g glucose per 100 g holocellulose, which was determined as described in Sect. 2.2.6 representing the cellulose as well as the hemicellulose content of the pulp. 2.2.6. Characterization of pulp To determine the moisture content the sample was weighed into a dry crucible and dried at 105 °C until constant mass is reached. The dried samples were further used to determine ash content at 525 °C again until constant mass is reached. The extractives were determined as ethanol/toluene-extractives in the ratio of 1:1 (v/v) in a Soxhlet extractor for 6 h. KLASON-lignin content (acid-insoluble lignin) was determined in accordance with TAPPI method T222 om-83 (1999). For the determination of holocellulose 0.3 g of extracted sample was dispersed in 45 mL water. 60 lL acetic acid (100%) and 0.3 g sodium chlorite were added. The mixture was kept at 75 °C in a shaking device for 5 h. Acetic acid and sodium chlorite were added after every hour. Afterwards the holocellulose was separated by filtration using a glass filter (40 lm), washed and dried at 105 °C. The intrinsic viscosity was determined according to ISO 5351-1 (1981). X-ray measurements were carried out in transmission mode by a STOE Stadi P system using Cu-radiation (k = 1.54 Angstroms), Ge (111)-monochromator and an Image Plate Position Sensitive Detector (IP-PSD) in Debye–Scherrer geometry. The data was collected at 0.01° resolution (2H), from 3 to 50° (2H). Peak fitting was conducted using the software Origin. Scanning electron microscopy (SEM) was performed using a Jeol JSM-T330 A, operated at 15 kV. 3. Results and discussion 3.1. Grinding of wheat straw Chopping, milling and thermomechanical pulp (TMP) refining were used to prepare wheat straw for the pulping procedures. Their influence has been studied by means of alkaline pulping using the following pulping parameters: concentration of sodium hydroxide of 3 wt% and pulping temperature of 160 °C for 30 min resulting in an H-factor of 320. Significant differences could be shown between the resulting pulps (Fig. 1). The KLASON-lignin content of all pulps was found to be very low with less than 5%. Especially the TMP refining showed a significant effect. Here a KLASON-lignin content of 0.3% could be achieved, thus a basically lignin free pulp, considering the limitation of the analytical method used. Whether the material is chopped or milled prior to pulping does
Fig. 1. Influence of grinding methods applied prior to alkaline pulping on lignin content and pulp hydrolysis.
not influence the KLASON-lignin content of the pulp significantly. However, it does influence the rate of hydrolysis, as it is considerably higher for pulp from milled wheat straw compared to pulp from chopped wheat straw. This can be explained due to the increased surface of the raw material, which results in a better accessibility of enzymes during hydrolysis. Nevertheless, yet another increase of surface area by refining does not lead to a further improvement of enzymatic hydrolysis. This finding is also supported by a study of Del Río et al. (2012), who did not find an influence of fiber length in the studied range between 0.2 and 4.5 mm. 3.2. Influence of pulping procedures and parameters In order to examine the influence of different pretreatment methods, alkaline and natural pulping as well as autohydrolysis was carried out. Parameters were varied with the aim of studying their effect on holocellulose yield based on the holocellulose content of wheat straw and the KLASON-lignin content found in the pulp. Fig. 2 shows the influence of temperature during (a) alkaline pulping (t = 180 min, c(NaOH) = 3 wt%) and (b) autohydrolysis. For natural pulping all experiments needed to be carried out at 101 °C (boiling point of formic acid). As can be seen, the yield of holocellulose obtained from the alkaline pulping procedure is not significantly influenced by the temperatures used in this study. However, a slight decrease could be observed for the KLASON-lignin content, probably due to cleavage of linkages between the lignin units and the resulting increase of solubility in the media (Gierer, 1985). Therefore, a pulping temperature of 160 °C is proposed, to provide the best holocellulose/lignin ratio. The opposite effect was observed for the hydrothermal treatment shown in Fig. 2b. Here the content of KLASON-lignin in the pulp increases due to the selective hydrolysis of hemicelluloses during the auto hydrolytic process. This effect can also be observed by the decreasing yield of holocellulose which is similar to the results reported previously by (Carvalheiro et al., 2009). However, at a temperature of 200 °C or higher, the yield of holocellulose is in the same range as for alkaline pulping. Fig. 2 further shows the influence of the medium concentration used in (c) alkaline pulping (h = 160 °C, t = 180 min) and (d) in natural pulping (t = 60 min). As can be seen, the yield of holocellulose is in a similar range between 55% and 65% for both kinds of pulping procedures. In contrast, the KLASON-lignin content is significantly lower after alkaline pulping compared to natural pulping as the lowest percentage reached is 0.7% and 10.8%, respectively. In both pulping procedures an increase in media concentration leads to a decreased KLASON-lignin content in the resulting pulp. Particularly
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Fig. 2. Influence of pulping temperature on the yield of holocellulose and KLASON-lignin content of pulp (a) alkaline pulping (b) autohydrolysis, Influence of medium concentration on the yield of holocellulose and KLASON-lignin content of pulp (c) alkaline pulping (d) natural pulping; Influence of pulping duration on the yield of holocellulose and KLASON-lignin content of pulp (e) alkaline pulping (f) natural pulping.
