Appl Biochem Biotechnol DOI 10.1007/s12010-014-1243-1
Enhanced Enzymatic Hydrolysis of Waste Paper for Ethanol Production Using Separate Saccharification and Fermentation Mohamed Guerfali & Adel Saidi & Ali Gargouri & Hafedh Belghith
Received: 22 April 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum-based liquid fuels. In this study, the feasibility of ethanol production from waste paper using the separate hydrolysis and fermentation (SHF) was investigated. Two types of waste paper materials, newspaper and office paper, were evaluated for their potential to be used as a renewable feedstock for the production of fermentable sugars via enzymatic hydrolysis of their cellulose fractions. Hydrolysis step was conducted with a mixture of cellulolytic enzymes produced locally by Trichoderma reesei Rut-C30 (cellulaseoverproducing mutant) and Aspergillus niger F38 cultures. Surfactant pretreatment effect on waste paper enzymatic digestibility was studied and Triton X-100 at 0.5 % (w w−1) has improved the digestibility of newspaper about 45 %. The effects of three factors (dry matter quantity, phosphoric acid pretreatment and hydrolysis time) on the extent of saccharification were also assessed and quantified by using a methodical approach based on response surface methodology. Under optimal hydrolysis conditions, maximum degrees of saccharification of newspaper and office paper were 67 and 92 %, respectively. Sugars released from waste paper were subsequently converted into ethanol (0.38 g ethanol g−1 sugar) with Saccharomyces cerevisiae CTM-30101. Keywords Bioethanol . Waste paper . Saccharification . Cellulases . Response surface methodology
Introduction Fuel ethanol production from renewable resources such as plant biomass is one of the most promising alternatives to conventional petroleum-based fuels. Currently, most bioethanol produced in the world is derived from starch and molasses. However, these sources are not sufficiently supplied for biofuel production due to the role they play in human and livestock M. Guerfali (*) : A. Saidi : A. Gargouri : H. Belghith Laboratory of Biomass Valorisation and Protein Production in Eukaryotes, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3038 Sfax, Tunisia e-mail: [email protected]
Appl Biochem Biotechnol
consumption. Therefore, the development of second-generation bioethanol from lignocellulosic biomass serves many advantages from both economic and environmental point of views [1]. Lignocellulosic biomass typically contains 50–80 % (dry basis) carbohydrates that are polymers of C5 and C6 sugar units. Most carbohydrates can be processed either chemically or biologically to yield biofuels such as ethanol [2]. One of the main economic factors in ethanol process is the cost of raw material which can be substantially reduced by utilization of industrial lignocellulosic wastes. However, due to structural complexity, pretreatment is required to disrupt the recalcitrant structure of lignocellulosic materials and to increase the accessibility of hydrolytic enzymes to the carbohydrates polymers. Until now, several types of pretreatment are used, including steam explosion, acid, alkali, organic solvent, alkaline hydrogen peroxide, ammonia and liquid hot water treatments [3]. The choice of pretreatment process is possibly the most important factor in the economics of ethanol production process because it influences waste treatment, cellulose conversion rates and mainly hydrolytic enzymes performance [4]. Employment of enzymes for lignocellulose hydrolysis is considered, prospectively, as the most viable strategy that offer advantages over chemical conversion routes of higher yields, minimal by-product formation, low energy requirements, mild operating conditions and environmentally friendly processing [5]. Enzymatic saccharification of lignocellulosic materials to soluble products can be carried out by a complex mixture of enzymes, amongst which cellulases (endoglucanase, exoglucanase and β-glucosidase) and hemicellulases (xylanase) acting in perfect synergy. Endoglucanases randomly attack cellulose chains and release cello-oligosaccharides, the ends of which are processed by exoglucanases to cellobiose, which is therefore converted by β-glucosidase to glucose. On the other hand, xylanases hydrolyze xylan (major component of hemicellulose) and facilitate the removal of lignin which blocks the action of cellulases [6]. Complete cellulase enzyme systems can be produced by a large variety of microorganisms. Amongst the best characterized and most widely studied ones are the inducible cellulases of the filamentous fungus Trichoderma reesei [7]. These cellulases have a high industrial interest as they are widely used in the food and feed industries and in the textile and pulp paper industries [8]. Waste paper is particularly attractive as feedstock for bioethanol production because it is readily available. Despite the growing awareness for recycling, the majority of waste paper will still end up in landfills. There is, therefore, an almost constant supply of large quantities of waste paper in many parts of the world. Previous researches have demonstrated the potential of bioethanol production from waste paper using a variety of process designs [9–11]. The cellulosic sugars obtained through acid and enzymatic hydrolysis can efficiently be used for ethanol fermentation either by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). However, the temperature optima for the yeast growth and hydrolytic enzymes differ, which means that the conditions used in SSF cannot be optimal for both (enzymes and yeast) and might result in lower efficiency and lower yield [12]. Hence, for better efficiency of ethanol production, the SHF approach was preferred in the present work. Enzymatic hydrolysis of lignocellulosic material is the critical step for sugar production as it can be affected by several factors at the same time such as enzyme loading, hydrolysis time, substrate concentration, cellulose crystallinity, and by lignin and hemicellulose content [13]. The conventional method (changing one independent variable while maintaining all of the others at a fixed level) to obtain the optimization of enzymatic hydrolysis is extremely time consuming.
Appl Biochem Biotechnol
Response surface methodology (RSM) is a time- and labour-saving method, which reveals the interaction between the factors and seeks the optimum levels [14]. In the present work, we tested the combinatorial effect of the production of ethanol from waste paper using the separate saccharification and fermentation (SHF). Enzymatic hydrolysis step was carried out by crude hydrolytic enzymes produced by T. reesei Rut C-30 (cellulase-overproducing mutant) and Aspergillus niger F38 cultures. The aim of this work was to apply response surface methodology (RSM) combined with a central composite design (CCD) to identify the optimal conditions for cellulose hydrolysis and mainly glucose production from waste paper by analyzing the relationships amongst a number of parameters that affect the overall process. Sugars released from selected waste paper, under optimal hydrolysis conditions, were subsequently converted into ethanol by Saccharomyces cerevisiae CTM-30101.
Materials and Methods Materials and Microorganisms Waste paper used in this study was collected locally. Tween 20, Tween 80, Triton X-100, p-nitrophenyl β- D -glucopyranoside (pNPG), 3,5-dinitrosalicylic acid, carboxymethyl cellulose (CMC) and glucose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SDS and PEG 6000 were supplied from Bio Basic Inc. The commercial cellulases tested for waste paper hydrolysis were purchased from Novo Nordisk, Copenhagen/Denmark (cellulase Novo 342 and Danimax 89-2). Kamaistone K-050 and Cellusoft cellulases both were obtained from local factories. The fungus T. reesei Rut C-30 (ATCC 56765) was obtained from the American Type Culture Collection (ATCC). A. niger F38 was obtained from LVBPPE collection [15]. S. cerevisiae CTM-30101 strain was purchased from the National Strains Collection of Centre of Biotechnology of Sfax, CBS, Tunisia. Cellulolytic Enzyme Production The spore suspensions of T. reesei Rut C-30 and A. niger (107 spores mL−1) were used as inoculums for production of cellulase and β-glucosidase (BGL), respectively. The mineral salt medium (modified Mandels medium) [16] used for both cellulase and BGL productions had a composition in grammes per liter: urea 0.3, KH2PO4 2, (NH4)2SO4 1.4, MgSO4 7H2O 0.3, peptone 0.75, yeast extract 0.25, CaCl2 2H2O 0.4 and supplemented with 1 mL L−1 of an oligoelement solution (MnSO4 1.6 g L−1, ZnSO4 1.4 g L−1, FeSO4 5 g L−1 and CoCl2 2 g L−1). Initial pH was adjusted at 4.8 and 3 % of wheat bran was used as a carbon source for enzyme production by both fungi. A. niger was cultivated in 500-mL Erlenmeyer flasks containing 120 mL of Mandels medium and incubated at 30 °C on a rotary shaker (160 rpm) for 5 days. On the other hand, the culture of T. reesei Rut C-30 was carried out in a 3.6-L stirred tank bioreactor (Infors, AG GH-4103 Bottmingen, Switzerland) with a 1.6-L working volume. The bioreactor (dished bottom glass-jacketed reactor) was equipped with instrumentation for measurement and/or control of agitation, temperature, pH and dissolved oxygen concentration. The cultivation temperature was maintained at 30 °C and dissolved oxygen was kept above 20 % of medium saturation by air supply. The pH was controlled at 5.0 by automatic addition of ammonium hydroxide
Appl Biochem Biotechnol
(4 M) and phosphoric acid (2 M). Production medium was sterilized by autoclaving at 120 °C for 20 min. At the end of the fermentation (5 days), the mycelium was harvested by centrifugation at 8000 rpm for 10 min, and the supernatant was employed for cellulase exploitation. Analytical Methods The cellulase activity was measured using the filter paper activity (FPA) assay, expressed in filter paper units (FPU) according to the method of Ghose [17]. This method measures the release of reducing sugars produced in 60 min from a mixture of enzyme solution (0.5 mL) and of citrate buffer (0.05 M, pH 4.8) in the presence of 50 mg of Whatman No.1 filter paper and incubated at 50 °C. The endo-β-1-4glucanase (or carboxymethyl cellulase, CMCase) was measured at 50 °C by determining the reducing sugars (RS) released after 30 min from a mixture of enzyme solution (0.5 mL) and 0.5 mL of a 2 % carboxymethyl cellulose (CMC) solution in 0.05 M sodium citrate buffer (pH 4.8). The filter paper and carboxymethyl cellulase assays are based on the same principle of estimating the amount of RS released from the relevant substrate [17]. Similarly, the xylanase activity was assayed by measuring the RS released from 1 % of Birchwood xylan [18]. The liberated reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) method [19]. One unit of enzyme activity was defined as the amount of enzymes releasing 1 μmol of reducing sugars per 1 min. The β-glucosidase activity was determined using pNPG as a substrate according to Yanai and Sato [20]. The assay mixture contained 200 μL of pNPG (2 mM) in 50 mM citrate buffer (pH 5.0) and 200 μL of diluted enzyme solution. After incubation for 10 min at 50 °C, the reaction was stopped by the addition of 1.6 mL of Na2CO3 (1 M). The absorbance was measured at 405 nm and 1 unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per minute in the reaction mixture. The waste paper (office paper and newspaper) was dried to a constant weight at 90 °C, followed by cooling in a desiccator and weighed. Cellulose, hemicellulose and lignin contents of the dried waste paper were assayed by means of a quantitative hydrolysis with sulphuric acid according to the method described by Browning [21]. Ash was determined by heating the dried waste paper in a muffle furnace at 500 °C. Ethanol and sugars were analyzed by high-performance liquid chromatography (Agilent, USA) equipped with a reflective index detector (RID) an Aminex HPX87H column. Ethanol and sugar identification was performed at 60 °C with 5 mM sulphuric acid as the mobile phase at a flow rate of 0.6 mL min−1. Waste Paper Preparation Office paper and newspaper were prepared as pieces of dimensions 1 cm×1 cm and presoaked in deionized water (10 g L−1) at 60 °C for 6 h with gentle stirring for ink removal [22]. The water was removed by squeezing and procedure was repeated several times. After the presoaking step, waste paper was dried overnight in an oven at 50 °C. Dry matter was then dray-defibrated with a mill designed to shear them by blades rotating at 1500 rpm for 5 min in order to remove the most physical barriers of cellulose structure [23]. Finally, waste paper was treated with diluted phosphoric acid (3 g L−1) for 2 h at 60 °C, washing with deionized water to a neutral pH and then dried again in an oven at 50 °C up to a constant weight.
Appl Biochem Biotechnol
Enzymatic Digestibility Test Crude enzymes recovered from T. reesei Rut C-30 and A. niger F38 cultures were, first, concentrated with lyophilization, resuspended in 20 mM citrate buffer (pH 4.8) and then mixed together to obtain a final enzyme concentration as follows: FP activity 3.5 U mL−1, endoglucanase 1.5 U mL−1, β-glucosidase 20 U mL−1 and xylanase 80 U mL−1. This mixture was tested simultaneously with other commercial and industrial cellulases (cellulase Novo 342, Danimax 89-2, Kamaistone K-050 and Cellusoft) for their ability to produce fermentable sugars from pretreated waste paper material. Enzymatic saccharification of biomass was done by incubating 1 g of pretreated waste paper (office paper, newspaper) with different cellulases at 40 °C, in stoppered 250-mL flasks in a total volume of 50 mL made up with 50 mM citrate buffer (pH 4.8). The flasks were agitated at 150 rpm in a controlled orbital shaker. Enzyme loading was fixed for all cellulases tested at 10 FPU g−1 of dry matter. After 48 h of incubation period, aliquots from each hydrolyzate were centrifuged at 6000 rpm for 10 min and the supernatant was analyzed for total reducing sugars, determined by DNS method. The degree of saccharification was calculated as follows [24]: Degree of saccharification ð%Þ ¼
Reducing sugar ðmg=mLÞ Hydrolysate volume 0:9 100 ð1Þ Potential sugar in the substrate
At least three parallel samples were used in all analytical determinations, and data are presented as the mean of three replicates. Effect of Surfactant Pretreatment on Enzymatic Digestibility The surfactant treatment was prepared according to Kim et al. [25] with some modifications. Ten grammes of pretreated office paper and newspaper were added to a 500-mL flask containing 200 g of deionized water. Of each surfactant (Tween 20, Tween 80, Triton X-100, SDS and PEG 6000), 0.5 % (w w−1) was added of the flasks agitated for 2 h at 250 rpm and 40 °C. The concentration of the surfactant was calculated as percentage (w w−1) based on the 10 g of the dry substrate. After pretreatment, the wet solid was centrifuged and washed with 3 L of deionized water to completely remove the surfactants. It was then dried in an oven at 50 °C overnight. Enzymatic digestibility was determined as described above. Response Surface Methodology To determine the best combination of parameters for optimizing the enzymatic saccharification of pretreated waste paper, a second-order central composite design (CCD) was employed. Dry matter quantity (X1), phosphoric acid concentration (X2) and hydrolysis time (X3) were selected as independent variables. Each variable was assessed at five coded levels (−1.682, −1, 0, +1 and +1.682). A total of 20 experiments were conducted including six replicates at the centre point. The response values (Y) used in each trial was the average of the replicates. Statistical Analysis and Modelling The data obtained from RSM with regard to saccharification degree were subjected to analysis of variance (ANOVA) to check for errors and the significance of each parameter. The degree of saccharification of office paper and newspaper was taken as responses (Y1) and (Y2),
Appl Biochem Biotechnol
respectively. The data were then subjected to a multiple regression analysis to obtain an empirical model that could relate the response measured to the independent variables. The behaviour of the system was explained by the following quadratic equation: Y ¼ β0 þ β 1 X 1 þ β 2 X 2 þ β 3 X 3 þ β 11 X 2 1 þ β 22 X 2 2 þ β 33 X 2 3 þ β 12 X 1 X 2 þ β 23 X 2 X 3 þ β 13 X 1 X 3
ð2Þ
where Y refers to the predicted response; X1, X2 and X3 to the independent coded variables; β0 to the offset term; β1, β2 and β3 to the linear effects; β11, β22 and β33 to the squared effects; and β12, β23 and β13 to the interaction terms. The statistical software package (Nemrod-W by LPRAI Marseilles, France) [26], was used to conduct a regression analysis on the experimental data and to plot the response surface graphs. The statistical significance of the model was determined by the application of Fisher’s F test. The two-dimensional graphical representation of the system behaviour, called the isoresponse contour plot, was used to describe the individual and cumulative effects of the variables as well as the possible correlations that existed between them. Ethanol Fermentation of Waste Paper Hydrolyzate The yeast S. cerevisiae CTM-30101 was used in all fermentation assays. A filter-sterilized hydrolyzate produced from office paper was used as a fermentation medium for bioethanol production using separate saccharification and fermentation (SHF). Experiments were performed in 1-L Erlenmeyer flasks containing 200 mL of fermentation medium supplemented with nitrogen sources that composed YMP medium (3 g L−1 of yeast extract, 3 g L−1 of malt extract and 5 g L−1 of peptone). Flasks were inoculated with 5 % (v v−1) of yeast suspension obtained from growth for 15 h on YPG medium composed of 10 g L−1 of yeast extract, 10 g L−1 of peptone and 20 g L−1 of glucose. Control fermentation was also run on YMP medium supplemented with reagent-grade glucose added at the same concentration present in the waste paper hydrolyzate used for the SHF process. Cultivations were carried out in duplicate at 30 °C with 150 rpm (orbital shaking). Samples were collected during 72 h and centrifuged at 12,000 rpm for 10 min. The cell-free supernatants were used for the determination of ethanol produced and sugar consumed. Cell growth was monitored directly by reading the optical density at 600 nm.