the increase from 1 to 3 wt% in sodium hydroxide concentration shows a significant effect. While a low lignin percentage is desirable for the subsequent saccharification, increasing the medium concentration also lowers the yield of holocellulose. After all, the best results were obtained for: c(NaOH) = 3 wt% and c(HCOOH) = 60 wt% for alkaline and natural pulping, respectively. Finally the effect of pretreatment duration has been examined for (e) alkaline pulping (h = 160 °C, c(NaOH) = 3 wt%) and (f) natural pulping (c(HCOOH) = 60 wt%) shown in Fig. 2. Increasing the pulping duration leads in both cases to a decrease in the yield of holocellulose. As for the alkaline pulping a very low KLASON-lignin content of the pulp is already achievable after only 30 min of pulping and high energy efficiency is desirable, short pulping periods should be used. In contrast, the pulp obtained from natural pulping still has a KLASON-lignin content of 13.7% after a pulping duration of 20 min. Thus the time had to be increased. A pulping duration of 60 min is proposed as an optimum, as a further increase has negative effects on the yield of holocellulose. 3.3. Enzymatic Hydrolysis The efficient saccharification process is one of the most important criteria for bioethanol production. Therefore different parameters, which could play a role during saccharification were studied. In order to study the effect of pulp storage, the pulp obtained from alkaline pulping was hydrolyzed fresh, after it was stored at
18 °C and after it was dried at 105 °C. The resulting glucose yield was 28.8, 37.9 and 78.1 g/100 g holocellulose, respectively. Thus, the highest yield of glucose could be obtained when the pulp was dried prior to hydrolysis. Hornification was previously reported as result of pulp drying, leading to pore shrinkage and hence to a low cellulase accessibility (Luo et al., 2011). However, in our study drying the pulp at 105 °C increased enzymatic hydrolysis significantly. Frozen pulp also leads to an increased glucose yield compared to never dried pulp, as the act of freezing probably leads to a disruption of cell walls and hence to an increased accessibility. As dried pulp is storable and leads to high hydrolyses rates, this procedure was used for all pulps discussed. In the following, the influence of three major parameters: viscosity, lignin content and hemicelluloses content, will be discussed for enzymatic hydrolysis using P. verruculosum. To allow comparison between different pulps, samples have been grouped in low and high viscosity (350–450 ml/g, 650–850 ml/mg); low, high and very high lignin content (65%, 10–15%, P17%) as well as low and high hemicelluloses content (68%, 12–18%). Not all combinations of groups are possible, as natural pulping generally leads to pulp with low intrinsic viscosity, high lignin and low hemicellulose content, alkaline pulping to pulp with high intrinsic viscosity, low lignin and high hemicellulose content and autohydrolysis to pulp with very high lignin and low hemicellulose content. The data of the samples studied in the following are listed in Table 1.
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C. Rossberg et al. / Bioresource Technology 169 (2014) 206–212 Table 1 Summary of samples chosen for investigation on hydrolysis rate. Sample code
Intrinsic viscosity (%)
Hemicellulose (%)
Lignin (%)
WS AP-1 AP-2 AP-3 AP-4 AP-5 AP-6 AP-7 AP-8 AP-9
Crystallinity (%)
Rate of hydrolysis (%)
n.d.
27.3
19.3
20.2
700 700 390 660 810 550 720 740 400
13.9 10.8 6.7 13.0 12.3 18.7 16.1 17.7 6.5
1.8 1.6 0.3 2.3 4.3 13.4 4.3 4.5 2.8
70.0 63.8 82.9 87.5 61.9 62.5 83.4 78.1 94.0
NP-1 NP-2 NP-3 NP-4 NP-5 NP-6 NP-7 NP-8
440 390 400 430 290 390 670 n.d.
5.7 4.0 8.1 7.9 5.6 6.0 6.7 12.0
11.0 10.8 13.8 11.3 13.7 14.6 13.7 17.0
AH-1
n.d.
7.9
32.9
69.2
72.2 61.3
74.0 74.2 63.4 73.2 58.3 77.0 75.3 45.6
58.0
63.3
100.0
WS – wheat straw, AP – alkaline pulping, NP – natural pulping, AH – autohydrolysis, n.d. – not determinable.
The intrinsic viscosity, which reflects the degree of polymerization of cellulose, was determined. As shorter polymer chains lead to lower viscosities, pulps with low intrinsic viscosities could be more accessible for enzymatic degradation. The intrinsic viscosities of two datasets are shown in Fig. 3a. As can be seen, the rate of enzymatic hydrolysis ranges from about 63% to 77% at a viscosity of 400 ml/mg as well as from 62% to 88% at a viscosity of around 700 ml/mg. Hence, no influence of the intrinsic viscosity on the enzymatic hydrolysis was found. Another parameter, which might influence the rate of hydrolysis, is the KLASON-lignin content of the pulp (Chang and Holtzapple, 2000; Chen et al., 2009). Fig. 3b shows two datasets for varying contents of hemicellulose. The intrinsic viscosities are similar within one set but differ between both groups, as there is no dataset with high contents of hemicellulose and low viscosities achievable for the pretreatments studied. The following will be discussed assuming that intrinsic viscosity is not significant for enzymatic hydrolysis as shown above. For the dataset with samples containing 12–18% hemicelluloses, no connection is observed between the lignin content and the rate of hydrolysis ranging between 62% and 88% for similar lignin contents. If the hemicellulose content is lower than
8% a slight trend is visible for four pulp samples. As their lignin content increases from 0.3% to 13.8% the rate of hydrolysis decreases from 94.0 to 63.4%. Thus, assuming that viscosity does not play a significant role, lignin content is important when hemicellulose is almost removed. The pulp obtained by autohydrolysis (circled black in Fig. 3b) however, does not at all fit in the described dataset. Although the lignin content is 32.9%, a complete conversion of holocellulose could be achieved. Hence, the lignin must have changed in a way that its presence does not have a negative effect. More details are given by scanning electron microscopy in Sect. 3.5. Finally, it should be pointed out that, though there is a slight correlation between lignin content and rate of hydrolysis for pulp with low hemicellulose content, pulp from alkaline pretreatment as well as from autohydrolysis can both yield a holocellulose to glucose conversion of more than 90% despite their differing lignin content. Hence, the lignin content is not the bottleneck for enzymatic hydrolysis, if an enzyme complex obtained from P. verruculosum is used. Fig. 3c shows the hydrolysis rate depending on the hemicellulose content of pulp, as it has been studied as a major factor influencing enzymatic hydrolysis (Leu and Zhu, 2013; Yang et al., 2011). Three datasets are shown for low, high and very high lignin
Fig. 3. Effect of (a) intrinsic viscosity (b) KLASON-lignin content and (c) hemicellulose content on hydrolysis rates.
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content. Again the intrinsic viscosity is given, but assumed to be of no significance as shown above. For pulps with a lignin content of below 5% no correlation is found between the hemicellulose content and the rate of hydrolysis. If the lignin content is between 10% and 15% the data points do not scatter as widely and in consideration of an error margin of ±1% for hemicellulose determination a slight correlation could be found for this set. With increasing lignin content the removal of hemicellulose is crucial, as shown for the third dataset (lignin P 17%). Chen et al. (2009) reported, that hemicellulose removal is not necessary for high hydrolysis yield. This statement is especially true for almost delignified samples, but cannot be agreed with for pulps containing higher amounts of lignin or when worked with enzymes obtained by P. verruculosum.
3.4. X-ray analysis The influence of the crystallinity of pulp on the hydrolysis rate has been in discussion, but without leading to clear results (Yang et al., 2011). Therefore samples obtained from alkaline pulping (AP-3, AP-7, AP-9), natural pulping (NP-6) and autohydrolysis (AH-1) were chosen for X-ray measurements (Table 1). Peaks were assigned according to the database PDF-2. Determination of crystallinity was done using peak deconvolution method modeling a broad peak at 21.5° and assigning it to the amorphous contribution (Park et al., 2010). For sample AT-9 it was of particular importance to fit peaks of both, cellulose Ib as well as cellulose II. Two pulps (AP-3 and AP-9), which were obtained from alkaline pulping of TMP-refined wheat straw, were chosen because of their similar KLASON-lignin content, hemicellulose content and intrinsic viscosity. Thus, only the concentration of sodium hydroxide, which was 3 wt% (AP-3) and 9 wt% (AP-9), could be responsible for the differing digestibility, being 82.9% for AP-3 and 94.0% for AP-9. Sample AP-3 shows a typical XRD pattern of cellulose Ib. If the pulping procedure is conducted with c(NaOH) = 9 wt% (AP-9), a conversion to cellulose II is observable, especially due to a new peak at 19.8°. However, the better enzymatic digestibility of AP-9 can also originate from the conversion of cellulose Ib to cellulose II, as the latter consists of an expanded crystal lattice due to the enclosure of sodium hydroxide. For no other sample such an observation was made. Crystallinity was found to be 69.2% for sample AP-3 and 61.3% for AP-9. Thus, as other pulp properties were similar, the lower crystallinity leads to a higher conversion rate. This finding is consistent with results from other studies (Chang and Holtzapple, 2000; Talebnia et al., 2010). To compare the different pretreatments AP-7, NP-6 and AH-1 were chosen, as they were obtained using milled wheat straw as raw material as well as the in Sect. 3.2 described optimum parameters. However, when comparing all studied pretreatments listed in Table 1, it is not possible to establish a relationship between crystallinity and hydrolysis rate. Thus, crystallinity was only shown to have an effect on enzymatic hydrolysis for pulp with a low KLASON-lignin and hemicellulose content and it cannot be used to explain the high hydrolysis rate achieved after autohydrolysis. Therefore, scanning electron microscopy (SEM) was used as another approach to attain more information.
3.5. Scanning electron microscopy SEM studies were undertaken to provide information about the morphology and surface properties of fibers. The pulps with the highest hydrolysis rate of each pretreatment method (AP-9, NP-6 and AH-1) were examined using a magnification of 3500 (image not shown).
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Fibers obtained from alkaline pulping (AP-9) were mostly undamaged and straight. Only furrows were observable along the fiber, probably due to drying effects. In contrast, fibers obtained from natural pulping (NP-6) appeared to be of more irregular length and structure, especially because milled wheat straw was used as raw material. Their surface appeared rough and scaled. When pretreated by autohydrolysis (sample AH-1), the sample again contains pieces and fibers of very irregular length and structure. In addition, the surface shows some special characteristics. Overall it appears smoother than the surface of fiber from natural pulping, but in some areas droplets of approximately 1 lm and smaller are found. According to studies of Donohoe et al. (2008) and Selig et al. (2007) these droplets consist of lignin. As autohydrolysis removes a large percentage of hemicellulose (Carvalheiro et al., 2009) and lignin has lost its supportive function, enzymes might be able to start hydrolysis also from inside the fiber. However, other publications describe a decrease in enzymatic hydrolysis as lignin droplets accumulate (Hansen et al., 2011; Selig et al., 2007) and hence further studies are needed to understand this effect. In conclusion, not the content of lignin is essential for the hydrolysis rate, but its change in morphology or chemistry. 4. Conclusions Alkaline pulping, natural pulping and autohydrolysis were compared regarding the separation of wheat straw, pulp properties and enzymatic hydrolysis. Best results for enzymatic hydrolysis were found for pulp obtained from autohydrolysis. Thus, when working with cellulase from P. verruculosum, the lignin content of pulp is no limiting factor for the hydrolysis rate, but the disruption of cell structure due to the removal of hemicellulose as well as the change in morphology or chemistry of lignin. These findings confirm assumptions made by Carvalheiro et al. (2009) and show the applicability of studies from Leu and Zhu (2013) to evaluate very different pretreatment methods. Acknowledgements Financial support by the German Ministry of Education and Research (grant No. 0315927B) and the European Union within the 6th Framework program (grant No. ERA-IB 100070654) is gratefully acknowledged. Authors thank Daniel Spindler (Saxon Institute for Applied Biotechnology) for his help and advice regarding Natural pulping and enzymatic hydrolysis, Holger Unbehaun (Institute of Wood and Paper Technology, TU Dresden) for preparing TMP refined wheat straw, Bertold Rasche (Department of Chemistry and Food Chemistry, TU Dresden) for conducting X-Ray measurements and Ernst Bäucker (Institute of Forest Utilization and Forest Technology, TU Dresden) for SEM imaging. The Penicillium verruculosum strain was kindly provided by Dr. Gerhard Kerns (Saxon Institute for Applied Biotechnology). References Carvalheiro, F., Duarte, L.C., Gírio, F.M., 2008. Hemicellulose biorefineries: a review on biomass pretreatments. J. Sci. Ind. Res. 67, 849–864. Carvalheiro, F., Silva-Fernandes, T., Duarte, L.C., Gírio, F.M., 2009. Wheat straw autohydrolysis: process optimization and products characterization. Appl. Biochem. Biotechnol. 153, 84–93. http://dx.doi.org/10.1007/s12010-008-8448-0. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting biomass enzymatic reactivity. Appl. Biochem. Biotechnol. 84–86, 5–37. http:// dx.doi.org/10.1385/ABAB:84-86:1-9:5. Chen, M., Zhao, J., Xia, L., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility. Biomass Bioenergy 33, 1381–1385. http://dx.doi.org/10.1016/j.biombioe.2009.05.025.