Results and Discussion Enzyme Production for Waste Paper Hydrolysis Enzymatic preparations used for waste paper saccharification were produced with individual cultures of the hyper-cellulolytic strain T. reesei Rut C-30 and A. niger F38, carried out in controlled 3.6-L fermentor and 500-mL Erlenmeyer flasks, respectively. The effect of carbon source on cellulase and β-glucosidase (BGL) production has been studied previously in our laboratory (unpublished results), and wheat bran was selected as the best enzymes inducer. Wheat bran is a cheap raw material and its use for enzyme induction can reduce the cost of lignocellulosic ethanol production [27]. Table 1 shows the production level of crude cellulase and BGL secreted by T. reesei Rut C-30 and A. niger F38, respectively, after 5 days of culture. Crude Rut C-30 cellulase preparation had appreciable levels of filter paper (1.14 U mL−1) and
Appl Biochem Biotechnol Table 1 Crude enzyme produced from T. reesei Rut C-30 and A. niger F38 after 5 days of culture Fungus
Type of culture
Protein (mg mL−1)
Enzyme activities (U mL−1) Total cellulase activity (FP)a
Endoglucanase activity (CMC)
β-glucosidase activity (pNPG)
Xylanase activity (xylan)
Trichoderma reesei Rut C-30
Fermentor 3.6 L
4.4±0.3
1.14±0.26
0.76±0.21
0.22±0.08
25±4
Aspergillus niger F38
Flasks 500 mL
1.5±0.1
0.24±0.1
0.33±0.17
6.8±1.3
8.8±1.6
a
FP, CMC, pNPG and xylan were used as substrates for the enzyme activity assays
CMCase activity (0.76 U mL−1) but lesser BGL activity (0.22 U mL−1). On the contrary, the crude A. niger preparation had a lesser filter paper (0.24 U mL−1) and CMCase activities (0.33 U mL−1) while the BGL activity was several fold higher and reach 6.8 U mL−1. T. reesei is a well-studied filamentous fungus for cellulase production, but it is endowed with a low BGL activity, causing accumulation of cellobiose, which produces repression and end-product inhibition during hydrolysis. Aspergillus sp. produces BGL in significant amounts as observed by Ahamed and Vermette [28]. It is worthy to note that adding external BGL decreases the inhibitory effect of cellobiose accumulated in the enzymatic hydrolysis [5]. On the other hand, xylanase activity was largely produced from both cultures. This enzyme may play an important role in hemicellulosic fraction degradation. It has been reported by many authors that xylanase supplementation clearly increased cellulose hydrolysis in xylancontaining lignocellulosic materials. The hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose and prevent the access of enzymes to cellulose surface [29]. Waste Paper Composition The biochemical characteristics of waste paper (office paper and newspaper) were analyzed (Table 2). Office paper is distinguished by high cellulose content (w w−1, 78.6 %) and a very low hemicellulose and lignin content (4.7 and 1.2 %, respectively). This characteristic is more suitable for fermentable sugar production because there will be less physical barrier during the enzymatic hydrolysis of cellulose fraction [2]. Table 2 shows that the level of cellulose in office paper, measured in different studies, ranged from 55.7 to 87.4 %. The low cellulose content (55.7 %) of the office paper tested by Wang et al. [30] is likely due to the presence of inorganic coatings (13 %) in the sample [31]. On the other hand, Foyle and co-workers [4] have demonstrated that not only the origin of pulp paper can affect the level of cellulose content but also the measurement method can also influence the biochemical composition. For this reason, it is necessary to choose the method that allows the minimum loss in carbohydrate content [4]. Compared to office paper, it is evident that newspaper has significantly more lignin (19.18 %) and hemicellulose (12.2 %) and substantially less cellulose (49.3 %). This structural feature of newspaper therefore controls the extent of cellulose hydrolysis, a limitation that can be overcome by using several hydrolytic enzymes acting synergistically [32]. Results obtained here were in agreement to earlier studies, where lignin, holocellulose
Appl Biochem Biotechnol Table 2 Office paper and newspaper composition Waste paper ingredient (content %)a Moisture Office paper
Newspaper
a
Cellulose
Hemicellulose
Reference Lignin
Ash
Others
3.2
78.6
4.7
1.2
9
3.3
This work
1.4
87.4
8.4
2.3
–
–
[33]
2.4
64.7
13
0.93
–
–
[34]
4.9
55.7
13
5.78
7.57
13
[30]
3
49.3
12.2
19.18
1.5
4.9
This work
1.5
48.5
9
23.9
–
–
[33]
2 7.25
48.3 43.7
18.1 16.7
22.1 16.8
– 9.5
– 6
[34] [30]
Data are presented as the mean of tow replicates
(cellulose and hemicellulose) and moisture content in newspaper were found to be 16–23, 57.5–66.4 and 3–7.2 %, respectively [30, 33, 34]. Enzymatic Digestibility Test It is necessary to evaluate the hydrolytic performance of any enzymatic preparation before its use. In this purpose and in order to produce fermentable sugars from waste paper, our enzyme mixture was compared with other commercial and industrial cellulases. Newspaper and office paper were hydrolyzed using the same enzyme activity (10 FPU g−1 of substrate). Figure 1 shows the degrees of saccharification after 48 h of hydrolytic reactions using different cellulases. Our local enzyme preparation and the industrial cellulase (Kamaistone K-050) provide highest degree of Office paper
Newspaper
Residual activity
80 70 70 60 60 50
50
40
40
30
30 20
20
10
10
0
0 Mixture
Denimax 98-2
Novo 342
Kamaistone
Cellusoft
Fig. 1 Saccharification degrees of office paper and newspaper using different enzyme preparations and their correspondent residual activities determined after 48 h of operating time. Mixture: crude enzyme obtained from T. reesei Rut C-30 and A. niger F38 (residual activities presented here are averages of three hydrolysis conditions)
Appl Biochem Biotechnol