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Luo, X.L., Zhu, J.Y., Gleisner, R., Zhan, H.Y., 2011. Effects of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses. Cellulose 18, 1055–1062. http://dx.doi.org/10.1007/s10570-011-9541-z. Morozova, V.V., Gusakov, A.V., Andrianov, R.M., Pravilnikov, A.G., Osipov, D.O., Sinitsyn, A.P., 2010. Cellulases of Penicillium verruculosum. Biotechnol. J. 5, 871– 880. http://dx.doi.org/10.1002/biot.201000050. Park, S., Baker, J.O., Himmel, M.E., Parilla, P.A., Johnson, D.K., 2010. Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol. Biofuels 3, 10. http://dx.doi.org/10.1186/ 1754-6834-3-10. Rahikainen, J., Mikander, S., Marjamaa, K., Tamminen, T., Lappas, A., Viikari, L., Kruus, K., 2011. Inhibition of enzymatic hydrolysis by residual lignins from softwood-study of enzyme binding and inactivation on lignin-rich surface. Biotechnol. Bioeng. 108, 2823–2834. http://dx.doi.org/10.1002/bit.23242. Selig, M.J., Viamajala, S., Decker, S.R., Tucker, M.P., Himmel, M.E., Vinzant, T.B., 2007. Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose. Biotechnol. Prog. 23, 1333– 1339. http://dx.doi.org/10.1021/bp0702018. Sewalt, V.J.H., Glasser, W.G., Beauchemin, K.A., 1997. Lignin impact on fiber degradation. 3. Reversal of inhibition of enzymatic hydrolysis by chemical modification of lignin and by additives. J. Agric. Food Chem. 45, 1823–1828. http://dx.doi.org/10.1021/jf9608074. Siegle, S., 2001. Method of producing a pulp from cellulosic material using formic acid and hydrogen peroxide. Patent US6183597. Solomon, B.D., Barnes, J.R., Halvorsen, K.E., 2007. Grain and cellulosic ethanol: history, economics, and energy policy. Biomass Bioenergy 31, 416–425. http:// dx.doi.org/10.1016/j.biombioe.2007.01.023. Talebnia, F., Karakashev, D., Angelidaki, I., 2010. Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 101, 4744–4753. http://dx.doi.org/10.1016/ j.biortech.2009.11.080. TAPPI method T222 om-83, 1999. Acid-insoluble lignin in wood and pulp. In: Test Methods 1998–1999. TAPPI Press. Wang, K., Sun, R., 2010. Biorefinery Straw for Bioethanol. In: Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels Chemistry, Extractives, Lignins, Hemicelluloses and Cellulose. Elsevier, Amsterdam, Boston. Wang, K., Xiong, X., Chen, J., Chen, L., Su, X., Liu, Y., 2012. Comparison of gamma irradiation and steam explosion pretreatment for ethanol production from agricultural residues. Biomass Bioenergy 46, 301–308. http://dx.doi.org/ 10.1016/j.biombioe.2012.08.013. Yang, B., Dai, Z., Ding, S.-Y., Wyman, C.E., 2011. Enzymatic hydrolysis of cellulosic biomass. Biofuels 2, 421–450. http://dx.doi.org/10.4155/bfs.11.116. Zeller, V., Weiser, C., Hennenberg, K., Reinicke, F., Thrän, D., Vetter, A., Wagner, B., 2011. Basisinformation fuer eine nachhaltige Nutzung landwirtschaftlicher Reststoffe zur Bioenergiebereitstellung. In: Energetische Biomassenutzung.
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Pulp properties resulting from different pretreatments of wheat straw and their influence on enzymatic hydrolysis rate Christine Rossberg a,⇑, Doreen Steffien b, Martina Bremer a, Swetlana Koenig c, Florbela Carvalheiro d, Luís C. Duarte d, Patrícia Moniz d, Max Hoernicke c, Martin Bertau b, Steffen Fischer a a
Institute of Plant and Wood Chemistry, Dresden University of Technology, Pienner Str. 19, 01723 Tharandt, Germany Institute of Chemical Technology, Freiberg University of Mining and Technology, Leipziger Str. 29, 09595 Freiberg, Germany Saxon Institute for Applied Biotechnology, Leipzig University, Permoserstr. 15, 04318 Leipzig, Germany d Laboratório Nacional de Energia e Geologia, I.P., Unidade de Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal b c
h i g h l i g h t s Process parameters for grinding and pulp storage were optimized. Comparison of pulp digestibility after three very different pretreatments. Pulp hydrolysis using the novel Penicillium verruculosum cellulase complex. Lignin removal is not crucial for enzymatic hydrolysis. Autohydrolysis pretreatment enables complete conversion of cellulose to glucose.
a r t i c l e
i n f o
Article history: Received 8 April 2014 Received in revised form 23 June 2014 Accepted 26 June 2014 Available online 3 July 2014 Keywords: Alkaline pulping Natural pulping Autohydrolysis Wheat straw Enzymatic hydrolysis
a b s t r a c t Wheat straw was subjected to three different processes prior to saccharification, namely alkaline pulping, natural pulping and autohydrolysis, in order to study their effect on the rate of enzymatic hydrolysis. Parameters like medium concentration, temperature and time have been varied in order to optimize each method. Milling the raw material to a length of 4 mm beforehand showed the best cost–value-ratio compared to other grinding methods studied. Before saccharification the pulp can be stored in dried form, leading to a high yield of glucose. Furthermore the relation of pulp properties (i.e. intrinsic viscosity, KLASON-lignin and hemicelluloses content, crystallinity, morphology) to cellulose hydrolysis is discussed. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Wheat straw is a fast-growing and abundant agricultural by-product. For instance, 8–13 million tons per year are readily available in Germany without risking humus reduction (Zeller et al., 2011). As it has no application in the food industry, it could serve as an excellent starting material for the production of cellulose, basic chemicals, lignin and bioethanol in the biorefinery framework. Especially the latter attains increasing importance, since the increasing level of greenhouse gases, the depletion of fossil fuels and the unstable oil market lead to a general interest
⇑ Corresponding author. Tel.: +49 (0) 35203 38 31877. E-mail address: [email protected] (C. Rossberg). http://dx.doi.org/10.1016/j.biortech.2014.06.100 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
in alternative transportation fuels (Solomon et al., 2007; Talebnia et al., 2010). For bioethanol production using lignocellulosic material at least four operations including pretreatment, hydrolysis, fermentation and distillation are necessary (Talebnia et al., 2010). Different pretreatment procedures have been developed in order to increase the accessibility of cellulose by removing hemicelluloses, by breaking down the lignin structure and disrupting the crystalline structure of cellulose (Wang et al., 2012). Furthermore, the removal of lignin can be of advantage, as it has been reported that high lignin contents in the substrates may exert inhibitory effect on the enzymes used for hydrolysis (Rahikainen et al., 2011; Sewalt et al., 1997). Thus different methods have been investigated in order to prepare the carbohydrate fraction for enzymatic hydrolysis, which can be classified into physical (e.g. milling), physico-chemical
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(e.g. autohydrolysis, steam explosion), biological (e.g. white rot-fungi) and chemical processes (e.g. organosolv, alkaline pulping) (Kumar et al., 2009; Talebnia et al., 2010). This study focuses on chemical and physico-chemical treatments. One of the advantages of using annual plants is that traditional pulping methods like Kraft-pulping are not necessary. Thus sulfurfree and hence more environment-friendly procedures can be applied. That is why several studies have been engaged with less severe pulping methods like alkaline pulping using sodium hydroxide or calcium hydroxide (Epelde et al., 1998; García et al., 2009; Khristova et al., 1998). The alkaline condition leads to the dissociation of phenolic hydroxyl groups in the lignin and increases its solubility. The latter is furthermore enhanced by lignin fragmentation and release of methanol, which, though released in small quantities, can work as an additional solvent (Epelde et al., 1998; Gierer, 1985). Due to this method a very good delignification of the pulp can be achieved and the accessibility of cellulose is increased due to swelling and removal of hemicelluloses. On the other hand fermentable sugars are lost and, because of the high silica content of annual plants, irrecoverable salts are formed and incorporated into the biomass (Kumar et al., 2009). However, Lei et al. (2010) showed that the silica content can be effectively lowered by a hot-water pretreatment. Thus, with further studies the process and the recovery of chemicals could still be improved. In contrast to alkaline pulping the use of organic solvents for pulping has the advantage of easily recovering the initial chemicals. Furthermore, very pure lignin can be obtained, so that a complete usage of the lignocellulosic material is possible. Among the organic solvents two main groups have been in the focus of study: organic acids (e.g. formic acid, acetic acid) and alcohols (e.g. ethanol). The so called NATURAL PULPING process, which is similar to the MILOX process, uses formic acid and hydrogen peroxide to generate peroxyformic acid in situ (Siegle, 2001). This leads to an oxidation and depolymerization of lignin and thus enhances its solubility (González et al., 2010; Ligero et al., 2010). However, the corrosive character of peroxyformic acid makes it necessary to work with glass or enameled steel. Compared to the pretreatments already described, autohydrolysis is the most environmental friendly procedure, as the biomass is treated only with hot water or steam at temperatures usually in the range of 150–230 °C for only short residence times (Carvalheiro et al., 2008; Garrote et al., 1999; Wang and Sun, 2010). Autohydrolysis leads to specific hemicellulose removal resulting in a cellulose and lignin rich solid fraction. Due to cell penetration and breakdown of the lignocellulosic structure the cellulose accessibility is increased. This process also has the advantage to produce a hemicellulosic liquid stream that can also be used for fermentation purposes or for recovering value-added compounds. However, a careful optimization of the operational conditions is important in order to minimize the formation of degradation compounds like furfural and hydroxymethylfurfural, which might act inhibitory on microorganisms used later on in the process (Gírio et al., 2010). After the pretreatment procedure the lignocellulosic material is prepared for the hydrolysis step. Two main procedures are used to hydrolyze cellulose: acid and enzymatic treatments. Unlike acid hydrolysis, enzymatic hydrolysis is highly specific and occurs under mild reaction conditions. Thus, it is considered to be more promising for the inclusion into a biorefinery concept (Wang and Sun, 2010). For this application a fungal cellulase complex is used. It includes endo-glucanase, exo-glucanase and b-glucosidase activities. They are known to be influenced by the pretreatment of cellulosic material as well as the hydrolyzing conditions. Most cellulase enzyme complexes show an optimum activity at h = 45–55 °C and pH = 4–5 (Talebnia et al., 2010). For this investigation an alternative enzyme complex obtained from Penicillium
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verruculosum strain was used. In comparison to the worldwide employed complex from Trichoderma reesei, P. verruculosum cellulases are characterized by higher content of b-glucosidase and higher resistance towards ethanol (Morozova et al., 2010). The objective of this work was to investigate the influence of pretreatment processes, i.e. alkaline pulping, natural pulping and autohydrolysis on the enzymatic hydrolysis of wheat straw pulp. Pulping parameters have been varied to optimize each method and to examine their impact on hydrolysis rate using an enzymatic complex of P. verruculosum. Furthermore the effect of a mechanical treatment of the wheat straw prior to pulping has also been investigated.
2. Experimental 2.1. Raw material The wheat straw used in this study was obtained from Agrargenossenschaft Rossau eG, Germany. It was prepared in three different ways: chopped to a length of 4 to 10 cm, milled to pass a 4 mm mesh and as thermo mechanical pulp (TMP) using a 12-zoll-refiner and a disk gap of 0.15 mm. The composition of the wheat straw is as follows: cellulose 44.2%, hemicellulose 27.3%, lignin 19.3%, extract 3.9% and ash 3.0% on a dry weight basis. With the parameters used, TMP refining was not found to have a significant effect on the chemical composition of wheat straw.
2.2. Methods 2.2.1. Alkaline Pulping Alkaline pulping was carried out in a 2 L digester. The cooking conditions were: liquid–solid ratio 6.2 mL/g, concentration of sodium-hydroxide (1, 3, 6, 9) wt%, maximum temperature (120, 140, 160) °C, time at maximum temperature (30, 90, 180) min. The pulp was separated from the black liquor over a Büchner funnel, washed with sodium hydroxide solution (0.1 M) and afterwards with deionized water. Enzymatic hydrolysis was conducted directly or after storing the pulp at 18 °C or after drying it at 105 °C to analyze the influence of different storage conditions.
2.2.2. Natural Pulping Natural pulping was carried out according to Siegle (2001) using a 3 L round bottom flask with reflux condenser. The following parameters were used: liquid–solid ratio 27 mL/g, concentration of formic acid (30, 50, 60, 70, 80) wt%, maximum temperature 101 °C, time at maximum temperature (20, 40, 60) min. After cooling down, the pulp was separated, washed and dried.
2.2.3. Autohydrolysis The hydrothermal treatments (autohydrolysis) were performed in a stainless steel reactor with a total volume of 2 L. The reactor was fitted with two four-blade turbine impellers, heated by an external mantle, and cooled by cold water circulating through an internal stainless steel loop. The wheat straw was mixed with water in the reactor in order to obtain a liquid-to-solid ratio of 10 mL/g. A maximum temperature of (180, 190, 200, 210, 220) °C at an average rate of temperature increase (from 100 °C) of approximately 4.7 °C/min was applied. When the desired temperature was reached, the reactor was cooled down and the liquid and solid phases were recovered by pressing in a hydraulic press up to 200 bar. The solid phase was washed with water, pressed again and dried.