saccharification using office paper (63 %, 68 %) or newspaper (29 %, 32 %) as substrate, respectively. This is due to the high level of BGL activity in Kamaistone K050 preparation and in our enzymatic mixture (85 U g−1 of substrate and 60 U g−1 of substrate, respectively). However, Denimax 98-2, Novo 342 and cells of cellulases contain relatively lower BGL activity (18, 22 and 12 U g−1, respectively) and consequently exhibit a low degree of saccharification. BGL activity is a key enzyme in the cellulolytic system of several microorganisms. Cellobiose, potential substrate for BGL activity, is a potent inhibitor of endo- and exoglucanases and therefore its accumulation significantly slows down the overall hydrolysis process [35]. Moreover, at the end of each hydrolytic reaction, residual activity of all enzymatic preparations was determined (Fig. 1). Our enzymatic mixture and Kamaistone K-050 cellulase showed better functional stability and are able to retain about 66 and 71 % of their initial activities, respectively, probably due to their good thermal stabilities. Operational stability is a much desired enzymatic characteristic for industrial applications especially in continuous saccharification process and subsequently can reduce significantly the cost of lignocellulosic ethanol production [36]. It is useful to note that the simultaneous use of the two enzyme preparations derived from T. reesei Rut C-30 and A. niger F38 improves the waste paper hydrolysis yield comparing at each preparation used alone (data not shown). All of this shows that our enzyme mixture is very suitable for lignocellulosic waste saccharification and will be employed in the following work. Effect of Surfactant Pretreatment on Enzymatic Digestibility It is a well-known fact that the addition of a surfactant into the enzymatic hydrolysis of lignocellulosic biomass increases its digestibility [32]. However, previous studies have used surfactants in the hydrolysis stage only. In this study, five different surfactants were evaluated for their ability to enhance enzymatic hydrolysis of waste paper (Fig. 2). All surfactants used increase the efficiency of hydrolysis of the newspaper but they have no noticeable effect on office paper. Triton X-100 at 0.5 % (w w−1) improved the digestibility of newspaper about 180
Hydrolysis yield (%)
160 140 120 100 80 60 40 20 0 Control
SDS
Tw een 20
Tw een 80 Triton X-100
PEG 6000
Surfactant (0.5 % w/w) Fig. 2 The effects of surfactant pretreatment on the enzymatic digestibility of newspaper and office paper
Appl Biochem Biotechnol
45 %. It has been demonstrated that the addition of surfactants, particularly the nonionic ones, can significantly improve the release of sugars from waste or recycled newspaper [25, 37]. Newspaper has a relatively high content of lignin compared to office paper, as shown in Table 2. Responsible of reducing cellulose accessibility to cellulase, lignin can impede cellulose hydrolysis via its ability to bend enzyme phenomenon which can be minimized through the addition of surfactant. It is believed that the surfactant adsorbs onto the lignin polymer, thereby preventing cellulase from adsorbing onto the same binding site [38]. Although several mechanisms have been proposed for the positive effect of surfactant pretreatment on cellulose hydrolysis, the enzyme-substrate interaction is one of the most supported [32, 38]. The relatively low lignin content of office paper (Table 2) could explain the insensitivity of sugar yield to a surfactant pretreatment. Triton X-100 will be maintained for only newspaper surfactant pretreatment. Response Surface Models A CCD was performed to determine the optimum levels of three independent variables (dry matter quantity (X1), H3PO4 concentration (X2) and hydrolysis time (X3)) and the effect of their interactions on the degree of saccharification of newspaper and office paper. The results obtained were subjected to an analysis of variance (ANOVA) to determine the significant differences. Table 3 represents the design matrix of the variables together with experimental results. Experiments 1–14 were performed at the different combinations and those from 15 to 20 were under the same conditions. By applying multiple regression analysis on the experimental data, the following second polynomial equations (Eq. (3) and Eq. (4)) were established to explain the degree of saccharification of each substrate, newspaper (Y1) and office paper (Y2), respectively. Y 1 ¼ 58:591 þ 8:953X 1 þ 7:158X 2 þ 3:406X −8:206X 1 2 −2:332X 2 2 −2:747X 3 2 þ 0:884X 1 X 2 þ 5:079X 1 X 3 −1:739X 2 X 3
ð3Þ Y 2 ¼ 85:807−4:162X 1 þ 6:317X 2 þ 10:685X 3 −15:861X 1 2 −7:465X 2 2 −5:414X 3 2 −0:812X 1 X 2 −5:762X 1 X 3 þ 6:737X 2 X 3
ð4Þ where Y is the predicted degree of saccharification; X1, X2 and X3 are the coded values of dry matter quantity, phosphoric acid concentration and hydrolysis time, respectively. The significance of the regression coefficients was given by a t test. The regression coefficients and corresponding P values for the models, corresponding to the degree of saccharification of newspaper and office paper, are presented in Table 4. The P values are used as a tool to check the significance of each of the coefficients which are, in turn, necessary to understand the pattern of the mutual interactions between variables. In fact, when the magnitude of the t test value is large and the P value is small, this indicates that the corresponding coefficient is highly significant [39]. As far as the current study, the estimated parameters and the corresponding P values suggest that, all independent factors, X1 (dray matter quantity), X2 (phosphoric acid concentration) and X3 (hydrolysis time) had a significant effect on the hydrolysis rate of newspaper and office paper. The quadratic term of the three factors, especially in the case of office paper saccharification, had a significant effect. On the other hand, the interactions between the factors do not always have a significant effect on the responses. The ANOVA for the quadratic regression model is given also in Table 4. These results show that the models predicted for the degree of saccharification of newspaper and office paper were adequate. The regression model
Appl Biochem Biotechnol Table 3 The central composite design matrix employed for three independent variables and experimental responses of the degree of saccharification of newspaper (Y1) and office paper (Y2) Number rune
Variables (X1) Dry matter (g L−1)a
Responses (X2) H3PO4 (g L−1)b
(X3) Time (h)c
Newspaper Y1
Office paper Y2
1
−1
−1
−1
29.86
56.90
2
1
−1
−1
40.05
52.50 55.50
3
−1
1
−1
50.36
4
1
1
−1
55.20
42.00
5
−1
−1
1
31.20
73.50
6
1
−1
1
52.82
40.20
7
−1
1
1
35.86
93.20
8 9
1 −1.681
1 0
1 0
69.90 19.55
62.50 30.00
10
1.681
0
0
50.22
44.90
11
0
−1.681
0
39.50
44.50
12
0
1.681
0
63.50
77.90
13
0
0
−1.681
40.75
42.20
14
0
0
1.681
59.90
91.80
15
0
0
0
60.22
88.32
16 17
0 0
0 0
0 0
55.50 59.50
86.22 87.50
18
0
0
0
60.50
84.50
19
0
0
0
55.60
86.00
20
0
0
0
60.40
83.50
a
X1: Dry matter (g L−1 ) (−1.681=5; −1=15; 0=30; +1=45; +1.681=55)
b
X2: H3PO4 (g L−1 ) (−1.681=2; −1=3; 0=5; +1=7; +1.681=8)
c
X3: Time (h) (−1.681=13; −1=20; 0=30; +1=40; +1.681=47)
was highly significant (P
Enhanced Enzymatic Hydrolysis of Waste Paper for Ethanol Production Using Separate Saccharification and Fermentation Mohamed Guerfali & Adel Saidi & Ali Gargouri & Hafedh Belghith
Received: 22 April 2014 / Accepted: 10 September 2014 # Springer Science+Business Media New York 2014
Abstract Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum-based liquid fuels. In this study, the feasibility of ethanol production from waste paper using the separate hydrolysis and fermentation (SHF) was investigated. Two types of waste paper materials, newspaper and office paper, were evaluated for their potential to be used as a renewable feedstock for the production of fermentable sugars via enzymatic hydrolysis of their cellulose fractions. Hydrolysis step was conducted with a mixture of cellulolytic enzymes produced locally by Trichoderma reesei Rut-C30 (cellulaseoverproducing mutant) and Aspergillus niger F38 cultures. Surfactant pretreatment effect on waste paper enzymatic digestibility was studied and Triton X-100 at 0.5 % (w w−1) has improved the digestibility of newspaper about 45 %. The effects of three factors (dry matter quantity, phosphoric acid pretreatment and hydrolysis time) on the extent of saccharification were also assessed and quantified by using a methodical approach based on response surface methodology. Under optimal hydrolysis conditions, maximum degrees of saccharification of newspaper and office paper were 67 and 92 %, respectively. Sugars released from waste paper were subsequently converted into ethanol (0.38 g ethanol g−1 sugar) with Saccharomyces cerevisiae CTM-30101. Keywords Bioethanol . Waste paper . Saccharification . Cellulases . Response surface methodology
Introduction Fuel ethanol production from renewable resources such as plant biomass is one of the most promising alternatives to conventional petroleum-based fuels. Currently, most bioethanol produced in the world is derived from starch and molasses. However, these sources are not sufficiently supplied for biofuel production due to the role they play in human and livestock M. Guerfali (*) : A. Saidi : A. Gargouri : H. Belghith Laboratory of Biomass Valorisation and Protein Production in Eukaryotes, Centre of Biotechnology of Sfax, University of Sfax, P.O. Box 1177, 3038 Sfax, Tunisia e-mail: [email protected]
Appl Biochem Biotechnol
consumption. Therefore, the development of second-generation bioethanol from lignocellulosic biomass serves many advantages from both economic and environmental point of views [1]. Lignocellulosic biomass typically contains 50–80 % (dry basis) carbohydrates that are polymers of C5 and C6 sugar units. Most carbohydrates can be processed either chemically or biologically to yield biofuels such as ethanol [2]. One of the main economic factors in ethanol process is the cost of raw material which can be substantially reduced by utilization of industrial lignocellulosic wastes. However, due to structural complexity, pretreatment is required to disrupt the recalcitrant structure of lignocellulosic materials and to increase the accessibility of hydrolytic enzymes to the carbohydrates polymers. Until now, several types of pretreatment are used, including steam explosion, acid, alkali, organic solvent, alkaline hydrogen peroxide, ammonia and liquid hot water treatments [3]. The choice of pretreatment process is possibly the most important factor in the economics of ethanol production process because it influences waste treatment, cellulose conversion rates and mainly hydrolytic enzymes performance [4]. Employment of enzymes for lignocellulose hydrolysis is considered, prospectively, as the most viable strategy that offer advantages over chemical conversion routes of higher yields, minimal by-product formation, low energy requirements, mild operating conditions and environmentally friendly processing [5]. Enzymatic saccharification of lignocellulosic materials to soluble products can be carried out by a complex mixture of enzymes, amongst which cellulases (endoglucanase, exoglucanase and β-glucosidase) and hemicellulases (xylanase) acting in perfect synergy. Endoglucanases randomly attack cellulose chains and release cello-oligosaccharides, the ends of which are processed by exoglucanases to cellobiose, which is therefore converted by β-glucosidase to glucose. On the other hand, xylanases hydrolyze xylan (major component of hemicellulose) and facilitate the removal of lignin which blocks the action of cellulases [6]. Complete cellulase enzyme systems can be produced by a large variety of microorganisms. Amongst the best characterized and most widely studied ones are the inducible cellulases of the filamentous fungus Trichoderma reesei [7]. These cellulases have a high industrial interest as they are widely used in the food and feed industries and in the textile and pulp paper industries [8]. Waste paper is particularly attractive as feedstock for bioethanol production because it is readily available. Despite the growing awareness for recycling, the majority of waste paper will still end up in landfills. There is, therefore, an almost constant supply of large quantities of waste paper in many parts of the world. Previous researches have demonstrated the potential of bioethanol production from waste paper using a variety of process designs [9–11]. The cellulosic sugars obtained through acid and enzymatic hydrolysis can efficiently be used for ethanol fermentation either by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). However, the temperature optima for the yeast growth and hydrolytic enzymes differ, which means that the conditions used in SSF cannot be optimal for both (enzymes and yeast) and might result in lower efficiency and lower yield [12]. Hence, for better efficiency of ethanol production, the SHF approach was preferred in the present work. Enzymatic hydrolysis of lignocellulosic material is the critical step for sugar production as it can be affected by several factors at the same time such as enzyme loading, hydrolysis time, substrate concentration, cellulose crystallinity, and by lignin and hemicellulose content [13]. The conventional method (changing one independent variable while maintaining all of the others at a fixed level) to obtain the optimization of enzymatic hydrolysis is extremely time consuming.
Appl Biochem Biotechnol
Response surface methodology (RSM) is a time- and labour-saving method, which reveals the interaction between the factors and seeks the optimum levels [14]. In the present work, we tested the combinatorial effect of the production of ethanol from waste paper using the separate saccharification and fermentation (SHF). Enzymatic hydrolysis step was carried out by crude hydrolytic enzymes produced by T. reesei Rut C-30 (cellulase-overproducing mutant) and Aspergillus niger F38 cultures. The aim of this work was to apply response surface methodology (RSM) combined with a central composite design (CCD) to identify the optimal conditions for cellulose hydrolysis and mainly glucose production from waste paper by analyzing the relationships amongst a number of parameters that affect the overall process. Sugars released from selected waste paper, under optimal hydrolysis conditions, were subsequently converted into ethanol by Saccharomyces cerevisiae CTM-30101.