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2.2.4. Enzymatic Hydrolysis For pulp hydrolysis 0.5 g biomass on dry weight base were incubated after adding 23.5 mL of citrate buffer solution (0.05 M, pH = 5) and an enzyme complex from Penicillium verruculosumM28-10b (DSM24317) (cellulase: 100 FPU/g dry feedstock and b-glucosidase: 600 IU/g dry feedstock). Incubation was carried out at 50 °C for 24 h in a laboratory shaking device (150 rpm). In terms of relevance for the SSF process, the hydrolysis time of 24 h was examined. 2.2.5. Analysis of hydrolyzate After 24 h of enzymatic hydrolysis 1 mL sample was centrifuged to remove insoluble material. 500 lL supernatant, 200 lL maltitol (20 g/L) and 500 lL acetone were mixed and filtered through 0.2 lm PVDF filter into vials. Soluble sugars were analyzed by HPLC using a NUCLEODURÒ100–5 NH2-RP column (Macherey Nagel). The analysis conditions were: mobile phase ACN:H2O 79:21 v/v, injection volume 5 lL, column temperature 30 °C, flow rate: 1 mL/min. The detector (ELSD) parameters were: evaporation temperature 94 °C, nebulizer temperature 60 °C, carrier flow (N2) 1 slm. The rate of hydrolysis was calculated as g glucose per 100 g holocellulose, which was determined as described in Sect. 2.2.6 representing the cellulose as well as the hemicellulose content of the pulp. 2.2.6. Characterization of pulp To determine the moisture content the sample was weighed into a dry crucible and dried at 105 °C until constant mass is reached. The dried samples were further used to determine ash content at 525 °C again until constant mass is reached. The extractives were determined as ethanol/toluene-extractives in the ratio of 1:1 (v/v) in a Soxhlet extractor for 6 h. KLASON-lignin content (acid-insoluble lignin) was determined in accordance with TAPPI method T222 om-83 (1999). For the determination of holocellulose 0.3 g of extracted sample was dispersed in 45 mL water. 60 lL acetic acid (100%) and 0.3 g sodium chlorite were added. The mixture was kept at 75 °C in a shaking device for 5 h. Acetic acid and sodium chlorite were added after every hour. Afterwards the holocellulose was separated by filtration using a glass filter (40 lm), washed and dried at 105 °C. The intrinsic viscosity was determined according to ISO 5351-1 (1981). X-ray measurements were carried out in transmission mode by a STOE Stadi P system using Cu-radiation (k = 1.54 Angstroms), Ge (111)-monochromator and an Image Plate Position Sensitive Detector (IP-PSD) in Debye–Scherrer geometry. The data was collected at 0.01° resolution (2H), from 3 to 50° (2H). Peak fitting was conducted using the software Origin. Scanning electron microscopy (SEM) was performed using a Jeol JSM-T330 A, operated at 15 kV. 3. Results and discussion 3.1. Grinding of wheat straw Chopping, milling and thermomechanical pulp (TMP) refining were used to prepare wheat straw for the pulping procedures. Their influence has been studied by means of alkaline pulping using the following pulping parameters: concentration of sodium hydroxide of 3 wt% and pulping temperature of 160 °C for 30 min resulting in an H-factor of 320. Significant differences could be shown between the resulting pulps (Fig. 1). The KLASON-lignin content of all pulps was found to be very low with less than 5%. Especially the TMP refining showed a significant effect. Here a KLASON-lignin content of 0.3% could be achieved, thus a basically lignin free pulp, considering the limitation of the analytical method used. Whether the material is chopped or milled prior to pulping does
Fig. 1. Influence of grinding methods applied prior to alkaline pulping on lignin content and pulp hydrolysis.
not influence the KLASON-lignin content of the pulp significantly. However, it does influence the rate of hydrolysis, as it is considerably higher for pulp from milled wheat straw compared to pulp from chopped wheat straw. This can be explained due to the increased surface of the raw material, which results in a better accessibility of enzymes during hydrolysis. Nevertheless, yet another increase of surface area by refining does not lead to a further improvement of enzymatic hydrolysis. This finding is also supported by a study of Del Río et al. (2012), who did not find an influence of fiber length in the studied range between 0.2 and 4.5 mm. 3.2. Influence of pulping procedures and parameters In order to examine the influence of different pretreatment methods, alkaline and natural pulping as well as autohydrolysis was carried out. Parameters were varied with the aim of studying their effect on holocellulose yield based on the holocellulose content of wheat straw and the KLASON-lignin content found in the pulp. Fig. 2 shows the influence of temperature during (a) alkaline pulping (t = 180 min, c(NaOH) = 3 wt%) and (b) autohydrolysis. For natural pulping all experiments needed to be carried out at 101 °C (boiling point of formic acid). As can be seen, the yield of holocellulose obtained from the alkaline pulping procedure is not significantly influenced by the temperatures used in this study. However, a slight decrease could be observed for the KLASON-lignin content, probably due to cleavage of linkages between the lignin units and the resulting increase of solubility in the media (Gierer, 1985). Therefore, a pulping temperature of 160 °C is proposed, to provide the best holocellulose/lignin ratio. The opposite effect was observed for the hydrothermal treatment shown in Fig. 2b. Here the content of KLASON-lignin in the pulp increases due to the selective hydrolysis of hemicelluloses during the auto hydrolytic process. This effect can also be observed by the decreasing yield of holocellulose which is similar to the results reported previously by (Carvalheiro et al., 2009). However, at a temperature of 200 °C or higher, the yield of holocellulose is in the same range as for alkaline pulping. Fig. 2 further shows the influence of the medium concentration used in (c) alkaline pulping (h = 160 °C, t = 180 min) and (d) in natural pulping (t = 60 min). As can be seen, the yield of holocellulose is in a similar range between 55% and 65% for both kinds of pulping procedures. In contrast, the KLASON-lignin content is significantly lower after alkaline pulping compared to natural pulping as the lowest percentage reached is 0.7% and 10.8%, respectively. In both pulping procedures an increase in media concentration leads to a decreased KLASON-lignin content in the resulting pulp. Particularly
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Fig. 2. Influence of pulping temperature on the yield of holocellulose and KLASON-lignin content of pulp (a) alkaline pulping (b) autohydrolysis, Influence of medium concentration on the yield of holocellulose and KLASON-lignin content of pulp (c) alkaline pulping (d) natural pulping; Influence of pulping duration on the yield of holocellulose and KLASON-lignin content of pulp (e) alkaline pulping (f) natural pulping.