Materials and Methods Materials and Microorganisms Waste paper used in this study was collected locally. Tween 20, Tween 80, Triton X-100, p-nitrophenyl β- D -glucopyranoside (pNPG), 3,5-dinitrosalicylic acid, carboxymethyl cellulose (CMC) and glucose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). SDS and PEG 6000 were supplied from Bio Basic Inc. The commercial cellulases tested for waste paper hydrolysis were purchased from Novo Nordisk, Copenhagen/Denmark (cellulase Novo 342 and Danimax 89-2). Kamaistone K-050 and Cellusoft cellulases both were obtained from local factories. The fungus T. reesei Rut C-30 (ATCC 56765) was obtained from the American Type Culture Collection (ATCC). A. niger F38 was obtained from LVBPPE collection [15]. S. cerevisiae CTM-30101 strain was purchased from the National Strains Collection of Centre of Biotechnology of Sfax, CBS, Tunisia. Cellulolytic Enzyme Production The spore suspensions of T. reesei Rut C-30 and A. niger (107 spores mL−1) were used as inoculums for production of cellulase and β-glucosidase (BGL), respectively. The mineral salt medium (modified Mandels medium) [16] used for both cellulase and BGL productions had a composition in grammes per liter: urea 0.3, KH2PO4 2, (NH4)2SO4 1.4, MgSO4 7H2O 0.3, peptone 0.75, yeast extract 0.25, CaCl2 2H2O 0.4 and supplemented with 1 mL L−1 of an oligoelement solution (MnSO4 1.6 g L−1, ZnSO4 1.4 g L−1, FeSO4 5 g L−1 and CoCl2 2 g L−1). Initial pH was adjusted at 4.8 and 3 % of wheat bran was used as a carbon source for enzyme production by both fungi. A. niger was cultivated in 500-mL Erlenmeyer flasks containing 120 mL of Mandels medium and incubated at 30 °C on a rotary shaker (160 rpm) for 5 days. On the other hand, the culture of T. reesei Rut C-30 was carried out in a 3.6-L stirred tank bioreactor (Infors, AG GH-4103 Bottmingen, Switzerland) with a 1.6-L working volume. The bioreactor (dished bottom glass-jacketed reactor) was equipped with instrumentation for measurement and/or control of agitation, temperature, pH and dissolved oxygen concentration. The cultivation temperature was maintained at 30 °C and dissolved oxygen was kept above 20 % of medium saturation by air supply. The pH was controlled at 5.0 by automatic addition of ammonium hydroxide
Appl Biochem Biotechnol
(4 M) and phosphoric acid (2 M). Production medium was sterilized by autoclaving at 120 °C for 20 min. At the end of the fermentation (5 days), the mycelium was harvested by centrifugation at 8000 rpm for 10 min, and the supernatant was employed for cellulase exploitation. Analytical Methods The cellulase activity was measured using the filter paper activity (FPA) assay, expressed in filter paper units (FPU) according to the method of Ghose [17]. This method measures the release of reducing sugars produced in 60 min from a mixture of enzyme solution (0.5 mL) and of citrate buffer (0.05 M, pH 4.8) in the presence of 50 mg of Whatman No.1 filter paper and incubated at 50 °C. The endo-β-1-4glucanase (or carboxymethyl cellulase, CMCase) was measured at 50 °C by determining the reducing sugars (RS) released after 30 min from a mixture of enzyme solution (0.5 mL) and 0.5 mL of a 2 % carboxymethyl cellulose (CMC) solution in 0.05 M sodium citrate buffer (pH 4.8). The filter paper and carboxymethyl cellulase assays are based on the same principle of estimating the amount of RS released from the relevant substrate [17]. Similarly, the xylanase activity was assayed by measuring the RS released from 1 % of Birchwood xylan [18]. The liberated reducing sugars were determined by the 3,5-dinitrosalicylic acid (DNS) method [19]. One unit of enzyme activity was defined as the amount of enzymes releasing 1 μmol of reducing sugars per 1 min. The β-glucosidase activity was determined using pNPG as a substrate according to Yanai and Sato [20]. The assay mixture contained 200 μL of pNPG (2 mM) in 50 mM citrate buffer (pH 5.0) and 200 μL of diluted enzyme solution. After incubation for 10 min at 50 °C, the reaction was stopped by the addition of 1.6 mL of Na2CO3 (1 M). The absorbance was measured at 405 nm and 1 unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per minute in the reaction mixture. The waste paper (office paper and newspaper) was dried to a constant weight at 90 °C, followed by cooling in a desiccator and weighed. Cellulose, hemicellulose and lignin contents of the dried waste paper were assayed by means of a quantitative hydrolysis with sulphuric acid according to the method described by Browning [21]. Ash was determined by heating the dried waste paper in a muffle furnace at 500 °C. Ethanol and sugars were analyzed by high-performance liquid chromatography (Agilent, USA) equipped with a reflective index detector (RID) an Aminex HPX87H column. Ethanol and sugar identification was performed at 60 °C with 5 mM sulphuric acid as the mobile phase at a flow rate of 0.6 mL min−1. Waste Paper Preparation Office paper and newspaper were prepared as pieces of dimensions 1 cm×1 cm and presoaked in deionized water (10 g L−1) at 60 °C for 6 h with gentle stirring for ink removal [22]. The water was removed by squeezing and procedure was repeated several times. After the presoaking step, waste paper was dried overnight in an oven at 50 °C. Dry matter was then dray-defibrated with a mill designed to shear them by blades rotating at 1500 rpm for 5 min in order to remove the most physical barriers of cellulose structure [23]. Finally, waste paper was treated with diluted phosphoric acid (3 g L−1) for 2 h at 60 °C, washing with deionized water to a neutral pH and then dried again in an oven at 50 °C up to a constant weight.
Appl Biochem Biotechnol
Enzymatic Digestibility Test Crude enzymes recovered from T. reesei Rut C-30 and A. niger F38 cultures were, first, concentrated with lyophilization, resuspended in 20 mM citrate buffer (pH 4.8) and then mixed together to obtain a final enzyme concentration as follows: FP activity 3.5 U mL−1, endoglucanase 1.5 U mL−1, β-glucosidase 20 U mL−1 and xylanase 80 U mL−1. This mixture was tested simultaneously with other commercial and industrial cellulases (cellulase Novo 342, Danimax 89-2, Kamaistone K-050 and Cellusoft) for their ability to produce fermentable sugars from pretreated waste paper material. Enzymatic saccharification of biomass was done by incubating 1 g of pretreated waste paper (office paper, newspaper) with different cellulases at 40 °C, in stoppered 250-mL flasks in a total volume of 50 mL made up with 50 mM citrate buffer (pH 4.8). The flasks were agitated at 150 rpm in a controlled orbital shaker. Enzyme loading was fixed for all cellulases tested at 10 FPU g−1 of dry matter. After 48 h of incubation period, aliquots from each hydrolyzate were centrifuged at 6000 rpm for 10 min and the supernatant was analyzed for total reducing sugars, determined by DNS method. The degree of saccharification was calculated as follows [24]: Degree of saccharification ð%Þ ¼
Reducing sugar ðmg=mLÞ Hydrolysate volume 0:9 100 ð1Þ Potential sugar in the substrate
At least three parallel samples were used in all analytical determinations, and data are presented as the mean of three replicates. Effect of Surfactant Pretreatment on Enzymatic Digestibility The surfactant treatment was prepared according to Kim et al. [25] with some modifications. Ten grammes of pretreated office paper and newspaper were added to a 500-mL flask containing 200 g of deionized water. Of each surfactant (Tween 20, Tween 80, Triton X-100, SDS and PEG 6000), 0.5 % (w w−1) was added of the flasks agitated for 2 h at 250 rpm and 40 °C. The concentration of the surfactant was calculated as percentage (w w−1) based on the 10 g of the dry substrate. After pretreatment, the wet solid was centrifuged and washed with 3 L of deionized water to completely remove the surfactants. It was then dried in an oven at 50 °C overnight. Enzymatic digestibility was determined as described above. Response Surface Methodology To determine the best combination of parameters for optimizing the enzymatic saccharification of pretreated waste paper, a second-order central composite design (CCD) was employed. Dry matter quantity (X1), phosphoric acid concentration (X2) and hydrolysis time (X3) were selected as independent variables. Each variable was assessed at five coded levels (−1.682, −1, 0, +1 and +1.682). A total of 20 experiments were conducted including six replicates at the centre point. The response values (Y) used in each trial was the average of the replicates. Statistical Analysis and Modelling The data obtained from RSM with regard to saccharification degree were subjected to analysis of variance (ANOVA) to check for errors and the significance of each parameter. The degree of saccharification of office paper and newspaper was taken as responses (Y1) and (Y2),
Appl Biochem Biotechnol
respectively. The data were then subjected to a multiple regression analysis to obtain an empirical model that could relate the response measured to the independent variables. The behaviour of the system was explained by the following quadratic equation: Y ¼ β0 þ β 1 X 1 þ β 2 X 2 þ β 3 X 3 þ β 11 X 2 1 þ β 22 X 2 2 þ β 33 X 2 3 þ β 12 X 1 X 2 þ β 23 X 2 X 3 þ β 13 X 1 X 3
ð2Þ
where Y refers to the predicted response; X1, X2 and X3 to the independent coded variables; β0 to the offset term; β1, β2 and β3 to the linear effects; β11, β22 and β33 to the squared effects; and β12, β23 and β13 to the interaction terms. The statistical software package (Nemrod-W by LPRAI Marseilles, France) [26], was used to conduct a regression analysis on the experimental data and to plot the response surface graphs. The statistical significance of the model was determined by the application of Fisher’s F test. The two-dimensional graphical representation of the system behaviour, called the isoresponse contour plot, was used to describe the individual and cumulative effects of the variables as well as the possible correlations that existed between them. Ethanol Fermentation of Waste Paper Hydrolyzate The yeast S. cerevisiae CTM-30101 was used in all fermentation assays. A filter-sterilized hydrolyzate produced from office paper was used as a fermentation medium for bioethanol production using separate saccharification and fermentation (SHF). Experiments were performed in 1-L Erlenmeyer flasks containing 200 mL of fermentation medium supplemented with nitrogen sources that composed YMP medium (3 g L−1 of yeast extract, 3 g L−1 of malt extract and 5 g L−1 of peptone). Flasks were inoculated with 5 % (v v−1) of yeast suspension obtained from growth for 15 h on YPG medium composed of 10 g L−1 of yeast extract, 10 g L−1 of peptone and 20 g L−1 of glucose. Control fermentation was also run on YMP medium supplemented with reagent-grade glucose added at the same concentration present in the waste paper hydrolyzate used for the SHF process. Cultivations were carried out in duplicate at 30 °C with 150 rpm (orbital shaking). Samples were collected during 72 h and centrifuged at 12,000 rpm for 10 min. The cell-free supernatants were used for the determination of ethanol produced and sugar consumed. Cell growth was monitored directly by reading the optical density at 600 nm.