the increase from 1 to 3 wt% in sodium hydroxide concentration shows a significant effect. While a low lignin percentage is desirable for the subsequent saccharification, increasing the medium concentration also lowers the yield of holocellulose. After all, the best results were obtained for: c(NaOH) = 3 wt% and c(HCOOH) = 60 wt% for alkaline and natural pulping, respectively. Finally the effect of pretreatment duration has been examined for (e) alkaline pulping (h = 160 °C, c(NaOH) = 3 wt%) and (f) natural pulping (c(HCOOH) = 60 wt%) shown in Fig. 2. Increasing the pulping duration leads in both cases to a decrease in the yield of holocellulose. As for the alkaline pulping a very low KLASON-lignin content of the pulp is already achievable after only 30 min of pulping and high energy efficiency is desirable, short pulping periods should be used. In contrast, the pulp obtained from natural pulping still has a KLASON-lignin content of 13.7% after a pulping duration of 20 min. Thus the time had to be increased. A pulping duration of 60 min is proposed as an optimum, as a further increase has negative effects on the yield of holocellulose. 3.3. Enzymatic Hydrolysis The efficient saccharification process is one of the most important criteria for bioethanol production. Therefore different parameters, which could play a role during saccharification were studied. In order to study the effect of pulp storage, the pulp obtained from alkaline pulping was hydrolyzed fresh, after it was stored at
18 °C and after it was dried at 105 °C. The resulting glucose yield was 28.8, 37.9 and 78.1 g/100 g holocellulose, respectively. Thus, the highest yield of glucose could be obtained when the pulp was dried prior to hydrolysis. Hornification was previously reported as result of pulp drying, leading to pore shrinkage and hence to a low cellulase accessibility (Luo et al., 2011). However, in our study drying the pulp at 105 °C increased enzymatic hydrolysis significantly. Frozen pulp also leads to an increased glucose yield compared to never dried pulp, as the act of freezing probably leads to a disruption of cell walls and hence to an increased accessibility. As dried pulp is storable and leads to high hydrolyses rates, this procedure was used for all pulps discussed. In the following, the influence of three major parameters: viscosity, lignin content and hemicelluloses content, will be discussed for enzymatic hydrolysis using P. verruculosum. To allow comparison between different pulps, samples have been grouped in low and high viscosity (350–450 ml/g, 650–850 ml/mg); low, high and very high lignin content (65%, 10–15%, P17%) as well as low and high hemicelluloses content (68%, 12–18%). Not all combinations of groups are possible, as natural pulping generally leads to pulp with low intrinsic viscosity, high lignin and low hemicellulose content, alkaline pulping to pulp with high intrinsic viscosity, low lignin and high hemicellulose content and autohydrolysis to pulp with very high lignin and low hemicellulose content. The data of the samples studied in the following are listed in Table 1.
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C. Rossberg et al. / Bioresource Technology 169 (2014) 206–212 Table 1 Summary of samples chosen for investigation on hydrolysis rate. Sample code
Intrinsic viscosity (%)
Hemicellulose (%)
Lignin (%)
WS AP-1 AP-2 AP-3 AP-4 AP-5 AP-6 AP-7 AP-8 AP-9
Crystallinity (%)
Rate of hydrolysis (%)
n.d.
27.3
19.3
20.2
700 700 390 660 810 550 720 740 400
13.9 10.8 6.7 13.0 12.3 18.7 16.1 17.7 6.5
1.8 1.6 0.3 2.3 4.3 13.4 4.3 4.5 2.8
70.0 63.8 82.9 87.5 61.9 62.5 83.4 78.1 94.0
NP-1 NP-2 NP-3 NP-4 NP-5 NP-6 NP-7 NP-8
440 390 400 430 290 390 670 n.d.
5.7 4.0 8.1 7.9 5.6 6.0 6.7 12.0
11.0 10.8 13.8 11.3 13.7 14.6 13.7 17.0
AH-1
n.d.
7.9
32.9
69.2
72.2 61.3
74.0 74.2 63.4 73.2 58.3 77.0 75.3 45.6
58.0
63.3
100.0
WS – wheat straw, AP – alkaline pulping, NP – natural pulping, AH – autohydrolysis, n.d. – not determinable.
The intrinsic viscosity, which reflects the degree of polymerization of cellulose, was determined. As shorter polymer chains lead to lower viscosities, pulps with low intrinsic viscosities could be more accessible for enzymatic degradation. The intrinsic viscosities of two datasets are shown in Fig. 3a. As can be seen, the rate of enzymatic hydrolysis ranges from about 63% to 77% at a viscosity of 400 ml/mg as well as from 62% to 88% at a viscosity of around 700 ml/mg. Hence, no influence of the intrinsic viscosity on the enzymatic hydrolysis was found. Another parameter, which might influence the rate of hydrolysis, is the KLASON-lignin content of the pulp (Chang and Holtzapple, 2000; Chen et al., 2009). Fig. 3b shows two datasets for varying contents of hemicellulose. The intrinsic viscosities are similar within one set but differ between both groups, as there is no dataset with high contents of hemicellulose and low viscosities achievable for the pretreatments studied. The following will be discussed assuming that intrinsic viscosity is not significant for enzymatic hydrolysis as shown above. For the dataset with samples containing 12–18% hemicelluloses, no connection is observed between the lignin content and the rate of hydrolysis ranging between 62% and 88% for similar lignin contents. If the hemicellulose content is lower than
8% a slight trend is visible for four pulp samples. As their lignin content increases from 0.3% to 13.8% the rate of hydrolysis decreases from 94.0 to 63.4%. Thus, assuming that viscosity does not play a significant role, lignin content is important when hemicellulose is almost removed. The pulp obtained by autohydrolysis (circled black in Fig. 3b) however, does not at all fit in the described dataset. Although the lignin content is 32.9%, a complete conversion of holocellulose could be achieved. Hence, the lignin must have changed in a way that its presence does not have a negative effect. More details are given by scanning electron microscopy in Sect. 3.5. Finally, it should be pointed out that, though there is a slight correlation between lignin content and rate of hydrolysis for pulp with low hemicellulose content, pulp from alkaline pretreatment as well as from autohydrolysis can both yield a holocellulose to glucose conversion of more than 90% despite their differing lignin content. Hence, the lignin content is not the bottleneck for enzymatic hydrolysis, if an enzyme complex obtained from P. verruculosum is used. Fig. 3c shows the hydrolysis rate depending on the hemicellulose content of pulp, as it has been studied as a major factor influencing enzymatic hydrolysis (Leu and Zhu, 2013; Yang et al., 2011). Three datasets are shown for low, high and very high lignin
Fig. 3. Effect of (a) intrinsic viscosity (b) KLASON-lignin content and (c) hemicellulose content on hydrolysis rates.