Results and Discussion Enzyme Production for Waste Paper Hydrolysis Enzymatic preparations used for waste paper saccharification were produced with individual cultures of the hyper-cellulolytic strain T. reesei Rut C-30 and A. niger F38, carried out in controlled 3.6-L fermentor and 500-mL Erlenmeyer flasks, respectively. The effect of carbon source on cellulase and β-glucosidase (BGL) production has been studied previously in our laboratory (unpublished results), and wheat bran was selected as the best enzymes inducer. Wheat bran is a cheap raw material and its use for enzyme induction can reduce the cost of lignocellulosic ethanol production [27]. Table 1 shows the production level of crude cellulase and BGL secreted by T. reesei Rut C-30 and A. niger F38, respectively, after 5 days of culture. Crude Rut C-30 cellulase preparation had appreciable levels of filter paper (1.14 U mL−1) and
Appl Biochem Biotechnol Table 1 Crude enzyme produced from T. reesei Rut C-30 and A. niger F38 after 5 days of culture Fungus
Type of culture
Protein (mg mL−1)
Enzyme activities (U mL−1) Total cellulase activity (FP)a
Endoglucanase activity (CMC)
β-glucosidase activity (pNPG)
Xylanase activity (xylan)
Trichoderma reesei Rut C-30
Fermentor 3.6 L
4.4±0.3
1.14±0.26
0.76±0.21
0.22±0.08
25±4
Aspergillus niger F38
Flasks 500 mL
1.5±0.1
0.24±0.1
0.33±0.17
6.8±1.3
8.8±1.6
a
FP, CMC, pNPG and xylan were used as substrates for the enzyme activity assays
CMCase activity (0.76 U mL−1) but lesser BGL activity (0.22 U mL−1). On the contrary, the crude A. niger preparation had a lesser filter paper (0.24 U mL−1) and CMCase activities (0.33 U mL−1) while the BGL activity was several fold higher and reach 6.8 U mL−1. T. reesei is a well-studied filamentous fungus for cellulase production, but it is endowed with a low BGL activity, causing accumulation of cellobiose, which produces repression and end-product inhibition during hydrolysis. Aspergillus sp. produces BGL in significant amounts as observed by Ahamed and Vermette [28]. It is worthy to note that adding external BGL decreases the inhibitory effect of cellobiose accumulated in the enzymatic hydrolysis [5]. On the other hand, xylanase activity was largely produced from both cultures. This enzyme may play an important role in hemicellulosic fraction degradation. It has been reported by many authors that xylanase supplementation clearly increased cellulose hydrolysis in xylancontaining lignocellulosic materials. The hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose and prevent the access of enzymes to cellulose surface [29]. Waste Paper Composition The biochemical characteristics of waste paper (office paper and newspaper) were analyzed (Table 2). Office paper is distinguished by high cellulose content (w w−1, 78.6 %) and a very low hemicellulose and lignin content (4.7 and 1.2 %, respectively). This characteristic is more suitable for fermentable sugar production because there will be less physical barrier during the enzymatic hydrolysis of cellulose fraction [2]. Table 2 shows that the level of cellulose in office paper, measured in different studies, ranged from 55.7 to 87.4 %. The low cellulose content (55.7 %) of the office paper tested by Wang et al. [30] is likely due to the presence of inorganic coatings (13 %) in the sample [31]. On the other hand, Foyle and co-workers [4] have demonstrated that not only the origin of pulp paper can affect the level of cellulose content but also the measurement method can also influence the biochemical composition. For this reason, it is necessary to choose the method that allows the minimum loss in carbohydrate content [4]. Compared to office paper, it is evident that newspaper has significantly more lignin (19.18 %) and hemicellulose (12.2 %) and substantially less cellulose (49.3 %). This structural feature of newspaper therefore controls the extent of cellulose hydrolysis, a limitation that can be overcome by using several hydrolytic enzymes acting synergistically [32]. Results obtained here were in agreement to earlier studies, where lignin, holocellulose
Appl Biochem Biotechnol Table 2 Office paper and newspaper composition Waste paper ingredient (content %)a Moisture Office paper
Newspaper
a
Cellulose
Hemicellulose
Reference Lignin
Ash
Others
3.2
78.6
4.7
1.2
9
3.3
This work
1.4
87.4
8.4
2.3
–
–
[33]
2.4
64.7
13
0.93
–
–
[34]
4.9
55.7
13
5.78
7.57
13
[30]
3
49.3
12.2
19.18
1.5
4.9
This work
1.5
48.5
9
23.9
–
–
[33]
2 7.25
48.3 43.7
18.1 16.7
22.1 16.8
– 9.5
– 6
[34] [30]
Data are presented as the mean of tow replicates
(cellulose and hemicellulose) and moisture content in newspaper were found to be 16–23, 57.5–66.4 and 3–7.2 %, respectively [30, 33, 34]. Enzymatic Digestibility Test It is necessary to evaluate the hydrolytic performance of any enzymatic preparation before its use. In this purpose and in order to produce fermentable sugars from waste paper, our enzyme mixture was compared with other commercial and industrial cellulases. Newspaper and office paper were hydrolyzed using the same enzyme activity (10 FPU g−1 of substrate). Figure 1 shows the degrees of saccharification after 48 h of hydrolytic reactions using different cellulases. Our local enzyme preparation and the industrial cellulase (Kamaistone K-050) provide highest degree of Office paper
Newspaper
Residual activity
80 70 70 60 60 50
50
40
40
30
30 20
20
10
10
0
0 Mixture
Denimax 98-2
Novo 342
Kamaistone
Cellusoft
Fig. 1 Saccharification degrees of office paper and newspaper using different enzyme preparations and their correspondent residual activities determined after 48 h of operating time. Mixture: crude enzyme obtained from T. reesei Rut C-30 and A. niger F38 (residual activities presented here are averages of three hydrolysis conditions)
Appl Biochem Biotechnol