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content. Again the intrinsic viscosity is given, but assumed to be of no significance as shown above. For pulps with a lignin content of below 5% no correlation is found between the hemicellulose content and the rate of hydrolysis. If the lignin content is between 10% and 15% the data points do not scatter as widely and in consideration of an error margin of ±1% for hemicellulose determination a slight correlation could be found for this set. With increasing lignin content the removal of hemicellulose is crucial, as shown for the third dataset (lignin P 17%). Chen et al. (2009) reported, that hemicellulose removal is not necessary for high hydrolysis yield. This statement is especially true for almost delignified samples, but cannot be agreed with for pulps containing higher amounts of lignin or when worked with enzymes obtained by P. verruculosum.
3.4. X-ray analysis The influence of the crystallinity of pulp on the hydrolysis rate has been in discussion, but without leading to clear results (Yang et al., 2011). Therefore samples obtained from alkaline pulping (AP-3, AP-7, AP-9), natural pulping (NP-6) and autohydrolysis (AH-1) were chosen for X-ray measurements (Table 1). Peaks were assigned according to the database PDF-2. Determination of crystallinity was done using peak deconvolution method modeling a broad peak at 21.5° and assigning it to the amorphous contribution (Park et al., 2010). For sample AT-9 it was of particular importance to fit peaks of both, cellulose Ib as well as cellulose II. Two pulps (AP-3 and AP-9), which were obtained from alkaline pulping of TMP-refined wheat straw, were chosen because of their similar KLASON-lignin content, hemicellulose content and intrinsic viscosity. Thus, only the concentration of sodium hydroxide, which was 3 wt% (AP-3) and 9 wt% (AP-9), could be responsible for the differing digestibility, being 82.9% for AP-3 and 94.0% for AP-9. Sample AP-3 shows a typical XRD pattern of cellulose Ib. If the pulping procedure is conducted with c(NaOH) = 9 wt% (AP-9), a conversion to cellulose II is observable, especially due to a new peak at 19.8°. However, the better enzymatic digestibility of AP-9 can also originate from the conversion of cellulose Ib to cellulose II, as the latter consists of an expanded crystal lattice due to the enclosure of sodium hydroxide. For no other sample such an observation was made. Crystallinity was found to be 69.2% for sample AP-3 and 61.3% for AP-9. Thus, as other pulp properties were similar, the lower crystallinity leads to a higher conversion rate. This finding is consistent with results from other studies (Chang and Holtzapple, 2000; Talebnia et al., 2010). To compare the different pretreatments AP-7, NP-6 and AH-1 were chosen, as they were obtained using milled wheat straw as raw material as well as the in Sect. 3.2 described optimum parameters. However, when comparing all studied pretreatments listed in Table 1, it is not possible to establish a relationship between crystallinity and hydrolysis rate. Thus, crystallinity was only shown to have an effect on enzymatic hydrolysis for pulp with a low KLASON-lignin and hemicellulose content and it cannot be used to explain the high hydrolysis rate achieved after autohydrolysis. Therefore, scanning electron microscopy (SEM) was used as another approach to attain more information.
3.5. Scanning electron microscopy SEM studies were undertaken to provide information about the morphology and surface properties of fibers. The pulps with the highest hydrolysis rate of each pretreatment method (AP-9, NP-6 and AH-1) were examined using a magnification of 3500 (image not shown).
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Fibers obtained from alkaline pulping (AP-9) were mostly undamaged and straight. Only furrows were observable along the fiber, probably due to drying effects. In contrast, fibers obtained from natural pulping (NP-6) appeared to be of more irregular length and structure, especially because milled wheat straw was used as raw material. Their surface appeared rough and scaled. When pretreated by autohydrolysis (sample AH-1), the sample again contains pieces and fibers of very irregular length and structure. In addition, the surface shows some special characteristics. Overall it appears smoother than the surface of fiber from natural pulping, but in some areas droplets of approximately 1 lm and smaller are found. According to studies of Donohoe et al. (2008) and Selig et al. (2007) these droplets consist of lignin. As autohydrolysis removes a large percentage of hemicellulose (Carvalheiro et al., 2009) and lignin has lost its supportive function, enzymes might be able to start hydrolysis also from inside the fiber. However, other publications describe a decrease in enzymatic hydrolysis as lignin droplets accumulate (Hansen et al., 2011; Selig et al., 2007) and hence further studies are needed to understand this effect. In conclusion, not the content of lignin is essential for the hydrolysis rate, but its change in morphology or chemistry. 4. Conclusions Alkaline pulping, natural pulping and autohydrolysis were compared regarding the separation of wheat straw, pulp properties and enzymatic hydrolysis. Best results for enzymatic hydrolysis were found for pulp obtained from autohydrolysis. Thus, when working with cellulase from P. verruculosum, the lignin content of pulp is no limiting factor for the hydrolysis rate, but the disruption of cell structure due to the removal of hemicellulose as well as the change in morphology or chemistry of lignin. These findings confirm assumptions made by Carvalheiro et al. (2009) and show the applicability of studies from Leu and Zhu (2013) to evaluate very different pretreatment methods. Acknowledgements Financial support by the German Ministry of Education and Research (grant No. 0315927B) and the European Union within the 6th Framework program (grant No. ERA-IB 100070654) is gratefully acknowledged. Authors thank Daniel Spindler (Saxon Institute for Applied Biotechnology) for his help and advice regarding Natural pulping and enzymatic hydrolysis, Holger Unbehaun (Institute of Wood and Paper Technology, TU Dresden) for preparing TMP refined wheat straw, Bertold Rasche (Department of Chemistry and Food Chemistry, TU Dresden) for conducting X-Ray measurements and Ernst Bäucker (Institute of Forest Utilization and Forest Technology, TU Dresden) for SEM imaging. The Penicillium verruculosum strain was kindly provided by Dr. Gerhard Kerns (Saxon Institute for Applied Biotechnology). References Carvalheiro, F., Duarte, L.C., Gírio, F.M., 2008. Hemicellulose biorefineries: a review on biomass pretreatments. J. Sci. Ind. Res. 67, 849–864. Carvalheiro, F., Silva-Fernandes, T., Duarte, L.C., Gírio, F.M., 2009. Wheat straw autohydrolysis: process optimization and products characterization. Appl. Biochem. Biotechnol. 153, 84–93. http://dx.doi.org/10.1007/s12010-008-8448-0. Chang, V.S., Holtzapple, M.T., 2000. Fundamental factors affecting biomass enzymatic reactivity. Appl. Biochem. Biotechnol. 84–86, 5–37. http:// dx.doi.org/10.1385/ABAB:84-86:1-9:5. Chen, M., Zhao, J., Xia, L., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility. Biomass Bioenergy 33, 1381–1385. http://dx.doi.org/10.1016/j.biombioe.2009.05.025.
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