saccharification using office paper (63 %, 68 %) or newspaper (29 %, 32 %) as substrate, respectively. This is due to the high level of BGL activity in Kamaistone K050 preparation and in our enzymatic mixture (85 U g−1 of substrate and 60 U g−1 of substrate, respectively). However, Denimax 98-2, Novo 342 and cells of cellulases contain relatively lower BGL activity (18, 22 and 12 U g−1, respectively) and consequently exhibit a low degree of saccharification. BGL activity is a key enzyme in the cellulolytic system of several microorganisms. Cellobiose, potential substrate for BGL activity, is a potent inhibitor of endo- and exoglucanases and therefore its accumulation significantly slows down the overall hydrolysis process [35]. Moreover, at the end of each hydrolytic reaction, residual activity of all enzymatic preparations was determined (Fig. 1). Our enzymatic mixture and Kamaistone K-050 cellulase showed better functional stability and are able to retain about 66 and 71 % of their initial activities, respectively, probably due to their good thermal stabilities. Operational stability is a much desired enzymatic characteristic for industrial applications especially in continuous saccharification process and subsequently can reduce significantly the cost of lignocellulosic ethanol production [36]. It is useful to note that the simultaneous use of the two enzyme preparations derived from T. reesei Rut C-30 and A. niger F38 improves the waste paper hydrolysis yield comparing at each preparation used alone (data not shown). All of this shows that our enzyme mixture is very suitable for lignocellulosic waste saccharification and will be employed in the following work. Effect of Surfactant Pretreatment on Enzymatic Digestibility It is a well-known fact that the addition of a surfactant into the enzymatic hydrolysis of lignocellulosic biomass increases its digestibility [32]. However, previous studies have used surfactants in the hydrolysis stage only. In this study, five different surfactants were evaluated for their ability to enhance enzymatic hydrolysis of waste paper (Fig. 2). All surfactants used increase the efficiency of hydrolysis of the newspaper but they have no noticeable effect on office paper. Triton X-100 at 0.5 % (w w−1) improved the digestibility of newspaper about 180
Hydrolysis yield (%)
160 140 120 100 80 60 40 20 0 Control
SDS
Tw een 20
Tw een 80 Triton X-100
PEG 6000
Surfactant (0.5 % w/w) Fig. 2 The effects of surfactant pretreatment on the enzymatic digestibility of newspaper and office paper
Appl Biochem Biotechnol
45 %. It has been demonstrated that the addition of surfactants, particularly the nonionic ones, can significantly improve the release of sugars from waste or recycled newspaper [25, 37]. Newspaper has a relatively high content of lignin compared to office paper, as shown in Table 2. Responsible of reducing cellulose accessibility to cellulase, lignin can impede cellulose hydrolysis via its ability to bend enzyme phenomenon which can be minimized through the addition of surfactant. It is believed that the surfactant adsorbs onto the lignin polymer, thereby preventing cellulase from adsorbing onto the same binding site [38]. Although several mechanisms have been proposed for the positive effect of surfactant pretreatment on cellulose hydrolysis, the enzyme-substrate interaction is one of the most supported [32, 38]. The relatively low lignin content of office paper (Table 2) could explain the insensitivity of sugar yield to a surfactant pretreatment. Triton X-100 will be maintained for only newspaper surfactant pretreatment. Response Surface Models A CCD was performed to determine the optimum levels of three independent variables (dry matter quantity (X1), H3PO4 concentration (X2) and hydrolysis time (X3)) and the effect of their interactions on the degree of saccharification of newspaper and office paper. The results obtained were subjected to an analysis of variance (ANOVA) to determine the significant differences. Table 3 represents the design matrix of the variables together with experimental results. Experiments 1–14 were performed at the different combinations and those from 15 to 20 were under the same conditions. By applying multiple regression analysis on the experimental data, the following second polynomial equations (Eq. (3) and Eq. (4)) were established to explain the degree of saccharification of each substrate, newspaper (Y1) and office paper (Y2), respectively. Y 1 ¼ 58:591 þ 8:953X 1 þ 7:158X 2 þ 3:406X −8:206X 1 2 −2:332X 2 2 −2:747X 3 2 þ 0:884X 1 X 2 þ 5:079X 1 X 3 −1:739X 2 X 3
ð3Þ Y 2 ¼ 85:807−4:162X 1 þ 6:317X 2 þ 10:685X 3 −15:861X 1 2 −7:465X 2 2 −5:414X 3 2 −0:812X 1 X 2 −5:762X 1 X 3 þ 6:737X 2 X 3
ð4Þ where Y is the predicted degree of saccharification; X1, X2 and X3 are the coded values of dry matter quantity, phosphoric acid concentration and hydrolysis time, respectively. The significance of the regression coefficients was given by a t test. The regression coefficients and corresponding P values for the models, corresponding to the degree of saccharification of newspaper and office paper, are presented in Table 4. The P values are used as a tool to check the significance of each of the coefficients which are, in turn, necessary to understand the pattern of the mutual interactions between variables. In fact, when the magnitude of the t test value is large and the P value is small, this indicates that the corresponding coefficient is highly significant [39]. As far as the current study, the estimated parameters and the corresponding P values suggest that, all independent factors, X1 (dray matter quantity), X2 (phosphoric acid concentration) and X3 (hydrolysis time) had a significant effect on the hydrolysis rate of newspaper and office paper. The quadratic term of the three factors, especially in the case of office paper saccharification, had a significant effect. On the other hand, the interactions between the factors do not always have a significant effect on the responses. The ANOVA for the quadratic regression model is given also in Table 4. These results show that the models predicted for the degree of saccharification of newspaper and office paper were adequate. The regression model
Appl Biochem Biotechnol Table 3 The central composite design matrix employed for three independent variables and experimental responses of the degree of saccharification of newspaper (Y1) and office paper (Y2) Number rune
Variables (X1) Dry matter (g L−1)a
Responses (X2) H3PO4 (g L−1)b
(X3) Time (h)c
Newspaper Y1
Office paper Y2
1
−1
−1
−1
29.86
56.90
2
1
−1
−1
40.05
52.50 55.50
3
−1
1
−1
50.36
4
1
1
−1
55.20
42.00
5
−1
−1
1
31.20
73.50
6
1
−1
1
52.82
40.20
7
−1
1
1
35.86
93.20
8 9
1 −1.681
1 0
1 0
69.90 19.55
62.50 30.00
10
1.681
0
0
50.22
44.90
11
0
−1.681
0
39.50
44.50
12
0
1.681
0
63.50
77.90
13
0
0
−1.681
40.75
42.20
14
0
0
1.681
59.90
91.80
15
0
0
0
60.22
88.32
16 17
0 0
0 0
0 0
55.50 59.50
86.22 87.50
18
0
0
0
60.50
84.50
19
0
0
0
55.60
86.00
20
0
0
0
60.40
83.50
a
X1: Dry matter (g L−1 ) (−1.681=5; −1=15; 0=30; +1=45; +1.681=55)
b
X2: H3PO4 (g L−1 ) (−1.681=2; −1=3; 0=5; +1=7; +1.681=8)
c
X3: Time (h) (−1.681=13; −1=20; 0=30; +1=40; +1.681=47)
was highly significant (P
